UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO DOCTORADO EN CIENCIAS BIOMÉDICAS INSTITUTO DE ECOLOGÍA ANÁLISIS DE LOS GENES DE AUTOFAGIA EN LAS RAÍCES DE Phaseolus vulgaris DURANTE LA NODULACIÓN TESIS QUE PARA OPTAR POR EL GRADO DE: DOCTORA EN CIENCIAS PRESENTA: BIÓL. EXP ELSA HERMINIA QUEZADA RODRÍGUEZ TUTORA PRINCIPAL: DRA.KALPANA NANJAREDDY ESCUELA NACIONAL DE ESTUDIOS SUPERIORES, LEÓN CIENCIAS AGROGENÓMICAS CIUDAD UNIVERSITARIA, CD.MX, 2022 UNAM – Dirección General de Bibliotecas Tesis Digitales Restricciones de uso DERECHOS RESERVADOS © PROHIBIDA SU REPRODUCCIÓN TOTAL O PARCIAL Todo el material contenido en esta tesis esta protegido por la Ley Federal del Derecho de Autor (LFDA) de los Estados Unidos Mexicanos (México). El uso de imágenes, fragmentos de videos, y demás material que sea objeto de protección de los derechos de autor, será exclusivamente para fines educativos e informativos y deberá citar la fuente donde la obtuvo mencionando el autor o autores. Cualquier uso distinto como el lucro, reproducción, edición o modificación, será perseguido y sancionado por el respectivo titular de los Derechos de Autor. 2 A MI FAMILIA Y SERES QUERIDOS POR SU INFINITO AMOR Y APOYO TO MY FAMILY AND MY LOVED ONES FOR THEIR INFINITE LOVE AND SUPPORT 3 ACADEMIC ACKNOWLEDGEMENTS THIS THESIS WAS REALIZED AT THE LABORATORY OF INTERDISCIPLINARY RESEARCH IN THE AREA OF AGRONOMIC SCIENCES, ENES-LEÓN-UNAM AND, ECOLOGY INSTITUTE UNDER MENTORING OF DR. KALPANA NANJAREDDY. I WOULD LIKE TO ACKNOWLEDGE TO POSTGRADUATE IN BIOMEDICAL SCIENCE AND CONACYT FELLOWSHIP (No. 409344/289810) AS WELL AS DGAPA/PAPIIT-UNAM (GRANT No. IN211218 AND IN216321 IN216321) SUPPORT FROM DRA. KALPANA NANJAREDDY AND CONACYTCF-MI- 20191017134234199/316538 FROM DR. MANOJ KUMAR ARTHIKALA AGRADECIMIENTOS ACADÉMICOS ESTA TESIS FUE REALIZADA EN EL LABORATORIO DE INTERDISCIPLINARIEDAD INVESTIGACIÓN EN EL ÁREA DE CIENCIAS AGRONÓMICAS, ENES-LEÓN-UNAM Y EL INSTITUTO DE ECOLOGÍA BAJO LA TUTORIA DE LA DRA. KALPANA NANJAREDDY. LE AGRADEZCÓ AL POSGRADO DE CIENCIA BIOMÉDICAS Y CONACYT POR LA BECA DE CONACYT (No. 409344/289810), ASI COMO EL APOYO DE /PAPIIT-UNAM (No. IN211218 E IN216321 IN216321) DE LA DRA. KALPANA NANJAREDDY Y CONACYTCF-MI-20191017134234199/316538 DEL DR. MANOJKUMAR ARTHIKALA. 4 ACKNOWLEDGEMENTS AGRADECIMIENTOS En el transcurso de este proyecto he conocido muchas personas que me han enseñado sobre la investigación científica, los métodos del laboratorio, los valores del investigador y la fuerza mental para continuar con ímpetu cada proyecto. Comienzo agradeciéndole a la Dra. Aurea Orozco Rivas quien fue coordinadora del Programa de Ciencias Biomédicas a la Dra. Laura Roxana Torres Avilés quien como responsable de posgrado del instituto de Ecología y a la Lic. Erika Rodríguez por el apoyo en los trámites requeridos en todo momento, así como el buen trato recibido en los momentos más delicados de mi trayecto en el posgrado. Le agradezco al Dr. Miguel Lara que me guío y apoyo desde el inicio para finalmente desarrollar esta tesis con una gran investigadora y persona, la Dra. Kalpana Nanjareddy a quien le agradezco profundamente por aceptarme en su laboratorio al igual que al Dr. Manoj Arthikala por enseñarme y motivarme al ver sus actitudes positivas, su pasión y energía al realizar su trabajo, ambos me llenaron de fuerza y seguridad para avanzar académicamente. También le agradezco a mi jurado: Dra. Helena Porta, Dr. Luis Cárdenas, Dra. María de la Paz, Dra. Ma. De la Paz, Dra. María Del Rocío Cruz y Dra. Esperanza Martínez por la revisión de esta tesis y sus comentarios que fueron valiosos aportes y enseñanzas. Durante el tiempo que realicé los estudios de posgrado recibí ayuda tanto académica como emocional para continuar y terminar el posgrado. Por lo que le agradezco a la Dra. Judith Márquez, Dra. Gladys Cassab, Dr. Felipe Jiménez, Paty Rueda, Dr. Erik Cruz, Dra. Alejandra Barrera, Dra. Larissa Tolalpa, Mtr. Marimar Garciadiego, Dr. Ubaldo, Dr. Homero Gómez y Dr. Luis Xoca por su tiempo y apoyo en general a todos los investigadores del laboratorio de ecofisiología del Instituto de Ecología, Instituto de Biotecnología y del Laboratorio de Agrogenómicas ENES-León. En el aspecto personal estoy eternamente agradecida por el apoyo brindado por mi familia, por sus bonitas palabras para seguir adelante, sus enseñanzas, por su amor y cariño, mis infinitas gracias a mis padres Mtr. Elsa Patricia Rodríguez Reyes y el Ing. Fernando Quezada Buendía, a mi hermano quien es un ejemplo de dedicación y lucha, Lic. Fernando Pascual Quezada Rodríguez, a Francisco Maldonado, Maura León, Paco y José por el apoyo que me dieron muchas gracias. También agradezco profundamente al Dr. Camilo Alcántara por construir y acompañarme en este trayecto académico, pero también por luchar por un mejor futuro, así como también por aprender juntos y compartirme experiencias académicas y de vida, gracias también a su familia a quienes aprecio mucho. No pueden faltar mis amigos con quienes compartí clases y el tiempo de laboratorio. Definitivamente fueron momentos muy especiales porque son muy buenas personas gracias, Mena, Martín, Ángel, Chocks, Humberto, Pale, Esther, Alexis, Diana, Laura, Laura Malagón, Betty, Guadalupe. A mis amigas Lucero y Andrea por escucharme y animarme a seguir. Gracias a mis compañeros y amigos del laboratorio de la ENES León a Caro, Salma, Paula, Ana, Gabriel, Carmen y Eli. Gracias por las enseñanzas y revisiones del inglés a mi profesora Rosy Bautista, David y Elisa. A mis amigos y familia Rod, Eddy y Ali con quien compartí el gusto y emoción por las plantas 24/7 y la atención de sus familias a quienes aprecio mucho, a la mamá de Ali por el cariño y apoyo. A todos los “canallitas” de las primeras generaciones de la UAM-I por inspirarme y sus familias, de quienes tengo buenos recuerdos. A mis compañeros del Movimiento por la Ciencia y Emisores de la ciencia a Mario, Dnefertém, Aldo, Guadalupe, Guillen, Luis, Fernando, Rodrígo, Ángel, Christian, Esteban, Ely, Rocio, Dany, Erick y José por animarme viendo sus ganas en la divulgación por la ciencia y por mejorar las condiciones de los trabajadores de la ciencia. 5 GENERAL ABSTRACT The present studies report the role of autophagy genes during symbiosis between Rhizobium and P. vulgaris. Nitrogen fixing symbiotic interaction between legumes- Rhizobium is very important as it contributes to the high nutritional status of the legumes. The host plant undergoes various physiological, biochemical, and developmental changes to accommodate the symbiont for the mutual benefit. Autophagy is one such biological process of cellular degradation to maintain homeostasis. However, the knowledge of autophagy genes in legumes is less explored and hence the possible role such important genes during symbiosis is even sparse. As presented in the chapter II of the present investigation, autophagy genes in Phaseolus vulgaris (common bean), Medicago truncatula and Glycine max (Soybean) across 17 families were identified and analyzed bioinformatically. Further, ATG18 family, a complex autophagy gene family was explored in-depth to understand the phylogeny, domain structures etc. to classify the ATG18 family into 3 subfamilies. Transcriptomic analysis of P. vulgaris roots inoculated with Rhizobium revealed PvATG9b as a candidate gene to carryout functional analysis under symbiotic conditions. In the chapter III, spatio-temporal studies of PvATG9b promoter revealed nodule specific expression. While RNAi silencing resulted in reduction of secondary roots and nodule numbers, overexpression reversed both the root and nodule phenotype. Subcellular localization of PvATG9b protein was found to be localized to the plasma membrane and nucleus. Chapter IV presents the Y2H interactions of PvATG9b and P. vulgaris cDNA library under symbiotic conditions. The Y2H showed a total of 24 interacting proteins and plant cysteine oxygen 2 (PCO2) was found to be an important partner among others. PCO2 is an element in the hypoxia response that is important in the functioning of nitrogenase during nitrogen fixation. Taken together, we identified autophagy genes in three legumes, and we explored in detail the ATG18 family. We also recognized that PvATG9b is highly expressed during symbiosis between bean and Rhizobium, an in-depth analyses revealed the role of PvATG9b in nodule development and nitrogen fixation probably by maintain hypoxic condition during nitrogen fixation through PCO2. 6 RESUMEN GENERAL La presente tesis estudia los genes de autofagia durante la simbiosis entre Rhizobium y Phaseolus vulgaris. La interacción simbiótica para la fijación de nitrógeno entre leguminosas-Rhizobium es muy importante ya que contribuye al alto estado nutricional de las leguminosas. La planta huésped sufre varios cambios fisiológicos, bioquímicos y de desarrollo para adaptarse al simbionte en beneficio mutuo. La autofagia es uno de esos procesos biológicos de degradación celular que contribuye a mantener la homeostasis. Sin embargo, poco se sabe de los genes de autofagia en las leguminosas y es aún menos explorado durante la simbiosis. En el capítulo II de la presente investigación, se identificaron y analizaron bioinformáticamente genes de autofagia en P. vulgaris (frijol común), Medicago truncatula y Glycine max (soja) en 17 familias. Además, la familia ATG18, una familia compleja de genes de autofagia se exploró con más detalle para comprender la filogenia, las estructuras de dominio, etc. para clasificar la familia ATG18 en 3 subfamilias. El análisis transcriptómico de raíces de P. vulgaris inoculadas con Rhizobium reveló que PvATG9b es un gen candidato para realizar análisis funcionales en condiciones simbióticas. En el capítulo III, los estudios espaciotemporales del promotor PvATG9b revelaron una expresión específica en el nódulo. Mientras que el silenciamiento con RNAi dio como resultado una reducción del número de nódulos y raíces secundarias, la sobreexpresión revirtió tanto el fenotipo de la raíz como el del nódulo. Se encontró que la localización subcelular de la proteína PvATG9b estaba localizada en la membrana plasmática y el núcleo. El Capítulo IV presenta las interacciones Y2H de PvATG9b y la biblioteca de ADNc de P. vulgaris en condiciones simbióticas. El Y2H mostró un total de 24 proteínas que interactúan y se descubrió que el oxígeno 2 de cisteína vegetal (PCO2) era un socio importante, entre otros. PCO2 es un elemento en la respuesta de hipoxia que es importante en el funcionamiento de la nitrogenasa durante la fijación de nitrógeno. En conjunto, identificamos genes de autofagia en tres leguminosas y exploramos en detalle la familia ATG18. También reconocimos que PvATG9b se expresa mucho durante la simbiosis entre el frijol y Rhizobium, un análisis detallado reveló el papel de PvATG9b en el desarrollo de nódulos y la fijación de nitrógeno, probablemente al mantener la condición hipóxica durante la fijación de nitrógeno a través de PCO2. 7 LIST OF TABLES TABLE 1.GENES OF AUTOPH AGY (ATGS) IN PLANTS. ..................................................................................................................................................................................... 44 TABLE 2. IDENTIF ICAT ION OF 17 GENE FAMILIES IN A. TH ALIAN A, P. VU LGARIS, M. TRUNCATU LA AND G. MAX........................................................................................................ 49 TABLE 3 ATG9 INTER ACTION S R EPORTED IN YEAST, MAMMALS AN D PLANTS. ................................................................................................................................................ 101 TABLE 4. 24 INTERACTING PARTNER S OF PVATG9B .................................................................................................................................................................................... 103 TABLE 5. ONTOLOGY ENRICHM ENT OF PVATG9B-IN TER ACTIN G PARTN ERS BY PANTHER. .............................................................................................................................. 105 LIST OF FIGURES FIGURE 1. TYPES OF AUTOPHAGY. (A) MACROAUTOPHAGY IS A PROCESS WHICH INVOLVES THE FORMATION OF THE AUTOPHAGOSOME, CHAPERONE-MEDIATED AUTOPHAGY (CMA) IS LEADS BY THE TRANSLOCATION OF PROTEIN BOUND AND MICROAUTOPHAGY IS A PROCESS WHICH SECLUDE THE TARGET COMPONENTS NEAR TO LYSOSOME OR VACUOLE. FINALLY, ALL OF THESE TYPES OF AUTOPHAGY END IN THE LYSOSOME OR VACUOLE (HO ET AL., 2019). (B) TYPES OF AUTOPHAGY CONFIRMED IN PLANTS. MACROAUTOPHAGY REQUIRE AUTOPHAGOSOME THAT FUSES INTO THE VACUOLE, MICROAUTOPHAGY COMPRISE A INVAGINATION OF THE TONOPLAST AND MEGA AUTOPHAGY IMPLY THE RUPTURED OR PERMEABLE TONOPLAST THAT RELEASE LYTIC CONTENTS INTO CYTOPLASM (WOJCIECHOW SKA ET AL., 2021) .................................................................................... 17 FIGURE 2 AUTOPH AGY DUR ING DEVELOPMEN T, HORM ONES, ABIOTIC STR ESSES, AND BIOT IC STRESSES REPORTED IN PLANTS (BASED ON GOU ET AL., 2019; FEDEROFF, 2012) ........ 23 FIGURE 3 NODULE DEVELOPMENT IN LEGUMES AND INFECTION THREAT. (A) THE FORMATION OF THE THREAD OF INFECTION BEGINS WITH THE CONTACT OF THE BACTERIA WITH THE ROOT HAIR (RH) (1A), CAUSING THE ROOT HAIR TO CURL (2A) AND THE NUCLEUS TO MOVE SURROUNDED BY A CYTOPLASMIC STREAMING (3A) THAT DIRECTS THE BACTERIA (4A) TOWARDS THE ROOT HAIR BASE NEAR TO CORTICAL CELLS (5-7A) AND THEN THE INFECTION THREATS BRANCHES. (B) DEVELOPMENTAL STAGES OF DETERMINATE LEGUME NODULES. ONCE THE ROOT HAIR CURVES, THE CORTICAL CELLS DIVIDED IN SUB-DERMICAL. BEGINNING WITH ANTICLINAL CORTICAL CELLS (1B) AND THE PERICLINAL CELL DIVISION (2B). THE INFECTION THREAT PROGRESS INTO OUTER CORTEX(3B) THEN INTO INNER CORTEX(4B). THE CELL LAYERS DIVIDED FORM THE NODULE PRIMORDIUM AND BEGAN TH E BAC TER OID D IFFER ENTIATION (6B) TO FOR A MATURE N-FIXIN G N ODULE (7B). (FER GUSON ET AL., 2010; RAE ET AL., 2021) .............................................. 32 FIGURE 4 MACROAUTOPHAGY OF YEAST AND ARABIDOPSIS. INDUCTION OF AUTOPHAGY IS REGULATED BY NUTRITIONAL STATUS. UNDER STARVATION ATG1 AND ATG13 ARE DEPHOSPHORYLATED AND PROMOTE THE ACTIVATION OF KINASE COMPLEX TO TRIGGER VESICLE NUCLEATION, VESICLE EXPANSION AND CLOSURE, FUSION, AND DIGESTION (THOMPSON ET AL., 2005; NAKATOGAWA ET AL., 2013)..................................................................................................................................................................... 47 FIGURE 5. PHYLOGENETIC TREE AND PROTEIN MOTIFS OF 17 ATG FAMILIES IN A. THALIANA, P. VULGARIS, M. TRUNCATULA AND G. MAX. CONSERVED MOTIFS ARE IDENTIFIED USING THE MEME SEARCH TOOL. TH E PHYLOGEN ETIC TREE W AS C ON STRUCTED USING TH E N EIGHBOR-JOININ G M ETHOD IN CLU STALW2 AND VISUALIZED U SING EVOLVIEW. .......... 51 FIGURE 6 THE CHROMOSOMAL LOCALIZATION, SYNTENY RELATIONSHIP AND GENE EXPRESSION OF AUTOPHAGY GENES WERE INTEGRATED INTO THE CIRCOS PLOT DESIGNED USING OMICCIRCOS. THE OUTERMOST CIRCLE SHOWS THE A. THALIANA (BLUE), P. VULGARIS (GREEN), M. TRUNCATULA (PINK) AND G. MAX (BROWN) CHROMOSOMES. THE INNER CIRCLE IS A HEATMAP THAT SHOWS THE LOG2 RPKM VALUES OF GENE EXPRESSION IN LEAVES AND ROOTS UNDER AMMONIA, NITRATE AND UREA TREATMENTS. THE INNERMOST LIN E IS TH E SYN TEN Y OF AUTOPHAGY GEN ES, BUT THE YELLOW, PUR PLE AND R ED LINES R EPR ESENT ATG18B SUBFAMILIES I, II AND III, RESPECTIVELY. ............ 52 FIGURE 7 3D REPRESENTATION OF 280 ATG18 PROTEINS FROM A DIFFERENT PLANT SPECIES ANALYZED BY MULTIDIMENSIONAL SCALING USING BIOS2MDS. ATG18 SUBFAMILIES COLORS CODE ARE SUBFAMILY I (YELLOW), SUBFAMILY II (PURPLE), SUBFAMILY III (RED). PC PRINCIPAL COMPONENT. AXIS ARE PRINCIPAL COMPONENTS (PC): THE X-AXIS (PC1); Y-AXIS (PC2); Z-AXIS (PC3). ................................................................................................................................................................................................ 54 FIGURE 8 PHYLOGENETIC TREE OF ATG18 PROTEINS IN PLANTS. PROTEIN SEQUENCES WERE ALIGNED USING CLUSTAL OMEGA AND THE PHYLOGENETIC TREE WAS CONSTRUCTED USING THE ML METHOD IN MEGA X SOFTWARE. 280 SEQUENCES OF ATG18 ARE DISTINGUISHED BY SUBFAMILIES: SUBFAMILY I (YELLOW), SUBFAMILY II (PURPLE), SUBFAMILY III(RED). THE PLANT SPECIES ARE DIFFERENTIATED BY LETTERS. A. THALIANA (AT), M.POLYMORPHA (MPO), O.SATIVA (OS), T. AESTIVUM (TA), ZEA MAYS (ZM), A. DURANENSIS (AD), A. IPAENSIS (AI), C. CAJAN (CC), L. JAPONICUS (LJ), C. ARIETINUM (CA), L. ANGUSTIFOLIUS (LA), P. SATIVUM (PS), V. ANGULARIS (VA), V. RADIATA (VR) AND TRIFOLIUM PRATENSE (TP), P. PATENS, C. BRAUNII (CB), C. REINHARDTII (CR), D. SALINA (DS), V. CARTERI (VC), K. NITENS (KN) , M. PUSILLA (MPU), O. LUCIMARINUS (OL), O. TAURI (OT) AND C. SUBELLIPSOID EA (CS). TH E BRAN CH LEN GTHS AR E LABELED. .................................................................................................................................................. 55 FIGURE 9 PROTEIN MOTIF OF ATG18 FAMILY FROM DIFFERENT PLANT SPECIES. CONSERVED MOTIFS ARE IDENTIFIED BY MEME. THE AMINO ACID SEQUENCE OF THE ATG18 FAMILY IS REPR ESENTED BY LIN ES AND MOTIFS BY BOXES USING TBTOOLS. MOTIF 1 (GREEN), M OTIF 2 (YELLOW), MOT IF 3 (D ARK GR EEN ), AND MOTIF 4 (PIN K). .................................. 57 FIGURE 10 TRANSCRIPT ION FACT OR BINDIN G SITES IN ATG PROM OTER S (2000PB) U SIN G PLATCARE. ................................................................................................................ 58 FIGURE 11 EXPRESSION PROFILES OF ATGS IN P. VULGARIS. EXPRESSION PROFILE IN DIFFERENT TISSUES AND ORGANS OBTAINED IN PHYTOZOME DATABASE. THE HEATMAP WAS BUILT WITH THE LOG2 OF FPKM VALUE AND ORD ERED BY DISTANC ES BETWEEN SAM PLES (R EPR ESENTED BY DENDR OGR AMS) ................................................................. 59 FIGURE 12 EXPRESSION PROFILES OF ATGS IN P. VULGARIS. HEAT MAP OF DIFFERENTIAL EXPRESSION OF ATGS IN TISSUES AND ORGANS DURING DIFFERENT STAGES OF DEVELOPM ENT AND DUR ING RH IZOBIA INFECTIONS OBTAINED IN P VGEA DATABASE. E XPR ESSION VALUES ARE FPKM NORM ALIZED WITH LOG2.......................................... 60 FIGURE 13 TRASNCRIPTOMIC DATA AND EXPRESSION PATTERNS OF P.VULGARIS NOLUDATED ROOTS (A) LOG2 RPKM AND FOLD CHANGE OF CONTROL AND NODULATED ROOTS. RED REPRESENT THE FC>2 AND BLUE FC<2. (B) FOLD CHANGE OF AUTOPHAGY CORE IN NODULATED ROOT OF P. VULGARIS. (C) EXPRESSION OF PVATG9 CONTROL BY RT- QPCR AN ALYSIS. TRAN SCRIPT ACCUMULATION W AS NORM ALIZED TO TH E EXPR ESSION OF METALLOPR OTEINASE AS R EFER ENCE GENE. .................................................... 61 FIGURE 14 AUTOPHAGOSOME MORPHOLOGY IN WILD TYPE AND ATG9 MUTANT OBTAINED IN ELECTRON MICROSCOPY (EM) OR FLUORESCENCE MICROSCOPY (FM)(ZHUANG ET AL., 2018).......................................................................................................................................................................................................................................... 75 FIGURE 15 SCHEMATIC REPRESENTATION OF PVATG9B (PHVUL.007G194300). PVATG9B CONTAINS 5.775KB WITH NINE EXONS AND EIGHT INTRONS. BLUE BOXES: EXONS; BLACK LIN E: INTR ONS. ............................................................................................................................................................................................................................. 76 FIGURE 16 PHYLOGENETIC TREE OF ATG9. NEIGHBOR-JOINING TREE USING PROTEIN SEQUENCES OF A. THALIANA, P. VULGARIS, M.TRUNCATULA, G. MAX, YEAST, AND HUMAN IN CLUSTAL OM EGA AND D ESIGN ED IN EVOLVIEW. ................................................................................................................................................................................. 77 FIGURE 17 ROOT EXPRESSION OF PVATG9B GENE PROMOTER. PROMOTOR ACTIVITY WAS DETECTED BY GUS STAINING (BLUE) DURING P. VULGARIS ROOTS OF 6 AN 10 DAYS USING OPTICAL MICROSCOPY. (A) UNINOCULATED ROOT ,6DAYS (B) INOCULATED ROOT ,6 DAYS.(C)UNINOCULATED ROOT, 10 DAYS. (D)INOCULATED ROOT, 10 DAYS. INOCULATED ROOT SHOWED MORE GUS STAINING THA UNINOCULATED ROOTS. ELONGATED ZONE (EZ), TRANSITION ZONE (TZ), MERISTEMATIC ZONE (MZ),LATERAL ROOT CAP (LRC) AND VASCULAR T ISSUE(V). SCALE BAR:1MM (A AND B), 2MM (C AND D). ................................................................................................................................................... 78 FIGURE 18 PVATG9B EXPRESSION PATTERNS DURING LATERAL ROOT FORMATION. PROMOTOR ACTIVITY WAS DETECTED BY GUS STAINING (BLUE) DURING P. VULGARIS LATERAL ROOT DEVELOPMENT USING OPTICAL MICROSCOPY. (A) AND (B) GUS STAINING WAS DETECTED IN CENTRAL CELLS OF LATERAL ROOT PRIMORDIUM IN STAGE VII (UNINOCULATED, 13DAYS). (C) LATERAL ROOT PRIMORDIUM IN STAGE VII (INOCULATED, 13 DPI) (D) EMERGENCE OF LATERAL ROOT (INOCULATED, 13DPI). (C) AND (D) SHOWED THE GUS STAIN ING IN PERIPH ERIAL C ELLS. EPID ERMIS (E);VASCU LATUR E (V) SCALE BARR: 1MM ............................................................................................. 78 FIGURE 19 EXPRESSION PATTERNS IN EARLY STAGES OF NODULE DEVELOPMENT. PROMOTOR ACTIVITY WAS DETECTED BY GUS STAINING (BLUE) DURING P. VULGARIS NODULE DEVELOPMENT USING OPTICAL MICROSCOPY (A) NODULATED ROOT AND CURLY HAIRY ROOT (HR), WHICH EXPRESSION WAS DETECTED IN TWO NODULE PRIMORDIUM AND VASCULATUR E. (B) NODU LE PRIM ORDIA OF 13 DPI HAS EXPRESSION IN VASCULATUR E AND AR OUND TH E INFECT ION ZON E. SCALE B ARR:1.25MM........................................ 79 FIGURE 20 EXPRESSION OF PVATG9B DURING NODULATION. PROMOTOR ACTIVITY WAS DETECTED BY GUS STAINING (BLUE) DURING P. VULGARIS NODULE DEVELOPMENT USING OPTICAL MICROSCOPY. (A) AND (B) RHIZOBIA INVASION INTO NODULE PRIMORDIA. (C) AND (D) YOUNG NODULE. (E) NODULE TRANSITION TO MATURATION. (F) MATURE N- FIXATION NODULE. THE EXPRESSION WAS MAINTAINED IN VASCULAR TISSUE. NODULE PRIMORDIUM(P); PROVASCULAR BUNDLE (PVB); VASCULATURE TISSUE (V); BACTERIA (B); NITR OGEN FIXIN G ZONE (NFZ) SCALE BARRS: 1.25MM(A);2MM(B,C,D);1MM(D) ............................................................................................................................ 79 FIGURE 21, PVATG9B EXPRESSION IN MATURE NODULES. PROMOTOR ACTIVITY WAS DETECTED BY GUS STAINING (BLUE) DURING P. VULGARIS NODULE DEVELOPMENT USING OPTICAL MICROSCOPY. (A) MATURE NODULE, LONGITUDINAL VIEW(B) MATURE NODULE- TRASNVERSAL VIEW. THE EXPRESSION APPEREAD IN VASCULATURE. NODULE CORTEX (NC);VASCULAR BUNDLE (VB);DEVELOPMENTAL ZON E (DZ); INFECTED ZONE (IZ); NODU LE PARENCH YMA (NP);NODULE M ERISTEM (NM). SCALE BARRS: 2MM ................... 80 8 FIGURE 22 TRANSCRIPT LEVELS OF 35S-PVATG9B-RNAI BY RT-PCR IN HAIRY ROOTS (DPI). RELATIVE TRANSCRIPT LEVELS WERE NORMALIZED WITH METALLOPROTEASE. WE COMPARED TRAN SFORM ED H AIR Y ROOTS OF SILENCIN G (35S-PVATG9B-RNAI) WITH EMPTY VECTOR (EV). ........................................................................................... 81 FIGURE 23 HAIRY ROOTS OF SILENCING OF PVATG9B. ROOTS OBSERVED UNDER OPTICAL MICROSCOPY WITHOUT STAINING. (A)EMPTY VECTOR (CONTROL-EV) AND (B) SILENCING OF PVATG9B ( PVATG9B-RNAI) WE OBSERVED THE REDUCED SIZE OF HAIRY ROOTS IN PVATG9B-RNAI TRANSFORMED ROOTS COMPARED WITH CONTROL-EV . SCALE BARR:2MM ................................................................................................................................................................................................................................... 81 FIGURE 24 SILENCING OF PVATG9B PHENOTYPE. (A) POTS AND (B) ROOTS OF P. VULGARIS PLANTS AT 35 DPI GROWN WITH NITROGEN-LIMITED B&D SOLUTION (KNO3 2NM) TO PROMOTE N ODU LAT ION. PVATG9B- IRNA SIZE IS R EDUCED COM PAR ED WITH THE CONTR OL-EV SCALE BAR: 7CM .................................................................................. 82 FIGURE 25 ROOT ARCHITECTURE OF PVATG9B SILENCING PLANTS. BAR PLOTS OF P. VULGARIS ROOTS AT 35 DPI GROWN WITH NITROGEN-LIMITED B&D SOLUTION (KNO3 2NM) TO PROMOTE NODULATION. (A)ROOT AND INTERNODE LENGTH.(B)ROOT AND PLANT WEIGHT.(C) PRIMARY, SECONDARY AND TERTIARY ROOTS. RED BOXES:CONTROL EMPTY VECTOR; BLU E BOXES:PVATG9B-IRNA. SIGNIFIC ATIVE DIFFERENC E VALU ES AT P < 0.05 STUDENT’S T TEST (***). .................................................................................. 82 FIGURE 26 LEAVES PHENOTYPE OF PVATG9B-RNAI. PLANTS OF P. VULGARIS PLANTS AT 35 DPI GROWN WITH NITROGEN-LIMITED B&D SOLUTION (KNO3 2NM) TO PROMOTE NODULATION. (A)LEAVES OF CONTROL-EV AND PVATG9B.(B) LENGTH AND (C) WIDTH.PVATG9B-RNAI SHOWED SMALLER AND YELLOWISH LEAVES COMPARED WITH CONTROL. RED BOXES:CONTROL EMPTY VEC TOR; BLU E BOXES:PVATG9B-RNAI. SIGNIF ICAT IVE DIFFER ENCE VALU ES AT P < 0.05 STUDENT’S T TEST (***)........................ 83 FIGURE 27. INFECTION THREAT OF PVATG9B SILENCING ROOTS. ROOTS OBSERVED UNDER OPTICAL MICROSCOPY WITH GUS STAINING. (A) CONTROL AND (B) INFECTION THREAT PVATG9B-IRNA IN TRANSGENIC ROOT. BOTH SHOWED TYPICAL CURLY HAIRY ROOTS ROOTS OF P. VULGARIS PLANTS AT 30 DPI GROWN WITH NITROGEN-LIMITED B&D SOLUTION (KNO3 2NM) TO PROM OTE NODU LAT ION. HAIR Y R OOTS (HR) SCALE BARR:2MM ................................................................................................................... 83 FIGURE 28. MATURE NODULES OF PVATG9B-IRNA AT 30 DPI. ROOTS OBSERVED UNDER OPTICAL MICROSCOPY WITH GUS STAINING. (A)CONTROL -EMPTY VECTOR (EV). (B) SILENCING OF PVATG9B WITH IRNA(PVATG9-IRNA). P. VULGARIS PLANTS AT 35 DPI GROWN WITH NITROGEN-LIMITED B&D SOLUTION (KNO3 2MM) TO PROMOTE NODULATION. PVATG9B-IRNA SH OWED LESS EXPRESSION IN INFECT ION ZON E. INFECTION ZONE (IZ),VASCU LAR BUND LE (VB), NODU LE CORTEX(NC). SCALE BARRS: 2MM . 84 FIGURE 29 OVEREXPRESSION OF PVATG9B PHENOTYPE. (A) POTS AND (B) ROOTS OF P. VULGARIS PLANTS AT 35 DPI GROWN WITH NITROGEN-LIMITED B&D SOLUTION (KNO3 2NM) TO PR OMOTE N ODULATION. PVATG9B-OE SIZE IS GR EATER COM PAR ED WITH THE CONTR OL-EV SCALE BAR: 7CM ................................................................................ 85 FIGURE 30 ROOT ARCHITECTURE OF PVATG9B-OE PLANTS. BAR PLOTS OF P. VULGARIS ROOTS AT 35 DPI GROWN WITH NITROGEN-LIMITED B&D SOLUTION (KNO3 2NM) TO PROMOTE NODULATION. (A)ROOT AND INTERNODES LENGTH.(B)ROOT AND TOTAL PLANT WEIGHT (C)PRIMARY, SECONDARY AND TERTIARY ROOTS. GREEN BOXES:OVEREXPR ESSION CONTROL; PURPLE BOXES:PVATG9B OVER EXPRESSION. SIGNIF ICAT IVE DIFFER ENCE VALU ES AT P < 0.05) STUDENT’S T TEST(***). .......................................................... 85 FIGURE 31 LEAVES PHENOTYPE OF PVATG9B OVEREXPRESSION PLANTS OF P. VULGARIS PLANTS AT 35 DPI GROWN WITH NITROGEN-LIMITED B&D SOLUTION (KNO3 2NM) TO PROMOTE NODULATION. (A)LEAVES OF CONTROL-EV AND PVATG9B-OE (B)BOXPLOT. SCALE BAR. 3CM. GREEN BOXES:OVEREXPRESSION CONTROL; PURPLE BOXES:PVATG9B O VER EXPR ESSION. SIGNIF ICAT IVE DIFFER ENCE VALU ES AT P < 0.05 STUDENT’S T TEST (***). ...................................................................................... 86 FIGURE 32 NODULES OF PVATG9B OVEREXPRESSION ROOTS AT 30DPI . ROOTS OBSERVED UNDER OPTICAL MICROSCOPY WITH GUS MAGENTA STAINING (A)CONTROL-OE, (B)OVER EXPRESSION OF PVATG9B (PVATG9-OE). P. VULGARIS PLANTS AT 30 DPI GROWN WITH LIMITED B&D SOLUTION (KNO3 2MM)TO PROMOTE NODULATION (NODULE CORTEX(NC); VASCU LAR BUND LE(VB); INFECTION ZONE(IZ); VASCULAR T ISSUE (V). SCALE 2MM. ........................................................................................................ 86 FIGURE 33 FOLD CHANGE OF RELATIVE EXPRESSION OF NIN, ENOD40 AND ERN1 IN PVATG9B OVEREXPRESSION ROOTS AND THE CONTROL. RELATIVE TRANSCRIPT LEVELS WERE NORMALIZED WITH METALLOPROTEASE. THE EXPRESSION OF NIN, ENOD40 AND ERN1 SHOWED HIGHER EXPRESSION THAN CONTROL. GREEN BOXES:OVEREXPRESSION CONTROL; PURPLE BOXES:PVATG9B OVER EXPRESSION. .................................................................................................................................................................. 87 FIGURE 34 ANALYSIS OF SUBCELLULAR LOCALIZATION OF PVATG9B FOR YFP FUSION PROTEIN IN P. VULGARIS. ROOTS AT 30D OBSERVED IN CONFOCAL MICROSCOPY. (A)PRIMARY ROOT AND LATER AL ROOT. (B)AND (C)PRIMAR Y R OOT. V ASCU LAR TISSU E (V) SCALE BAR: 1MM............................................................................................................. 87 FIGURE 35 ATG9 PROTEIN STRUCTURE AND ATG2-ATG18 COMPLEX. A) ATG9 CONTAIN TRANSMEMBRANE HELICES AND FORMS A PORE. B)ATG9- MEDIATE LIPID TRANSFER FROM ER TO TH E ISOLAT ION MEM BRAN E FOR EXPAN SION TOGETHER W ITH ATG2 AND ATG18.FIGURE BASED ON MATOBA & NODA,2020; LAI ET AL., 2020........................................ 99 FIGURE 36. ATG9 IN VESICULAR TRAFFICKING AND AUTOPHAGOSOME FORMATION. ATG9 IS INTERNALIZED FROM THE PLASMA MEMBRANE, VAMP3-MEDIATED FUSION BETWEEN THE ATG16L1 AND ATG9 VESICLES. ATG9 CYCLES BETWEEN THE TGN AND A PERIPHERAL POOL, IN RECYCLING ENDOSOMES THAT IS MEDIATED BY TRAPPII-LIKE COMPLEX AND RAB1. ATG9 VESIC LES FORM TH E AUTOPHAGOSOM E (SØREN G ET AL., 2018). ................................................................................................................................... 100 FIGURE 37 Y2H OF ATG9 AND CU PIN, CDO PROTEIN S GR OWN FOR 3-5 DAYS ON THE SELECTIVE MEDIUM SYNTHETIC (SD) DDO(−LEU/−TRP) AND QDO (SD/-ADE/-TR P/-LEU/-H IS).103 FIGURE 38 PVATG9B-INTERACTING PARTNERS. 24 PROTEINS INTERACT DIRECTLY WITH PVATG9B IN Y2H SCREENING (BLUE POINTS; REPRESENT THE PROTINES), THE LIST OF THEM CONTAIN A BR IEFLY DESCRIPTION. ................................................................................................................................................................................................ 104 FIGURE 39 PVATG9 NETWORK. PVATG9 INTERACT WITH 10 PROTEIN RESULTS BASED ON COOEXPRESSION AND TEXMINING FROM STRING DATABASES. (PURPLE SQUARE: NODES; BLUE SQU ARE: P VATG9B)........................................................................................................................................................................................................... 106 FIGURE 40. EXPANDED NETWORK OF PVATG9B. PVATG9 EXPANDED NETWORK CONTAIN 241 NODES THAT INCLUDE THE STRING RESULTS AND Y2H SCREENING. (BLUE POINT:N ODES) ............................................................................................................................................................................................................................ 107 FIGURE 41 PVATG9B-INTERACTING PARTNERS COEXPRESSION DURING NODULATION. NINE PVATG9B-INTERACTING PARTNERS INCREASED THEIR EXPRESION WHILE EIGHT DECREASED TH E EXPRESSION DURING R. TR OPICI SYM BIOSIS IN P. VU LGARIS.................................................................................................................................... 108 FIGURE 42 EXPR ESSION PR OFILE OF PVATG9B-INTERAC TING PARTNER S IN P. VU LGARIS R OOTS AND N ODULES. ............................................................................................... 109 FIGURE 43 PLANT CYSTEINE OXIGENASE 2 (PCO2) NETWORK. (A) 10 NODES ARE INTERACTING WITH PCO2 OF WHICH HRA1, ERF71, VPS39 AND HRA1 INCREASED THEIR EXPRESSION DURING SYMBIOSIS BETWEEN R. TROPICI AND P. VULGARIS.(B) NORMOXIA PATHWAY THAT INVOLVES THE PLANT CYSTEINE OXIGENASE 2 (PCO2) NETWORK (TAYLOR‐KEARNEY ET AL. 2022). ................................................................................................................................................................................................. 110 9 LIST OF SUPPLEMENTARY MATERIAL SUPPLEMENTAL MATERIAL S1. AUTOPHAGY PATHWAY. CANONICAL AUTOPHAGY PATHWAY WHERE PARTICIPATE THE AUTOPHAGY CORE. RIGHT SCHEMES SHOW THE STAGES OF AUTOPHAGOSOME. ........................................................................................................................................... 130 SUPPLEMENTAL MATERIAL S2 . ANALYSIS OF ATG GENES HOMOLOGS IN P. VULGARIS, M. TRUNCATULA AND G. MAX USING BASIC LOCAL ALIGNMENT SEARCH TOOL (BLAST). WE OBTAINED THE QUERY COVER AND PERCENTAGE OF IDENTITY VALUE COMPARED A. THALIANA PROTEIN SEQUENCE WITH LEGUMES: (A) P. VULGARIS; (B) M.TRUNCATULA; (C) G.MAX. ..................................................................................... 130 SUPPLEMENTAL MATERIAL S3 PERCENTAGE OF LEGUME ATG HOMOLOGS IN DIFFERENT SOFTWARES. BAR GRAPH SHOWING THE P. VULGARIS (RED BAR), M. TRUNCATULA (ORANGE BAR), G. MAX (PINK BAR) RESULTS USING BLAST, EGGNOG, ENSEMBL, HMMER, INPARANOID,AND KEGG. . 133 SUPPLEMENTAL MATERIAL S4 LIST OF ACCESSION NUMBERS OF ATG (A) GENES, (B) TRANSCRIPTS AND (C)PROTEINS IN P.VULGARIS .................................... 134 SUPPLEMENTAL MATERIAL S5 IDENTIFICATION OF ATG8 IN 13 LEGUMES ........................................................................................................................................ 136 SUPPLEMENTAL MATERIAL S6 IDENTIFICATION OF ATG18 PROTEINS IN 15 PLANTS........................................................................................................................ 136 SUPPLEMENTAL MATERIAL S7 SEQUENCE AND STRUCTURE OF ATG9A (PHVUL.001G159900) (A) DNA SEQUENCE OBTAINED IN PHYTOZOME. GREEN HIGHLIGHT:5’UTR, GREENBLUE; HIGHLIGHT:5’UTR , GREEN EXONS; PINK HIGHLIGHT:5’UTR 3’UTR (B)CDS STRUCTURE OF 7 T STRUCTURES OF ATG9A DESIGNED IN GSDS.V2: DARK BLUE BOXES: CDS; LINES: INTRONS; DARK GREEN BOXES: UPSTREAM /DOWNSTREAM. (C) PROTEIN SEQUENCE FEATURES CARRIED OUT BY HMMER. GREEN BOXES: PFAM DOMAIN; PURPLE BOXES: DISORDER REGIONS OBTAINED BY IUPRED; RED BOXES: TRANSMEMBRANAL REGION AND SIGNAL PEPTIDE OBTAINED BY PHOIBUS. ................................................................................................................................................................................................................. 137 SUPPLEMENTAL MATERIAL S8. SEQUENCE AND STRUCTURE OF ATG9A (PHVUL.007G194300) (A) DNA SEQUENCE OBTAINED IN PHYTOZOME. GREEN HIGHLIGHT:5’UTR, BLUE; HIGHLIGHT: EXONS; PINK HIGHLIGHT: 3’UTR (B)CDS STRUCTURE OF 7 T STRUCTURES OF ATG9A DESIGNED IN GSDS.V2: DARK BLUE BOXES: CDS; LINES: INTRONS; DARK GREEN BOXES: UPSTREAM /DOWNSTREAM. (C) PROTEIN SEQUENCE FEATURES CARRIED OUT BY HMMER. GREEN BOXES: PFAM DOMAIN; PURPLE BOXES: DISORDER REGIONS OBTAINED BY IUPRED; RED BOXES: TRANSMEMBRANAL REGION AND SIGNAL PEPTIDE OBTAINED BY PHOIBUS. .......................................................................... 138 SUPPLEMENTAL MATERIAL S9 OLIGONUCLEOTIDES SEQUENCES ................................................................................................................................................... 138 SUPPLEMENTAL MATERIAL S10 THERMOCYCLING PROGRAM FOR PCR AND QRT-PCR .................................................................................................................. 139 SUPPLEMENTAL MATERIAL S11. MAP OF VECTORS USED IN GATEWAY CLONING (INVITROGEN). LEFT VECTOR WAS USED AS ENTRY VECTOR. RIGHT VECTOR WAS USED AS DESTINATION VECTOR IN PLANT CONSTRUCTION OF PVATG9 PROMOTER. ................................................................. 139 SUPPLEMENTAL MATERIAL S12 SUPPLEMENTAL FIGURE S5. MAP OF VECTORS USED IN GATEWAY CLONING (INVITROGEN). LEFT VECTOR WAS USED TO IRNA CONSTRUCTION. RIGHT VECTOR WAS USED TO OBTAIN THE OVEREXPRESSION CONSTRUCTION(EARLEY ET AL., 2006; VALDÉS-LÓPEZ ET AL., 2008). ............................................................................................................................................................................................ 140 SUPPLEMENTAL MATERIAL S13CLONING REACTIONS ...................................................................................................................................................................... 140 SUPPLEMENTAL MATERIAL S14 BACTERIA AND PLANT TRANSFORMATIONS. .................................................................................................................................. 140 SUPPLEMENTAL MATERIAL S15 (LIRUA-BERTANI) LIQUID AND SOLID MEDIUM. ................................................................................................................................ 141 SUPPLEMENTAL MATERIAL S16 PY LIQUID......................................................................................................................................................................................... 141 SUPPLEMENTAL MATERIAL S17 B&D NUTRIENT SOLUTION COMPOSITION (BROUGHTON & DILWORTH, 1971) ............................................................................... 141 SUPPLEMENTAL MATERIAL S18. CLONING OF PVATG9B: (A) CDNA, AQUAPORINE OLIGONUCLEOTIDES (B) PROMOTOR AMPLIFICATED.(C)PLASMID PENTR WITH M13 OLIGONUCLEOTIDES. (D) PLASMID PBGWF7.0 WITH THE PROMOTOR OF PVATG9B...................................................................... 141 SUPPLEMENTAL MATERIAL S19 CLONING OF PVATG9B SILENCING. (A) FRAGMENT AMPLIFICATED (B) FRAGMENT ENTRY VECTOR PENTR/D-TOPO (C) PLASMID PTDT WITH THE PROMOTOR OF PVATG9B .............................................................................................................................................. 142 SUPPLEMENTAL MATERIAL S20 OVER EXPRESSION AND LOCALIZATION ISOLATED FRAGMENT TO ENTRY VECTOR. (A) FRAGMENT AMPLIFICATED OF PVATG9B (B) ISOLATED FRAGMENT TO ENTRY VECTOR PENTR/D-TOPO (C)COLONIES IN FINAL VECTOR. (D)LOCALIZATION ............................ 142 SUPPLEMENTAL MATERIAL S21 GUS ESSAY (JEFFERSON, 1987) ...................................................................................................................................................... 142 SUPPLEMENTAL MATERIAL S22 T- TEST OF PVATG9B SILENCING ROOTS........................................................................................................................................ 142 SUPPLEMENTAL MATERIAL S23 T- TEST OF PVATG9B SILENCING LEAVES....................................................................................................................................... 143 SUPPLEMENTAL MATERIAL S24 T TEST OF OVEREXPRESSION OF PVATG9B ROOTS...................................................................................................................... 143 SUPPLEMENTAL MATERIAL S25 T- TEST OF PVATG9B OVEREXPRESSION ROOTS........................................................................................................................... 143 SUPPLEMENTAL MATERIAL S26 ANOVA OF PVATG9B OVEREXPRESSION LEAVES .......................................................................................................................... 144 SUPPLEMENTAL MATERIAL S27 PVATG9 PHENOTYPE PVATG9 PHENOTYPE: SILENCING AND OVEREXPRESSION PLANTS.(A)POTS(B)ROOTS(C)LEAVES(D)LENGTH AND WIDTH OF LEAVES.SCALE BAR: A & B: 7CM; C:3CM ......................................................................................................................... 144 SUPPLEMENTAL MATERIAL S28 PVATG9B-INTERACTING PARTNERS ............................................................................................................................................... 145 SUPPLEMENTAL MATERIAL S29 PROTEIN FEATURES OF PVATG9B-INTERACTING PARTNERS........................................................................................................ 145 SUPPLEMENTAL MATERIAL S30 TRANSMEMBRANE DOMAINS OF PVATG9B-INTERACTING PARTNERS PHVUL.001G18101.1 (NO. 3), PHVUL.004G102800.1 (NO. 9), PHVUL.006G125700.1 (NO. 12), PHVUL.006G203200.1 (NO. 13), PHVUL.007G0053500.1 (NO. 14), PHVUL.008G290800.1 (NO. 17) ............ 146 SUPPLEMENTAL MATERIAL S31 PVATG9 NETWORK AND SUMMARY STATISTICS............................................................................................................................. 146 SUPPLEMENTAL MATERIAL S32 PVATG9B-INTERACTING PARTNERS IN STRING DATA .................................................................................................................... 147 SUPPLEMENTAL MATERIAL S33 PVATG9B NETWORK INCLUDING THE 24 INTERACTING PARTNERS............................................................................................... 147 SUPPLEMENTAL MATERIAL S34 . HOMOLOGS IN A. THALIANA OF PVATG9B-INTERACTING PARTNERS........................................................................................... 148 10 ABBREVIATIONS 3-AT 3-Aminotriazol ABA Abscisic Acid ABS3 Abnormal Shoot 3 ACR4 Arabidopsis Crikly 4 AgNps Silver Nanoparticules ANOVA Análisis of Variance AON Autoregulation Of Nodulation ARF Auxine Response Factor ARK/ATPUB Arabidopsis Receptor Kinase 2/E3 Ligase Plant U-Box Armadillo Repeat Protein9 ARP2 Actin-related protein 2 AS Aspargine Synthetase ASN Asparagines ASP Aspartic Acid ASPAT Asparate Aminotransferase ATE1 Arginyl Transferase ATG Autophagy Axr5 Auxin Resistant 5 BCAS3 Breast Carcinoma Amplified Sequence 3 BLAST Basic Local Alignment Search Tool BLOSUM62 BLOcks of Amino Acid SUbstitution Matrix BRS Brassinosteroids BZR1 Brassinazol-Resistant 1 CCZ Vacuolar fusion protein CCZ1 CDC48 Cell division control protein 48 CHR Chalcone Reductase CHS Chalcone Synthase CIAT Centro Internacional de Agricultura Tropical CK Cytokinin CLE12 CLAVATA/ESR-related CMA Chaperone-Mediated Autophagy COG3 Conserved oligomeric Golgi complex subunit 3 CPSASE Carbamoylphosphate Synthase CVT Cytoplasm-To-Vacuole Targeting DMI2/SYMRK “Does Not Make Infections 2” /Symbiosis Receptor Kinase DMI3/CCAMK Nuclear Calcium-Calmodulin Kinase DAS Days After Sowing dpi days post inoculation dTRAF2 Drosophila tumor necrosis factor receptor-associated factor 2 EFD Ethylen Response Factor EMSA Electrophoretic Mobility Shift Assay EIN2 Ethylene-insensitive protein 2 ENOD11 Early nodulin 11 ENOD40 Early nodulin 40 ER Endoplasmatic Reticulum ERE Ethylene Response Elements ERES ER-Exit Sites ERF5 Ethylene Response Factor 5 ERF7 Ethylene-Esponsive Factor 71 ERGIC Golgi Intermediate Compartment ERN1 Endoplasmatic Reticulum To Nucleus Signalling 1 ET Ethylene EV Empty Vector Faa1 Long-Chain-Fatty-Acid--Coa Ligase 1 FC Fold Change FLOT2 Flotillin 2 FLOT4 Flotillin 4 FNS Flavone Synthase FPKM Fragments Per Kilobase GA Gibberellin GA2ox10 Giberrellin 2-Oxidase 10 GAPCS Glyceraldehyde-3-Phosphate Dehydrogenases Gate-16 Golgi-Associated Atpase Enhancer Of 16 GFP Green Fluorescence Protein GLN Glutamine GLU Glutamic Acid Glo3 GLyOxalase 3 GO Gene ontology GOGAT Glutamine 2-Oxoglutarate Amidotransferase GRAVY Grand Average Of Hydropathicity GS Glutamine Synthetase GUS β-glucuronidase HDA9 Histone Deacetylase 9 HMM Hidden Markov Model HRA1 Hypoxia Response Attenuator1 HSFA1 Heat-Shock Trasncription Factor HY5 Elongated Hypocotyl IM Initial Isolation Membranes iRNA RNA interferent JA Jasminic Acid K Potassium KA Synonymous Substitutions KAPP1 Kinase-associated protein phosphatase KEGG Kyoto Encyclopedia of Genes And Genomes KNAT Homeobox protein knotted-1-like1 KOG eukaryotic Orthologue KS Nonsynonymous Ones KSP1 Serine/threonine-protein kinase KSP1 LHB Leghemoglobin LHK1 Lotus Histidine Kinase LysM-RLK Lysin Motif Receptor -Like Kinase Map1lc3 Or Lc3 Microtubule-Associated Protein 1 Light Chain 3 MDR4/PGP4 Multidrug Resistance 4/P- Glycoprotein 4 MEME Multiple Expectation Maximization For Motif Elicitation MTC Microtubule center mya million year ago N2 Dinitrogen NADPH Nicotinamide Adenine Dinucleotide Phosphate NAP1 Nucleosome Assembly Protein NS-LRR Nucleotide-binding site and leucin-rich repeat NCBI National Center for Biotechnology Information NENA Nucleoporin-Localized Protein NF Nod Factor NFR1/LYK3 Nod Factor Receptor/Lysm Receptor Kinase NFR5/NFP Nod Factor Receptor 5 /Nod Factor Perception NH3 Ammonia NIC1 High-affinity Nickel Transport Protein NIN Nodule Inception NOX Nicotinamide Adenine Dinucleotide Phosphate Oxidase NPL N acetylneouraminate lyase NR Nitrate Reductase 2 NSP1 & NSP2 Nodulation Signaling Pathway 1 And 2 NUP85 Nucleoporin Subunits Nucleoporin 85 NUP133 Nucleoporin Subunits Nucleoporin 133 ODO Doble Drop Out OE Overexpression P Phosphorus p38IP p38-interacting protein PANTHER Protein Analysis Through Evolutionary Relationships PAS Phagophore Assembly Site PATJ PALS1-Associated Tight Junction Protein PCD Plant cell Death PCO2 Plant Cysteine Oxygenase PE Phosphotidylethanolamine PHO23 Transcriptional regulatory protein PHO23 PHO80 Phosphate system cyclin PHO80 PI3K Phosphoinositide 3-Kinases PI(4)KIIα Phosphatidylinositol-4-kinase type II alpha PIR1 Protein with Internal Repeats PLENTY Hydroxyproline O-Arabinosyltransferase PLR Primordium of Lateral Roots PRT6 Proteolysis 6 ptdIns3K Phosphatydylinositol Three Kinase Complex PTC2 Protein Phosphatase 2C Homolog 2 PTC3 Protein Phosphatase 2C Homolog 3 PTI Pathogen- Associated Modecular Pattern- Triggered Immunity PUB1 Plant U-Box Protein 1 PvGEA Phaseolus vulgaris Gene Expression Atlas PWR Powerdress QC Quiescent Center QDO Quarter Drop Out Rab1B Ras-related protein Rab-1B RBOH Respiratory Burst Oxidase Homolog rh Root hair RIC1 & RIC2 Rhizobial-Induced Cle Peptide RIP Receptor-Interacting Protein RNA-SEQ Rna Sequencing ROS Reactive Oxygen Species ROP6 Rholike GTPase6 S6K Ribosomal Protein Six Kinase SA Salicylic Acid SAG12 Cysteine Protease SAGS Senescence-Associated Genes SCS7 Ceramide very long Chain Fatty Acid Hydroxylase SC-LTHA Screening on plates lacking Leucine, Tryptophane, Histindine and Adenine SDI Shoot-Derived Inhibitor SEC1A Secretory protein 1A SEC22 Secretory protein 22 SEC4 Secretory protein 4 SEC18 Secretory protein 18 SEC17 Secretory protein 17 SFN Nitrogen- Fixing Symbiosis SIE2 Symrk Interacting E3 Ubiquitin Ligase SIPS Symrk Interactinf Proteins SNARE Soluble NSF attachment protein receptor SNRK1 Sucrose Nonfermenting1-Related Protein Kinase 1 STRING Search Toll for the Retrieval of Interacting Genes/ Proteins SSA1 Heat shock protein SSA1 SYMREM1 Simbiotic Remorin 1 SUI2 Eukaryotic translation initiation factor 2 subunit TAIR The Arabidopsis Information Resource TBC1D5 TBC1 Domain Family Member 5 TF Transcription Factor TfR Transferrin receptor (recycling endosome marker) TGN Trans-Golgi Network TLG2 t-SNARE affecting a late Golgi compartment protein 2 TMEM74 Transmembrane Protein 7 TMV Tobacco Mosaic Virus TOR Target Of Rapamycin TRAF6 Tumor necrosis Factor Receptor-Associated Factors 6 TRAPPIII Trafficking Proteins Particle Trs85 Trafficking Protein Particle Complex III-specific Subunit 85 UIM Ubiquitin-interacting motives VAMP3 Vesicle Associated Membrane Protein 3 VAMP7 Vesicle Associated Membrane Proteins 7 VPE-1 Proteases Vacuolar Processing Enzyme Gamma VPS Vesicular Proteins Sorting VPS15 Vesicular Protein Sorting 15 VPS34 Vesicular Protein Sorting 34 VPS38 Vesicular Protein Sorting 38 VPS39 Vacuolar Sorting Protein 39 VPS9 Vacuolar Protein Sorting 9 VPS21 Vacuolar Protein Sorting 21 VSV Vista Synteny Viewer VTI1 Vesicle transport v-SNARE protein WAG Whelan And Goldman WAK4 Wall Associated Kinase 4 Y2H Yeast Two Hybrid YFP Yellow Fluorescence protein YPT7 Ypt/Rab-type GTPase YPT7 YPT31 GTP-binding protein YPT31/YPT8 12 INDEX ACADEMIC ACKNOWLEDGEMENTS ............................................................................................... 3 GENERAL ABSTRACT ....................................................................................................................... 5 LIST OF TABLES................................................................................................................................ 7 LIST OF FIGURES.............................................................................................................................. 7 LIST OF SUPPLEMENTARY MATERIAL ............................................................................................ 9 ABBREVIATIONS ........................................................................................................................... 10 CHAPTER I. General Introduction ................................................................................................. 15 ANALYSIS OF AUTOPHAGY GENES IN ROOTS OF Phaseolus vulgaris DURING THE NODULATION ....................................................................................................................................................... 15 AUTOPHAGY ................................................................................................................................ 16 Genes of autophagy (ATGs) ...................................................................................................................................... 18 AUTOPHAGY IN PLANTS................................................................................................................ 19 Proteins involved in regulation of macroautophagy in plants ............................................................................... 19 Role of autophagy in plants .......................................................................................................... 21 Plant Development and hormones .......................................................................................................................... 21 LEGUME NODULATION AND AUTOPHAGY IN P. vulgaris ................................................................ 30 Legumes nodulation .................................................................................................................................................. 30 Autophagy in P. vulgaris and legumes nodulation .................................................................................................. 35 PROPOSAL OF THE PROBLEM ........................................................................................................ 36 AIM OF THIS THESIS ...................................................................................................................... 37 GENERAL OBJECTIVES ................................................................................................................... 37 Research questions that will be answered ..................................................................................... 37 References ................................................................................................................................... 38 CHAPTER II .................................................................................................................................... 41 IDENTIFICATION OF AUTOPHAGY GENES IN Phaseolus vulgaris AND OTHER LEGUME ............ 41 ABSTRACT .................................................................................................................................... 42 INTRODUCTION ............................................................................................................................ 43 Genes of autophagy (ATGs) in plants ....................................................................................................................... 43 RESULTS ....................................................................................................................................... 48 Identification of ATG families in 3 legumes ............................................................................................................. 48 Phylogenetic relationships, chromosome localization of ATG families ................................................................. 50 13 Identification of ATG18 family in plants .................................................................................................................. 52 Principal components analysis for ATG18 family .................................................................................................... 53 Phylogenetic relationship of ATG 18 family in plants ............................................................................................. 54 Conserved protein motif analysis of ATG18 family. ................................................................................................ 56 Promoter analysis, Expression profiling and Transcriptome of ATGs families...................................................... 58 DISCUSSION ................................................................................................................................. 61 MATERIAL AND METHODS ............................................................................................................ 66 Identification of ATG families in legumes. ............................................................................................................... 66 Alignment and Phylogenetic tree analysis ............................................................................................................... 67 Chromosome localization. ........................................................................................................................................ 67 Promoter analysis, Expression profiling and Transcriptome of ATGs families...................................................... 68 Quantitative Real Time PCR Analysis ....................................................................................................................... 68 Principal components analysis for ATG18 family .................................................................................................... 69 Conserved motif detection of ATG18 family ........................................................................................................... 69 REFERENCES ................................................................................................................................. 70 CHAPTER III ................................................................................................................................... 72 UNDERSTANDING THE ROLE OF ATG9 DURING SYMBIOSIS ....................................................... 72 BETWEEN Phaseolus vulgaris AND Rhizobium tropici ................................................................ 72 ABSTRACT .................................................................................................................................... 73 INTRODUCTION ............................................................................................................................ 74 RESULTS ....................................................................................................................................... 76 Structure and Phylogenetic analysis of PvATG9 ...................................................................................................... 76 Expression of PvAtg9b gene in roots and nodules .................................................................................................. 77 Transcript downregulation of ATG9b in P. vulgaris hairy roots ............................................................................. 80 Overexpression of PvATG.......................................................................................................................................... 84 DISCUSSION ................................................................................................................................. 88 MATERIAL AND METHODS ............................................................................................................ 90 Plant Material ............................................................................................................................................................ 90 Bacteria Material ....................................................................................................................................................... 90 Structure and Phylogenetic analysis ........................................................................................................................ 91 Plasmid construction and transformation ............................................................................................................... 91 Plant transformation ................................................................................................................................................. 93 Histochemical GUS staining ...................................................................................................................................... 93 REFERENCES ................................................................................................................................. 95 CHAPTER IV. .................................................................................................................................. 96 DECIPHERING THE PVATG9B INTERACTION NETWORK DURING SYMBIOSIS BETWEEN Phaseolus vulgaris AND Rhizobium tropici.................................................................................. 96 ABSTRACT .................................................................................................................................... 97 INTRODUCTION ............................................................................................................................ 98 The Atg1–kinase complex tethers Atg9-vesicles to initiate autophagy ................................... 101 14 Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy ................................................................................................... 101 The Atg1–kinase complex tethers Atg9-vesicles to initiate autophagy ................................... 102 dTRAF2/TRAF6 ............................................................................................................................ 102 The Golgi as an Assembly Line to the Autophagosome ............................................................ 102 RESULTS ..................................................................................................................................... 103 PvATG9b protein interactions during nodulation in P. vulgaris ...........................................................................103 Identification of PvATG9b-interacting partners during nodulation in P. vulgaris...............................................103 Interaction Network of PvATG9b in P. vulgaris .....................................................................................................106 Expression profile of PvATG9b-interacting partners.............................................................................................108 DISCUSSION ............................................................................................................................... 110 MATERIAL AND METHODS .......................................................................................................... 114 Yeast two-hybrid screening ....................................................................................................................................114 Interaction network construction ..........................................................................................................................116 Expression profiling and transcriptome .................................................................................................................117 REFERENCES ............................................................................................................................... 118 CHAPTER V .................................................................................................................................. 120 GENERAL DISCUSSION AND CONCLUSION ................................................................................. 120 General Discussion ..................................................................................................................... 121 Conclusions ................................................................................................................................ 124 Discusión General....................................................................................................................... 125 Conclusiones .............................................................................................................................. 128 SUPPLEMENTAL MATERIAL ........................................................................................................ 130 PUBLICATIONS AND MATERIALS OBTEINED .............................................................................. 149 PUBLICATION .............................................................................................................................. 150 EXPLORATION OF AUTOPHAGY FAMILIES IN LEGUMES AND DISSECTION OF THE ATG18 FAMILY WITH A SPECIAL FOCUS ON Phaseolus vulgaris ........................................................... 150 15 CHAPTER I. General Introduction ANALYSIS OF AUTOPHAGY GENES IN ROOTS OF Phaseolus vulgaris DURING THE NODULATION Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 16 AUTOPHAGY Autophagy is an essential degradation process for maintaining cellular homeostasis and is also related to various physiological and pathophysiological roles, such as host defense mechanisms, development, infection, and tumorigenesis (King, 2012; Sirko & Masclaux- Daubresse, 2021). During the autophagy process, the cytosolic components fall inside the double-membrane vesicles that fuse with lysosomes or vacuoles. This process consists of several sequential steps that finally will be delivered to end up in lysosomes or vacuoles for degradation of organelles and misfolded, proteins (Hughes & Rusten, 2007). The eukaryotic cells have other degradation mechanisms, such as the protease complex named proteasome. The proteasome’s mission is to carry out selective proteins and participate in protein quality control, regulation of proliferation, DNA repair, and signal transduction (Tanaka, 2009). The autophagy and proteasome are the conserved mechanisms of degradation involved in cellular homeostasis. The main differences between both are that autophagy is an exclusive mechanism present in eukaryotic cells, advocated to recycle, and degrade a bulk of proteins and organelles in response to stress. At the same time, the proteasome appears within prokaryotic and eukaryotic cells. In eukaryotic cells, the proteasome is in-charge of fast protein elimination (Hughes & Rusten, 2007). In terms of localization, the proteasome is present in the nucleus and cytoplasm, whereas autophagy functions only in the cytoplasm (Zientara-Rytter & Sirko, 2016). Both processes are complementary to maintain homeostasis in eukaryotic cells (Lilienbaum, 2013). Autophagy is classified into three major types namely, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), have been elucidated, and these differ in the mode of cargo delivery to the lysosome or vacuole (Xie et al, 2007; González-Polo et al, 2016). Macroautophagy can be nonselective or selective: Nonselective autophagy is a cellular response to nutrient deprivation that involves the random uptake of cytoplasm into phagophores (precursors to autophagosomes) (Thompson et al, 2005), and selective Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 17 autophagy is responsible for the specific removal of certain components, such as protein aggregates and damaged or superfluous organelles (Li et al, 2012; Marshall et al,2018) . Selective autophagic degradation has been observed with several organelles, such as mitochondria (Ashrafi et al, 1999), peroxisomes (Hutchins et al, 1999) lysosomes (Hung et al, 2013), endoplasmic reticulum and nucleus (Nakatoga et al. 2015). In contrast, microautophagy is the least characterized type of autophagy; during this nonselective process, smaller molecules acting as substrates and the cargo for degradation are transferred into vacuole via invagination of the tonoplast membrane. CMA involves molecular chaperones in the cytosol that unfold proteins and translocate them through the lysosomal membrane (Dice et al. 2007). The fourth type of autophagy is mega- autophagy which is a massive degradation at the end of one type of programmed cell death (PCD) (Doorn & Papini, 2013) (Fig. 1). Given the complexity and significant variation in the autophagy process, we had to focus our research only on a subtype of autophagy. We consider that macroautophagy in legumes is poorly known to date, and it is a crucial mechanism to understand the formation and the function of cellular structures such as the autophagosome, connected with many other cellular processes. In this investigation, we will focus and use the term autophagy to refer to macroautophagy. Figure 1. Types of autophagy. (A) Macroautophagy is a process which involves the formation of the autophagosome, chaperone-mediated autophagy (CMA) is leads by the translocation of protein bound and microautophagy is a process which seclude the target components near to lysosome or vacuole. Finally, all of these types of autophagy end in the lysosome or vacuole (Ho et al., 2019). (B) Types of Autophagy confirmed in plants. Macroautophagy require autophagosome that fuses into the vacuole, Microautophagy comprise a invagination of the tonoplast and mega Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 18 autophagy imply the ruptured or permeable tonoplast that release lytic contents into cytoplasm (Wojciechowska et al., 2021) Genes of autophagy (ATGs) Autophagy is a conserved catabolic pathway present in all eukaryotic organisms from yeast to mammals. To dissect the process of autophagy, current study first focuses on identification and validation of AuTophaGy genes (ATG). Several groups that work on autophagy across different organisms found a highly conserved core (King, 2012). The core autophagy machinery is constituted of 18 proteins in yeast (Suzuki et al., 2017), subdivided into distinct stages. There are initiation (ATG1 and ATG13), autophagosome formation (ATG2, ATG9, ATG18), nucleation (PAS (PRE AUTOPHAGOSOME- STRUCTURE), ATG6/VPS30, ATG14, VPS34, VPS15), cargo recognition (ATG11, ATG19), expansion and completion of autophagosome (ATG12 system, ATG8 lipidation), fusion with vacuoles digestion and recycling and efflux of macromolecules and amino acids (SNARE proteins such as Vam3 (Qa), Vam7 (Qc), Ykt6 (R), and Vti1 (Qb))(Suzuki et al., 2010, Wang et al.,2016).  The mammal's autophagy genes are 33, distributed in several subgroups. Of the 33 genes, only 17 genes constitute the core (Braschi et al., 2019). Initial step involves the ATG1/ UNC-51-LIKE KINASE (ULK) complex (ULK1, ULK2, mATG13, FIP200, mATG101) then complex ATG2-ATG18 that include ATG9 (Subramani & Malhotra, 2013). During nucleation, the participant proteins include the class III PHOSPHATIDYLINOSITOL THREE KINASE COMPLEX (ptdIns3K/VPS34) and p150, BECLIN1, ATG14, AMBRA. During the elongation stage, the main proteins participating are the two ubiquitin-like protein conjugation systems ATG12 and ATG8/LC3 (ATG7, ATG10, ATG5, ATG16L1, ATG4A-D, and ATG3). It is claimed that some proteins have other functions in addition to playing a role in autophagy (Levine & Kroemer, 2019). For instance, the protein ATG9 and complex Atg2-Atg18, ATG9 contributes to the membrane and transport’s mechanism from trans-Golgi network (TGN) to late endosome (He & Klionsky, 2009; Mizushima & Levine, 2010; Z. Yang & Klionsky, 2010). Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 19 AUTOPHAGY IN PLANTS In plants, autophagy studies focus on the importance of autophagy in biotic and abiotic stress, salt salinity, drought, heat, oxidative stress, hypoxia, pathogen attack, endoplasmic reticulum stress during plant development (Soto-Burgos et al., 2018). Unlike yeast and animals, chaperone-mediated autophagy has not been reported in plants so far, but the other types of autophagy are present including mega autophagy. This autophagy type is a massive degradation process at the end of the programmed cell death process (Van Doorn & Papini, 2013). We also know that the membrane of macroautophagy is provided by multiple sources such as the endoplasmic reticulum or mitochondria. In the case of microautophagy, the membrane comes from the tonoplast (Tooze & Yoshimori, 2010). We still do not have a full grasp of the role of autophagy in plants, but by looking at the consequences in mutants on the various stages of the plant life cycle, we can understand its relevance. We have observed that the defects in macroautophagy are displayed in abnormal embryonic development, disrupted root growth, shoot growth and flowering, lower seed yield, leaf chlorosis, poor seed germination, and senescence (Zientara-Rytter & Sirko, 2016). Whereas the defects of the microautophagy mechanism mediate on the flavonoid aggregates into the vacuole (Chanoca et al., 2015).  Proteins involved in regulation of macroautophagy in plants Macroautophagy has been studied in transcriptional, post-transcriptional, and post translate levels. The transcriptional mechanism of autophagy in plants involves the HsfA1a, WRKY33, WRKY20, BZR1, ERF5TRANSCRIPTIONAL FACTORS (TF). The TF makes more accessible or more complex the binding of RNA polymerase in the promoter. Tomato HsfA1a induces autophagy and acts as a positive regulator of the ATG10, ATG18f, and autophagosome formation under drought (Wang et al., 2015; Cai, et al., 2015). WKRY33 induces the early steps of autophagy in wild type and botrytis-infected tomatoes (Zhou et al., 2014). In Manihot esculenta, it has been found MeWRK20 and MeATG8a/8f/8h interaction is necessary for sensitivity to bacterial diseases and Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 20 autophagy activity. The transcription factor BRASSINAZOL- RESISTANT 1 (BZR1), a positive regulator of the brassinosteroids pathway and autophagy can bind to the promoters of ATG2 and ATG6 in response to nitrogen starvation in tomatoes (Wang et al, 2018). Another study in tomato explores the DRE-Binding site (ACCGAC) in ATGs by ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) suggest the interaction between ATG8D, ATG18H, and the transcription factor ETHYLENE RESPONSE FACTOR -5 (ERF5) (Zhu et al., 2018). The HISTONE DEACETYLASE 9 (HDA9), together with WRK53 and POWERDRESS (PWR), bind to W-box of the Atg9 promotor. The HDA9 and PWR mutations provoke the H3K27 hyperacetylation at Atg9 genomic region, consequently the upregulation of the ATG9 (Chen et al., 2016; Yang et al., 2020).  At the posttranslational level, phosphorylation, acetylation, ubiquitination, and lipidation of ATG proteins control their activity. The phosphorylation of TOR regulates ribosomal protein Six Kinases (S6K), PP2A regulatory subunit TAP46, LIPIN, and ATG1. The TOR sensitivity and pathway modulation depend on the substrate phosphorylation sites (Kang et al., 2018). ATG1 has kinase activity through ATG11 interaction during nutrient rich medium, but during starvation the dephosphorylation of ATG1 and ATG13 by type 2C protein phosphatases Ptc2 and Ptc3 happens and triggers autophagy (Memisoglu & Haber, 2019; Puente et al., 2016). BECN1/ATG6 phosphorylation and ubiquitination at several residues respond to distinct autophagy modulating stimuli and control the balance between pro-survival autophagy and pro-apoptotic response (Menon & Dhamija, 2018). ATG9 phosphorylation regulates the rate of autophagosome formation and phagophore assembly site (Feng et al., 2016). Finally, the modulation of ATG18 phosphorylation by nutrients regulates the vacuolar dynamics. Only, dephosphorylation of ATG18 is required to associate with the vacuolar membrane and rephosphorylation of ATG18 allows the vacuoles to fuse and form a single rounded structure (Tamura et al., 2013). With respect to acetylation, during stressful conditions, the α‐tubulin acetylation stimulates the autophagy in A. thaliana (Olenieva et al., 2019). In mammals, NAD- Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 21 dependent deacetylase Sirt1 increases basal autophagy, forming the complex ATG5, ATG7, and ATG8, but formation of such a complex is not known in the plants (Lee et al., 2008). Other relevant post transcriptional activity is the ubiquitination that can be generated modification to bring selectivity (Yin et al., 2020). Role of autophagy in plants Plant Development and hormones During plant development, autophagy plays an essential role from the seedling stage until the cell death. In algae, microautophagy is reported as lipophagy for triacylglycerol degradation during seed germination (Heredia-Martínez et al., 2018; Yoshitake et al., 2019). In Arabidopsis, lipophagy is involved in early seedling development (Kurusu et al., 2017). During the Arabidopsis seed development, ATG gene expression increases more in the maturation phase, where the oil and proteins bodies are formed. ATG5 was reported to affect the storage protein deposition in A. thaliana seeds (Di Berardino et al., 2018). Macroautophagy and microautophagy participate in programmed cell death and lipid metabolic regulation in several developmental stages in Arabidopsis and rice tapetum while mega autophagy was detected in Citharexylum myrianthum during nectar development (Hanamata et al., 2014; Machado & Rodrigues, 2019). Autophagy relation in primary, secondary root and stem development was analyzed in Populus trichocarpa where ATG8 participates during the differentiation and early xylem and phloem development (including xylary and extra xylary fibers) (Wojciechowska et al., 2019). Recently, researchers demonstrated the participation of autophagy in vacuole formation during cortical tissue development, i.e., vascular differentiation and root senescence (Wojciechowska et al., 2019, 2021).  During plant senescence, autophagy mutants show hypersensitivity to starvation conditions and early senescence. For instance, ATG8A, ATG8B, ATG8H, and ATG9 were identified as senescence-associated genes (SAGs) (Buchanan-Wollaston et al., 2005; Lan & Miao, 2019). During leaf senescence, the ATG5, ATG4, ATG7, ATG10 mutants and Atg12a/Atg12b double mutant show premature leaf senescence (Doelling et al., 2002; Lan & Miao, 2019). Also, leaf senescence induced by methyl jasmonate has been Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 22 reported to increase autophagosomes (Yin et al., 2020). In addition, the interaction of Atg8 and ABNORMAL SHOOT 3 (ABS3) control the senescence in a non-autophagy interaction (Jia et al., 2019). Furthermore, as we have seen before, plant hormones have been studied in relation to autophagy when they respond to environmental challenges (Fig. 2). Plant hormones or phytohormones are signaling molecules that can act at low concentrations. Some of the well-studied phytohormones are Abscisic Acid (ABA), Auxin, Brassinosteroids (BRS) Cytokinin (CK), Gibberellin (GA), Ethylene (ET), Jasmonic Acid (JA), and Salicylic Acid (SA). ABA regulates stomal opening and adaptations to drought, salt, and cold stress (Sah et al., 2016). During the stress condition, ABA provoked TOR complex inhibition triggering autophagy (Kravchenko et al., 2015; Wang & Zhang, 2019). TOR inhibition is due to Raptor phosphorylation by SNRK2 activation. Now, ABA and Auxin are recognized as a regulator of TOR-dependent pathways (Avin-Wittenberg, 2019; Wang et al., 2018). Auxins are involved in cell division, apical dominance, differentiation of vascular tissue that imply cell growth and development. Natural auxins such as Indole-3-acetic acid (IAA) has long been studied for its role in agronomy, PHENYLACETIC ACID (PAA), and INDOLE-3-BUTYRIC ACID (IBA) and some synthetic auxins such as 1- NAPHTHALENEACETIC ACID (NAA) (Piotrowska-Niczyporuk & Bajguz, 2014; Zhao, 2010). NAA could activate TOR under salt and osmotic stresses. Brassinosteroids play a role in plant growth, development, and during extreme temperatures and drought and are related to selective degradation because these hormones activate TOR and NEIGHBOR OF BRCA (NBR), promoting selective autophagy (Chi et al., 2020). Gibberellins are responsible for stem elongation, seed germination, dormancy, flowering, leaf, and fruit senescence. GA inhibit the SnrK2 activity, which means autophagy could not counteract the effect of ABA (Li et al., 2020). Another hormone is the citokinins that promote cell division in shoot and root cells. This hormone and GA show a decline in Osatg7-1 mutants suggesting that ATG7 is involved in plant hormones metabolism Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 23 (Kurusu et al., 2017). Also, AtATG8f regulates cytokinin’s effect on root architecture that was suggested by fusion protein experiments (Slavikova et al., 2008). Ethylene is another interesting hormone which activity also appears in plant growth and development. Several transcription factors of this hormone have been found to be binding to the promoters of some ATG genes. ETHYLENE RESPONSE FACTOR 5 (ERF5) binds to ATG8 and ATG18h promoter in tomato and leads to ET-mediated drought tolerance (Yang et al., 2019; Zhu et al., 2018). On the other hand, jasmonic acid and salicylic acid induce WRKY33, promoting autophagy in plant resistance to a necrotrophic fungal pathogen. WRKY33 interacts with ATG18a to regulate the autophagy process (Lai et al., 2011). These three last hormones are required against biotic stress, but SA is essential in early leaf senescence and PROGRAMMED CELL DEATH (PCD) (Li et al., 2020; Rigault et al., 2021; Yoshimoto et al., 2009).  Figure 2 Autophagy during development, hormones, abiotic stresses, and biotic stresses reported in plants (Based on Gou et al., 2019; Federoff, 2012) Biotic stress Biotic stress is the damage caused by pathogens (bacteria, viruses, parasites, fungi, etc.). The plant pathogen has been divided into biotrophic, hemibiotrophic, and necrotrophic. The biotrophic pathogens do not kill the cell contrary to the necrotrophic, while hemibiotrophic keep its host alive while establishing itself within the host tissue, taking up the nutrients with brief biotrophic-like phase (Lai et al., 2011). The defense mechanisms Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 24 against pathogens are mainly PAMP‐triggered immunity (PAMP; Pathogen-Associated Molecular Pattern) and EFFECTOR-TRIGGERED IMMUNITY (ETI). The salicylic acid and jasmonic acid/ethylene pathways are associated downstream to PTI and ETI (W. Zhang et al., 2018). ETI is mediated by the NUCLEOTIDE-BINDING DOMAIN (NB- LRR). Occasionally, NB-LRR is accompanied by programmed cell death called hypersensitive response (HR PCD). atg5, atg10, and atg18a mutants show defects in basal plant immunity against pathogen by PAMP‐triggered immunity (Leary et al., 2018). ATGs are upregulated during PCD; PI3K/VPS34, ATG3, and ATG7 are expressed during uncontrolled cell death in response to TMV infection. Autophagy leads to cell death in damaged tissue and promotes survival in uninfected tissue (Seay et al., 2006). Abiotic stress Plants have developed mechanisms that allow them to perceive and respond to a stress condition due to the constant changes in the environment. Thus, besides being a mechanism that enables nutrients to be recycled and remobilized, autophagy can also respond to the abiotic response, as we have already seen in the hormones section (Akpinar et al., 2012). Salt stress is one of the severe problems that affect agriculture because it causes growth inhibition and inadequate development in plants. The RNAi-AtATG18a plants show a sensitive phenotype in salt and drought conditions than wild-type Arabidopsis. In these studies, they realized the NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH) oxidase inhibitors block autophagy by nutrient starvation and salt but not by osmotic stress (Liu et al., 2009). ATG8 overexpression performs better germination assay in salt and osmotic stress. Studies with quantification of osmolytes confirmed the autophagy is relevant in salt stress adaptation (Luo et al., 2017), and the ATG8 overexpression confers tolerance to drought and nutrient stress in Foxtail millet (Setaria italica L.) in Arabidopsis (Li et al., 2015). In wheat, TdATG8 silencing showed ATG8 as a positive regulator of osmotic and drought response (Kuzuoglu-Ozturk et al., 2012). Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 25 Under abiotic stresses of drought, heat, cold, and carbohydrate starvation in pepper (Capsicum annuum L.) increases the autophagosome numbers. CaATG6 interacts with CaHSP90 (Heat-Shock protein) family indicating its role in heat tolerance (Zhai et al., 2016). Salt, drought, and heat stresses result in unfolded or misfolded proteins in the endoplasmic reticulum (ER). This ER stress induces autophagy in Arabidopsis (Liu et al., 2012). In oxidative stress, RNAi-AtATG18 Arabidopsis plants cannot degrade the oxidized proteins suggesting the role of ATG18 in oxidative stress (Xiong et al., 2007). Under waterlogging, the plants induce hypoxia-responsive genes and respiratory burst oxidase homolog (RBOH)-mediated REACTIVE OXYGEN SPECIES (ROS) production in roots. Furthermore, ATG mutants under waterlogging respond with higher ROS and cell death levels suggesting the autophagy attenuating effect on programmed cell death in roots (Guan et al., 2019).  Nutrition Starvation During starvation of nutrients, autophagy maintains homeostasis with bulk degradation to facilitate nutrient mobilization in plants. ATG1 was analyzed under carbon starvation in mutants of ATG1a, ATG1b, ATG1c, ATG1t and a quadruple mutant to understand the essential part of ATG1 under carbon starvation and nitrogen deprivation. While analyzing the role of PI3K complex and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE 1 (SnRK1), the possible mechanism is phosphorylation of ATG6 as a subunit of P13K complex by KIN10 subunit of SnRK1 (Huang et al., 2019). Lipidomic, proteomic, and metabolomic analysis show altered lipid composition in ATG mutants and the increase of respiration in etiolated ATG mutants and under carbon and nitrogen starvation (Avin-Wittenberg et al., 2015). Also, Aubert results suggest that the mitochondria along with the respiratory substrates control the induction of autophagy during carbohydrate starvation, unlike the idea that the decrease of sucrose induces autophagy (Aubert et al., 1996).  Under phosphate starvation, the inhibition of lateral root and auxin accumulation is mediated by autophagy. For example, the ARABIDOPSIS RECEPTOR KINASE 2 / E3 LIGASE PLANT U-BOX/ARMADILLO REPEAT PROTEIN 9 (ARK2/AtPUB) module Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 26 regulates the lateral root development through autophagy (Sankaranarayanan & Samuel, 2015). Also, mutant atg5-4 has a reduction of lateral root development under low phosphate (Sakhonwasee & Abel, 2009).  In relation to the micronutrient potassium and autophagy have not been any reported in plants while yeast and mammals during starvation, the deacetylation of ATG3 reduced the expression of the ” potassium dependence 3” (Kondratskyi et al., 2018; Yi & Yu, 2012). On the contrary, the K homeostasis with K selective ionophore valinomycin and salinomycin promotes autophagy in several cell types where salinomycin induces ROS generation (Klein et al., 2011; Rigault et al., 2021).  In addition, calcium regulates autophagy to maintain the mammalian cell survival implied in the life and death decision, but in plants, exogenous calcium increases autophagy, providing resistance to Botryosphaeria dothidea infection in pear (Harr & Distelhorst, 2010; Sun et al., 2020). Concerning zinc and sulfur, some experiments show that cells accumulate autophagosomes during zinc limitation. ATG5 and ATG10 mutants accelerate senescence under zinc deprivation; hence autophagy is essential to zinc recycling, zinc-deficient conditions mainly (Eguchi et al., 2017). Moreover, atg5 mutants had lower S remobilization than control lines under high Sulphur conditions than under Sulphur limitations (Lornac et al., 2020). Finally, magnesium, boron, and molybdenum have not yet been analyzed, but if there are studies of copper stress in Vitis vinifera. After four copper stress treatments, VvATG8a and VvATG8i had more expression compared to the control. This suggests ATG8 is involved in copper stress (Shangguan et al., 2018).  Nitrogen metabolism and starvation Nitrogen is an essential element for life on the earth. Nitrogen constituent 80% of the atmosphere, primarily elemental, is di-nitrogen (N2) and other nitrogen gases such as ammonia (NH3), nitric oxide (NO), nitrogen dioxide (NO2), and nitrous oxide (N2O). In contrast, aquatic systems contain nitrate (NO3 -) and ammonia/ammonium (NH4 +). Cycling nitrogen is the dynamic exchange of chemical species between the atmosphere and the surface landmasses and ocean (Polacco & Todd, 2011). The nitrogen cycle is composed Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 27 of nitrogen fixation, ammonification, nitrification, and denitrification (Byrne et al., 2019). Usually, the plant nitrogen metabolism looks at uptake and transport of nitrogen in nitrate assimilation, amino acid biosynthesis, protein synthesis, and ammonium assimilation but is more complex. The storage, remobilization, recycling ammonia, nitrogen acquisition efficiency, and nitrogen interaction with carbon metabolism are significant aspects of understanding the nitrogen in the plant (Kiba et al., 2012; Stitt et al., 2002). This understanding of the nitrogen metabolism is crucial to plant sciences. Plant growth is limited by nitrogen because it is fundamental for the amino acids as GLUTAMIC ACID (Glu), GLUTAMINE (Gln), ASPARTIC ACID (Asp), and ASPARAGINES (Asn), enzymes such as NITRATE REDUCTASE (NR), GLUTAMINE SYNTHETASE (GS), GLUTAMATE DEHYDROGENASE (GDH), GLUTAMINE SYNTHASE (GOGAT), ASPARGINE SYNTHETASE (AS), and ASPARATE AMINOTRANSFERASE (AspAT), also coenzymes, phospholipids, nucleic acids, chlorophyll, and more molecules which influence in root architecture, senescence and flowering (Fredes et al., 2019; Hörtensteiner & Feller, 2002; Weber & Burow, 2018; Zhang et al., 2007). As previously mentioned, nitrogen is one of the essential elements for plants. In the morphological aspects, it is known that the plants present symptoms such as impaired plant development, leaf chlorosis, and reduced quality and quantity crop production during nitrogen starvation (Massaro et al., 2019). Primary and lateral root length is increased under minimal N limitation in Arabidopsis (López-Bucio et al., 2003). The root length increment may happen because of the induction of WALL ASSOCIATED KINASE 4 (WAK4) and shootward auxin transporter MULTIDRUG RESISTANCE 4/P- GLYCOPROTEIN 4 (MDR4/PGP4) in Arabidopsis (Lally et al., 2001).  The root development is retarded under severe N limitation; therefore, the primary root is short, and the lateral roots are scarce in Arabidopsis (Araya et al., 2016). This root phenotype is caused by ARABIDOPSIS CRIKLY 4 (ACR4) and AUXIN RESISTANT 5 (AXR5) downregulation (De Smet et al., 2008). The GLUTAMATE DEHYDROGENASE (GHD) is also downregulated under nitrogen starvation roots affecting the carbon and nitrogen metabolism (Hirai et al., 2004). Nitrogen deficiency modulates the localization of ROS into epidermis and regulates the gene expression in response to this macronutrient. Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 28 Deficiency. (Shin et al., 2005). In rice, the shoot biomass and NITRATE REDUCTASE (NR) decrease during the nitrogen starvation (Li et al., 2006).  In tomato, ROS increased, the photosynthesis and leaf expansion were reduced after one day under N starvation, considering the critical concentration for optimum growth rate is 3.8% reached before three days of N starvation (Martínez-Romero et al., 1991; Dong et al., 2021). The chloroplast carries out photosynthesis and stores the nitrogen in leaves used under N stress with the help of autophagy genes (Makino & Osmond, 1991; Ren et al., 2014; Wada & Ishida, 2019). ATG is responsible for chloroplast and rubisco degradation in senescent leaves (Ishida et al., 2014). Studies on the carbohydrate connection with N deficiency signaling in tobacco demonstrated that, “the response of photosynthesis to the early effects of N deficiency is identical to the response of photosynthesis elevate carbohydrate” (Paul & Driscoll, 1997).   MYB48, NF-Y, WRKY, and BHLH are upregulated in response to N starvation (Curci et al., 2017). Many transcription factors are activated after one hour, and few continue after seven days, and transporters are activated along with the N stress progression (Cai et al., 2012). The Durum wheat transcriptome reveals the upregulation of the N transporter. The N remobilization is higher in N starvation in leaves and PROTEASES VACUOLAR PROCESSING ENZYME GAMMA (VPE-γ), metacaspase, asparaginase, and one cysteine protease, cysteine protease (SAG12), explaining the senescence and remobilization in leaves (Curci et al., 2017). Meanwhile, microRNAs are small RNAs with a negative regulator of genes and a functional role in N starvation. The identification of microRNAs involves N starvation, for example, mR169 in Medicago is a key regulator of a nodule. In Chrysanthemum nankingense, 81 miRNAs in roots and 101 in leaves were found under N starvation; among these, miR156, miR169, and miR393 are notable (Song et al., 2015). Recent studies found the participation of autophagy genes in nitrogen metabolism during average growth and under starvation (Ren et al., 2014). Several authors have reported Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 29 analyses of trends in autophagy that demonstrated the autophagy genes play an essential role in nitrogen starvation. The epigenetic network of ELONGATED HYPOCOTYL (HY5) and HISTONE DEACETYLASE 9 (HDA9) regulates autophagy responses to nitrogen starvation and light to dark conversion (Yang et al., 2020). In terms of global expression of ATG genes are correlated with assimilation of ammonium genes. AtATG3, AtATG5, AtATG9, and AtATG10 are expressed genes with most responses shown in N starvation, followed by ATG1, ATG4a, ATG4b, ATG18f, and five members of ATG8 (Bedu et al., 2020). During nitrogen uptake, MdATG10 overexpression construction in apple promotes the uptake of limited nitrogen nutrients, and MdATG9 overexpression in callus enhances tolerance to nitrogen depletion stress (Huo et al., 2020, 2021). Besides, autophagy is correlated with nitrogen storage; some evidence is considered with the atg5 mutant in Arabidopsis seeds (Di Berardino et al., 2018). In nitrogen flow and nitrogen remobilization, some ATG was analyzed, such as overexpression of AtATG8 in Arabidopsis stimulating autophagic activity and nitrogen remobilization under N starvation. In the same plant, atg5 mutants had defects in nitrogen remobilization; here, the authors suggest that it is for premature cell death in leaves (Guiboileau et al., 2012). As was mentioned before, in senescent leaves the autophagy was studied under N starvation, and the biological process is implied in the protein aggregations, chloroplast, and rubisco degradation, which induce the nitrogen recycling and nitrogen remobilization (Feller et al., 2008; Havé et al., 2017; Ishida & Yoshimoto, 2008; Toyooka et al., 2006).  In seeds and roots, autophagy was reported under N starvation, and results are related to nitrogen remobilization directly. OsATG8b contributes to nitrogen remobilization and rice grain quality (Fan et al., 2020). In Triticum aestivum, autophagy is regulated by H2O2 and O2, which are produced by NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE OXIDASE (NOX) under nitrogen deficiency (Jing et al., 2020).Also, CsATG8e, in Camellia sinensis, has been related in nitrogen remobilization under deficient N conditions (Huang et al., 2020).  Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 30 The role of ATG genes in nitrogen use efficiency (NUE) was demonstrated by silencing atg5-1, atg9-2, and atg18a (Hanaoka et al., 2002; Masclaux-Daubresse & Chardon, 2011; Thompson et al., 2005; Xiong et al., 2007), OsATG8a is considered relevant to increase NUE and rice yield (Yu et al., 2019). Recent studies demonstrate autophagy as a response to nitrogen starvation because it is essential in nitrogen metabolism (Sirko & Masclaux-Daubresse, 2021). LEGUME NODULATION AND AUTOPHAGY IN P. vulgaris Legumes nodulation . Legumes cover 18,000 to 19,000 species, which are identified in warm temperature regions of both the northern and southern hemisphere (Nassar et al.,2010 and Polhill et al., 1981). Papilionoideae subfamily of Fabaceae is one of the biggest subfamilies, diverse and widely distributed around the world. Papilionoideae contains important plants for food, genomic models (Gepts et al., 2005). This subfamily also contains economically important legume crops such as Phaseolus vulgaris (Common bean), Medicago truncatula, Glycine max (Soybean), Arachis duranensis, (Peanut), Arachis ipaensis, Cajanus cajan, Lotus Japonicus, Cicer arietinum, Lupinus angustifolius, Pisum sativum (Pea), Vigna angularis, Vigna radiata, and Trifolium pratense (red clover). Legumes establish a nitrogen-fixing root nodule by symbiosis with bacteria. The mutualistic relationship between Rhizobium and legumes is categorized into nutritional mutualism and a bidirectional consumer-resource (Jones et al., 2012). The complex relation is explained in sections, nodule organogenesis, plant immunity and host rage restriction, Rhizobial infection, nodule autoregulation, bacteria release, symbiotic metabolism, and transport, senescence, and defense (Roy et al., 2020). Overall, legumes develop determinate and indeterminate nodule. Tropical and subtropical develop determinate and legumes from temperate climates develop an indeterminate nodule. The indeterminate nodules initiate the cell division, inner cortex and cell division is persistent while the determinate nodules have the initial cell divisions, outer cortex and the nodule growth is bases of expansion (Hirsch, 1992). Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 31 The symbiosis initiates when the root exudate the phenolic flavonoid compounds which determine the specificity of the symbiosis (Fig. 3) (Redmond et al., 1986). These flavonoid compounds attract the bacteria to the legume roots and triggers the nod gene expression to produce LIPO-CHITO-OLIGOSACCHARIDES (LCO) known as NOD FACTOR (NF) in Rhizobia. Plant perception of the compatible Rhizobia species and NF stimulates the re- arrangement of microtubules of the root hair that deform the structure, and allows the penetration of Rhizobia encapsulated (Bhuvaneswari, 1981; Yao & Vincent, 1969). The structure where it is encapsulated is known as the infection chamber, which expands inwards as the bacteria start to divide, and here is when the infection threads initiate (Fournier et al., 2015). The infection thread is a transcellular tubular structure that grows and moves behind the nucleus which moves down the root hair (Brewin, 2004; Cole & Fowler, 2006; Nutman, 1959). Then the nod factor from bacteria provokes the sub- epidermal cell division in the outer or middle cortex next to the xylem pole and after ramify (Ferguson et al., 2010). These divisions initiate with anticlinal cortical cell division and then periclinal cell division. While the progression infection thread progression into inner cortex and towards nodule primordium (Ferguson et al., 2010). When the Rhizobia are released intracellularly, form symbiosomes (specialized compartments of periplasm membrane in a host cell) finally differentiate into nitrogen-fixing bacteroids (Liu et al., 2019). Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 32 Figure 3 Nodule development in legumes and Infection threat. (A) The formation of the thread of infection begins with the contact of the bacteria with the root hair (rh) (1A), causing the root hair to curl (2A) and the nucleus to move surrounded by a cytoplasmic streaming (3A) that directs the bacteria (4A) towards the root hair base near to cortical cells (5-7A) and then the infection threats branches. (B) Developmental stages of determinate legume nodules. Once the root hair curves, the cortical cells divided in sub-dermical. Beginning with anticlinal cortical cells (1B) and the periclinal cell division (2B). The infection threat progress into outer cortex(3B) then into inner cortex(4B). The cell layers divided form the nodule primordium and began the bacteroid differentiation (6B) to for a mature N-fixing nodule (7B). (Ferguson et al., 2010; Rae et al., 2021) The flavonoids are produced under low N and trigger Nod factor production in bacteria (Liu & Murray, 2016). Flavonoid synthesis in legumes involves CHALCONE SYNTHASE (CHS), CHALCONE REDUCTASE (CHR), FLAVONE SYNTHASE (FNS) and Nod factor in bacteria imply the expression of nod genes, such as nodABC and nodD. NodABC that determines the synthesis of the lipochito-oligosaccharide core common in Nod factor (Subramanian et al., 2006; Wasson et al., 2006; Zhang et al., 2009). These nod factors induce the nodulation at low concentrations (down to 10-12 mol l-1) and are perceived by nod factor receptors where are in the plasma membrane of epidermal cells. Nod factor receptors containing oligosaccharide-binding LysM domains to recognize the LCOs some receptors are NOD FACTOR RECEPTOR/LYSM RECEPTOR KINASE (NFR1/LYK3), NOD FACTOR RECEPTOR 5 /NOD FACTOR PERCEPTION (NFR5/NFP) and “DOES NOT MAKE INFECTIONS 2” /SYMBIOSIS RECEPTOR KINASE (DMI2/SYMRK) (Dénarié, 2001; Dénarié & Cullimore, 1993). The receptor DMI2/SYMRK is essential for Rhizobial and Arbuscular mycorrhizal symbiosis and interacts with other co-receptors at cell membrane, for example NFR1 and NFR5 (Geurts et al., 2016). SYMRK can autophosphorylate, this receptor conserves three Ser/Thr residues as phosphorylated sites which are crucial in kinase activity (Yoshida & Parniske, 2005). SYMRK INTERACTING PROTEINS (SIPs) are part of nod factor receptor complex in nodule organogenesis some reports involve ARID domain- containing protein and SYMRK INTERACTING E3 UBIQUITIN LIGASE (SIE2) (Wang et al., 2013; Zhu et al., 2008). The perception of Nod factors for plants allows depolymerization of cell membranes and changes in ion fluxes. For instance, “calcium spiking” is the oscillations in calcium concentration in nuclei of epidermal root hair cells driving changes in gene expression Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 33 related with nodulation (Charpentier & Oldroyd, 2013). Some nuclear envelope proteins are required in calcium spiking, such as calcium channels CASTOR and POLLUX/DMI1 encode potassium-permeable channels essential in calcium spiking, NUCLEOPORIN SUBUNITS NUCLEOPORIN 85 AND 133 (NUP85 and NUP133), NUCLEOPORIN- LOCALIZED PROTEIN (NENA), cyclic nucleotide gates channels a, b, c (CNGC) permeable cation transport channel implicate in the uptake Ca2+(Charpentier et al., 2008; Nawaz et al., 2014). The nuclear calcium spiking signal is deciphered by the NUCLEAR CALCIUM-CALMODULIN KINASE (DMI3/CCaMK), CYCLOPS and calmodulin. CCaMK is required to transduce the signal to effect changes in gene expression in legumes (Mitra et al., 2004). The transcription factor CYCLOPS interacts with CCaMK and is a phosphorylation target of CCaMK (Yano et al., 2008). Besides, NODULATION SIGNALING PATHWAY 1 AND 2 (NSP1 and NSP2) and CCaMK-CYCLOPS are some regulators involved in expression of NIN transcription factor and noduline genes necessary for bacterial infection (Verma et al., 1986; Xiao et al., 2020). Also, CCaMK leads activate cytokinin signaling. The cytokinin signal moves by diffusion and selective transport from epidermis to cortex (Frugier et al., 2008). In cortex, cytokinin signal via CRE1 mediates NIN, NF-Y and ERN regulation that provoke the upregulation of ENOD expression which controls the cortical division and nodule organogenesis (Chaulagain & Frugoli, 2021). Also, some microRNAs are reported during organogenesis miR167 acts up stream of NIN, NSP1, NF-YA1, NF-YA2 and ENOD40, miR160 maintain the balance between auxin and cytokinin during nodule inception (Nizampatnam et al., 2015; Wang, Li et al., 2015). Moreover, immune system and host range restriction are triggered and employs checkpoints to differ between pathogen and symbiont. LRR-RLK and LysM-RLK identify the bacteria molecules which are neutralized by NBS-LRR and R proteins (Cao et al., 2017). Rhizobial infection is established after attachment of Rhizobia to root hair and form the infection pockets via infection threat. At this point, reports identified proteins such a multiple hormonal regulation (e.g., EIN2, ERN1, ARF8a, ARF8Bb, ARF16, LHK1, GA2ox10), cytoskeleton orientation (e.g., NAP1, PIR1, SCAR/WAVE, ARPC1, SARN), cell wall (e.g., NPL, ENOD11, ENOD12), membrane (e.g., FLOT2, FLOT4, SYMREM1), Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 34 autophagy pathway (e.g., P13K, TOR, BECLIN), reactive oxygen species (e.g., RIP, ROP6, RHOB), cell division (e.g., PLT, KNAT) (Roy et al., 2020). The continues cell divisions in the cortex and pericycle prompted nodule organogenesis. Simultaneously to organogenesis, the plants maintain a long-distance systemic signaling regulatory system called AUTOREGULATION OF NODULATION (AON). The nodule induces a translocatable signal Q which shift toward to leave through the root-shoot xylem pathway (Oka-Kira & Kawaguchi, 2006). In AON, we can find the shoot-dependent components (e.g., NOD4, NOD5, NOD6) and the root-dependent components (e.g., NIC1, CLE12, RIC1, PLENTY, EFD) (Han et al., 2010; Reid et al., 2011). During AON, nitrate induces CLE peptides (e.g., NIC1 and CLE-RS2) in the epidermis that operate in cortex via the NARK receptor. CLE peptides are putative ligands for the autoregulation LRR receptor kinase within NARK (Oka-Kira & Kawaguchi, 2006). NARK inhibits the nodule progression, but in the shoot recognizes a Rhizobial-induced CLE peptide (RIC1 and RIC2) which are transported via the xylem to the shoot. NARK acts in shoot with CLV2, KLV and CRN likely to recognize RICS but also NARK phosphorylates KAPP1 and KAPP2. The equilibrium of phosphorylation between these NARK and KAPP1/2 requires the system require SHOOT-DERIVED INHIBITOR (SDI) which is transported by phloem to roots where it inhibits the cell division and nodulation (Reid et al., 2011). On the other hand, Rhizobia, after being released divides, differentiates into N-fixing bacteroid that releases ammonia into the plant cell in exchange for reducing carbon (Patriarca et al., 2002). Differentiation is accompanied by a decrease in free oxygen that is to prevent the inactivation of nitrogenase, and the color of the nitrogen fixation zone is converted in pink. Nitrogenase is a metalloenzyme system in bacteria that catalyzes the ATP- dependent reduction of DINITROGEN (N2) to AMMONIA (NH3) and is protected by LEGHEMOGLOBIN (LHb) (encoded in host plant) from being inactivated by oxygen (Masepohl & Forchhammer, 2007; Sudhakar et al., 2016). In nitrogen fixation, the molybdenum, the oxygen, carbon and nitrogen ratio, Fe availability, temperature, and light intensity, among other variables allow better performance. Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 35 When the plant uptake nitrogen, nitrate reduction is catalyzed by NITRATE REDUCTASE (NR) and is translocated to the chloroplast where NITRATE REDUCTASE (NiR) allows the reduction to ammonium (NH4) (Meyer & Stitt, 2001). The major NH4 assimilation pathway consists in GLUTAMINE SYNTHETASE/GLUTAMINE: 2-OXOGLUTARATE AMINOTRANSFERASE (GS/ GOGAT) cycle (Lea & Miflin, 1974; Kojima et al., 2014). Here the glutamine synthetase fixes ammonium; this is the condensation of the glutamate and ammonia to form glutamine. Two GOGAT isoenzymes (NADH-GOGAT & Fd- GOGAT) transfer the amido nitrogen of glutamine to 2-oxoglutarate. GOGAT requires energy, reductant, and cytoskeleton in the form of 2OG and NADH or Fd as reductances (Lancien et al., 2000). Additionally, ASPARAGINE SYNTHETASE (AS), CARBAMOYLPHOSPHATE SYNTHASE (CPSase) and NADH-glutamate dehydrogenase participate in ammonium assimilation (Masclaux-Daubresse et al., 2010). Finally, During the nodule senescence the pink nodule changes to green color because of the nitration reaction for the heme group of leghemoglobins. Reports suggest that the leghemoglobins with modifications and aberrant O2 binding results in senescence of nodule (Navascués et al., 2012). Furthermore, the structure changes defined by different studies are the decrease of electron density, increase the vesicle number in cytoplasm and peroxisomes, the mitochondria form an elongated complex, damaged cell wall and lysis of bacteroid, and symbiotic membrane disintegration (Puppo et al., 2005; Van de Velde et al., 2006). Autophagy in P. vulgaris and legumes nodulation The relationship between autophagy and root nodule symbiosis is scarcely studied. First legume explored was soybean (G. max) which was inoculated with Bradyrhizobium japonicum. The studies reveal activation of autophagy process at level of symbiosomes during senescence induced by dark (Vauclare et al., 2010). Then, Faba bean (Vicia faba) inoculated with Rhizobium leguminosarum and/or Glomus aggregatum autophagy was induced by during application of silver nanoparticules (AgNps) in soil (Abd-Alla et al., Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 36 2016). Also, the identification of 39 ATGs in Medicago companying with expression profile during seed development, response to salt and drought stress (Yang et al., 2020). The autophagy genes have been studied in P. vulgaris. PHOSPHOPHATIDYLINOSITOL 3-KINASE (PI3K) which participates in the immune response, intracellular trafficking, autophagy, and senescence was analyzed in P. vulgaris. PI3K was downregulated and results show significant decrease in root hair growth and curling at the same time BECLIN1/ATG6, VPS15 and ATG8 which interact with PI3K showed reduced expression. Results suggest the autophagy provides precursors during Rhizobium tropici and Rhizophagus irregularis penetration (Estrada-Navarrete et al., 2016). Another relevant work for this study is the deep exploration of TOR in legume where ATG1, ATG13 and ATG8 were analyzed. These ATG genes increase their expression in TOR RNAi. TOR is a negative regulator of autophagy but also is essential for the nodule development (Nanjareddy et al., 2016). PROPOSAL OF THE PROBLEM Legumes are considered an alternative way to help in economic and ecological situations such as in agriculture crises providing nitrogen and an important source of protein (Triboi & Triboi-Blondel, 2021). The regulation of legume-Rhizobium symbiotic association is very intricate, and many biochemical processes have been attributed to play an indispensable role during this association. Autophagy is an important phenomenon in the successful establishment of host-microbe interactions not only in pathogenesis, but also in symbiotic interactions, as demonstrated in several species (Wang et al., 2021; Tang et al., 2016). However, little is known about autophagy as a regulator of symbiotic associations in plant-microbe interactions. Therefore, a better understanding of autophagic processes and their involvement in host- symbiont interactions will allow us to generate new knowledge and insight in this field of research. Recent studies demonstrate that the autophagy-associated kinase Beclin1/Atg6 in Phaseolus is involved in root hair growth. In addition, it is also found to be essential for nitrogen-fixing symbiosis (SFN) by regulating the growth of the infection Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 37 thread, the number of root nodules, and the formation of symbiosomes in the root nodule cells. The present project is mainly focused on the identification of nodulation specific ATG genes and subsequent functional characterization of these ATG genes during symbiosis AIM OF THIS THESIS Role of autophagic process during symbiosis remains to be elucidated. In economically important legume such as P. vulgaris, such studies will contribute not only for the understanding of the regulation of symbiotic association but may also help improve the biological nitrogen fixation. Herein, the objective is to identify the autophagy (ATG) genes in P. vulgaris and understand the role of candidate ATG in root nodule symbiosis. GENERAL OBJECTIVES 1. Identification of ATG genes in P. vulgaris and transcriptional analysis (RNA-seq) of P. vulgaris roots inoculated with Rhizobium tropici to find those genes with differential expression (candidate genes) in the P. vulgaris-Rhizobium interaction. 2. Functional characterization of candidate genes using transgenic bean roots and gene silencing (RNAi) and gene overexpression techniques, to understand the dynamic expression of genes during P. vulgaris-Rhizobium interaction 3. Physical interaction between the candidate ATGs based on studies in other eukaryotes and using the yeast two-hybrid system to predict the role during P. vulgaris-Rhizobium interaction. Research questions that will be answered 1. What is the role of autophagy during nodulation in P. vulgaris? (General) 2. Which ATG genes are conserved in legumes? (Chapter II) 3. What are the features of ATG gene families in legumes? (Chapter II) 4. Which ATG participate in the legume-rhizobium symbiotic interaction? (Chapter III & Chapter IV) 5. What is the role of candidate ATG genes in P. vulgaris root nodule symbiosis? (Chapter III) 6. What are the interacting proteins of ATG genes in P. vulgaris under Rhizobial symbiotic conditions? (Chapter IV) Chapter I. General Introduction: Analysis of autophagy genes in roots of P. vulgaris during nodulation 38 References Abd-Alla, M. H., Nafady, N. A., & Khalaf, D. M. (2016). 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However, ATG genes are poorly known in legumes and here we contribute to detect the autophagy core, which are 32 genes in P. vulgaris, 38 genes in M. truncatula and 61 genes in G. max. Besides, we explored the chromosome localization, phylogenetic relationships. Then we examined the ATG18 family which is one of the largest family. Based on the phylogenetic tree analysis, principal components analysis, and primary structure analysis, we proposed 3 subfamilies using the proteins sequences of 27 photosynthetic organisms including legumes. In addition to understand the autophagy genes in legumes we performed promoter analysis, expression profiling, transcriptome, qRT-PCR of P. vulgaris nodulation, we found a particular set of ATG genes which show high expression in P. vulgaris during symbiotic relation with Rhizobium, they are PvATG9b and PvATG18g.II. Finally, we demonstrate the autophagy core is conserved and some autophagy genes could play an important role in symbiosis. Chapter II. identification of autophagy genes in P. vulgaris and legumes 43 INTRODUCTION Genes of autophagy (ATGs) in plants The interest in plant autophagy genes has been growing recently. After the initial studies in rice and Arabidopsis reported in 2002, there has been an increase in number of studies to decipher the role of ATG genes in plants (Hanaoka et al., 2002). Further, advancements in ‘omics’ such as transcriptomics, proteomics and metabolomic studies have contributed tremendously to autophagy studies (Liu et al., 2018). Conserved ATG core of A. thaliana, Barley, Grapevine, Maize, Rice, Tobacco, Tomato, Banana, Foxtail millet, Pepper, Wheat, Cassava have been understood by carrying out omics analysis (Table.1). This plant core comprises ATG1, ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG8, ATG9, ATG10, ATG11, ATG12, ATG13, ATG16 and ATG18. Each of these ATG genes belong to different families, which in turn are grouped based on the autophagy process they are involved in (Supp. S1). So far, autophagy genes in plants are analyzed in response to development, nutrient starvation, senescence, pathogens, ROS, drought, salinity, heat stress or hypoxia in different plants. Autophagy signaling pathway is regulated by various upstream kinase cascades, one of them is TOR kinase (Kunz et al., 1993; Díaz-Troya et al., 2008; Rabinowitz & White, 2010). Target of rapamycin (TOR) is a conserved central growth regulator in eukaryotes that has a key role in maintaining cellular nutrient and energy status (Arthikala et al. , 2021). For this reason, TOR is a highly conserved kinase across the eukaryotes and the process of autophagy may be dependent or independent of TOR pathway (Pu et al., 2017). Autophagy activation for nutrient starvation, salt and osmotic stress is TOR- dependent and independent of oxidative and ER stress (Pu et al., 2017). Chapter II. identification of autophagy genes in P. vulgaris and legumes 44 Table 1.Genes of autophagy (ATGs) in plants. *(Avila-Ospina et al., 2016; Inoue et al., 2006; Klionsky et al., 2003; Li et al., 2015; Norizuki et al., 2019; Shangguan et al., 2018; Tang & Bassham, n.d.; Wei et al., 2017; Xia et al., 2011; Yang et al., 2019; Zhou et al., 2015) Under nutrient-rich conditions, the TOR hyperphosphorylates ATG13 affecting the formation of the complex ATG1/ATG13. ATG1 encodes Ser/Thr protein kinase domain at their N terminal. In Arabidopsis, The ATG1 family is comprised of ATG1a, ATG1b, ATG1c and ATG1t. The Atg1a and b/c are paralogs, and they are identified in many species such as bryophytes, eudicot, and monocot. ATG1t is unique in monocots and gymnosperm but has been not found in bryophytes. ATG1 phylogenetic studies showed that ATG1 and ATG13 are two parallel gene families. Upon TOR inhibition, these ATGs interact and form the ATG1/13 kinase complex as autophagic inductors, accompanied by ATG11 and ATG101 in Arabidopsis. The complex forming genes in other organisms are ATG17, ATG29 and ATG31 that have not been found in Arabidopsis (Kang et al., 2018; Li et al., 2014; Suttangkakul et al., 2011). ATG11 may play an important role in the initiation of autophagy because the atg11-1 mutants were defective in ATG1 phosphorylation and Chapter II. identification of autophagy genes in P. vulgaris and legumes 45 thus autophagy. In the same studies have also demonstrated ATG11 interaction with ATG101 (Li et al., 2014). Once the complex ATG1/ATG13 formed with the accessory proteins ATG11 and ATG101 in the phagophore assembly site or phagophore assembly site (PAS), it induces the phagophore nucleation through PI3K complex (Phosphatidylinositol 3-P). ATG2/ATG18 complex and ATG9 are also involved along with PI3K in the nucleation stage. PI3K complex contains the ATG6/BECLIN-1/VPS30 as one of the most important components which is accompanied by a VESICULAR PROTEIN SORTING 34 (VPS 34), VESICULAR PROTEIN SORTING 38 (VPS 38), VESICULAR PROTEIN SORTING 15 (VPS15) and ATG14 proteins. Arabidopsis contains a single homologue of VPS34, VPS15, ATG6 and ATG14 (Bassham et al., 2006; Tang & Bassham et al., 2018). The CYTOPLASM-TO- VACUOLE TARGETING (CVT) pathway is another sequestration mechanism mediated by cytosolic double membrane vesicle but operates under nutrient-rich condition, PI3K complex with some VPSs take part in this process (Klionsky & Emr, 2000). The VPS34 has a kinase site with ATP- binding domain near to C terminus and the putative lipid binding domain near the N terminus which appear in proteins involved in vesicle transporting (Welters et al., 1994). VPS34 interacts directly with VPS15. The findings in Vps15 implied the post transcriptional lipid modifications by myristylation of VPS15 (Turnbull & Hemsley, 2017; Wang et al., 2012). The last member in this PI3K complex is ATG14, which has been recently identified in barley, grapevine, maize, rice, tobacco and tomato as ATG14a and ATG14b (Tang & Bassham et al., 2018). On the other hand, ATG2/ATG18 complex and ATG9 as part of nucleation stage where ATG2 structure has been poorly understood in plants but in yeast this protein binds two membranes at the same time transferring phospholipids from the endoplasmic reticulum (ER) to the autophagosome (Osawa & Noda, 2019) and interacts with ATG18. ATG18 contains two WD-40 domains that form a propel structure (Dove et al., 2004). ATG18 is required for ATG2 and ATG9 interaction (Gómez-Sánchez et al., 2018). ATG9 has six transmembrane and the C- and N- terminal are exposed into cytosol. This protein is the only transmembrane protein in ATGs and plays a role in the progression of Chapter II. identification of autophagy genes in P. vulgaris and legumes 46 autophagosome from ER (Zhuang et al., 2017). During the process of autophagy, ATG9 is phosphorylated in multiple serine residues (six consensus sites) by ATG1 for the recruitment of ATG18 and allows the recruitment of ATG8 (Papinski & Kraft, 2014). After the nucleation, the expansion and enclosure take place with ubiquitin complexes or also called conjugation systems and these processes are made up of ATG5- ATG12 and ATG8-PE (LC3 system). In plants, ATG5 has is placed onto the curvature edge of early phagophore (Le Bars et al., 2014). ATG5 is conjugated in lysine residue by isopeptide bonds with the C-terminal glycine of ATG12 (George et al., 2000; Hanada & Ohsumi, 2005). Before ATG5 and ATG12 interaction, ATG7 (E1 like protein) activates and ATG10 (E2 like protein) transfers to reaching the interaction with ATG12. The null mutants of ATG10 in Arabidopsis cannot form ATG5-ATG12 complex and the same phenotype is also seen in ATG5 and ATG7 mutants (Phillips et al., 2008). ATG7 is an E1 like protein that catalyzes the ATG12 and ATG8 conjugation using ATP (Yamaguchi et al., 2010). ATG5 also interacts with ATG16 through the N- terminal region, together with ATG12 form ATG12-ATG5-ATG16. The structure of the complex is 2:2:2 heterohexamer (Nakatogawa & Mochida, 2015). ATG16 is a conserved E3 like protein which links the autophagy and the ubiquitin- proteasome system (Xiong et al., 2019). Complex ATG12-ATG5 is being formed, the complex ATG8-PE is ensembled as well. In relation to the ATG8-PE complex formation, ATG4 process a ATG8 exposing the Gly, where ATG7 (E1 like protein) is active and ATG3 (E2 like protein) to conjugates with PE and once again ATG4 deconjugates ATG8 (Nakatogawa & Mochida, 2015). ATG4 is involved in ATG8 recycling by hydrolysis reaction between ATG8 and PE (Kirisako et al., 2000). ATG3 is an E2 like protein, overexpression of ATG3 can induce the autophagy in plants. Also, the studies demonstrated that the ATG3 is inhibit by glyceraldehyde-3- phosphate dehydrogenases (GAPCS) in Nicotiana benthamiana (Han et al., 2015). ATG8 in Arabidopsis has 9 isoforms and is divided into 3 subfamilies GABA type A receptor- associated protein (GABARAP), microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16) (Ryabovol & Minibayeva, 2016). The fusion of autophagosome to vacuole is carried out Chapter II. identification of autophagy genes in P. vulgaris and legumes 47 by Rab GTPases and SNARE proteins. These proteins are evolutionarily conserved in plants (Fig. 4.) (Zhuang et al., 2015). Figure 4 Macroautophagy of yeast and Arabidopsis. Induction of autophagy is regulated by nutritional status. Under starvation ATG1 and ATG13 are dephosphorylated and promote the activation of kinase complex to trigger vesicle nucleation, vesicle expansion and closure, fusion, and digestion (Thompson et al., 2005; Nakatogawa et al., 2013) Furthermore, in this chapter we explore ATG18 protein, that is one of the most abundant autophagy core. The members in ATG8 and ATG18 families comprise between 2 - 10 genes. Many isoforms are non-redundant in their expression patterns and may have different functions (Suttangkakul et al., 2011). In plants, Arabidopsis contains eight ATG18 homologs which are classified as AtATG18a through AtATG18h, with multiple splice variants (Bassham et al., 2006; Xiong et al., 2005). ATG18 was also explored in Sweet Orange (Citrus sinensis), tomato (Solanum lycopersicum), rice (Oriza sativa) and apple (Malus domestica). These recent findings suggest that AtATG18a regulates autophagy under ER stress by reversible persulfidation of the protein at Cys103 site (Aroca et al., 2021). In Sweet Orange, CsATG18a showed enhanced tolerance to osmotic Chapter II. identification of autophagy genes in P. vulgaris and legumes 48 stress, salt and drought while CsATG18b showed cold tolerance (Fu et al., 2020). In tomato, the Heat-shock transcription factor (HsfA1) induces the drought tolerance by activating ATG10 and ATG18f and inducing autophagy (Wang et al., 2015). In apple, MdATG18a has a positive influence on drought tolerance, enhanced antioxidant activity, reduced chloroplast damage and minimizes the impact of Diplocarpon mali pathogen (Sun et al., 2018). Trying to give an ATG18 identifier letter allow us to understand the family and propose a classification based in protein features. Thus, in this chapter we explored the autophagy process identifying autophagy genes and understanding the ATG18 family. RESULTS Identification of ATG families in 3 legumes In A. thaliana, a total of 39 ATG sequences divided into 17 families have been reported. In the present study, we identified a total of 32 genes in P. vulgaris (2n), 39 genes in M. truncatula (2n) and 61 genes in G. max (4n) (Table. 2). A BLAST- NCBI analysis of Arabidopsis sequences returned 19 (59.37%) homologs in P. vulgaris, 28 (77.77%) homologs in M. truncatula and 30 (48.38%) homologs in G. max with a query coverage of 93–94% and 66–77% identity (Supp. S2). For this reason, other ortholog analysis databases were used to identify any missing ATG members. The KEGG orthology table for the autophagy pathway was the second main tool because it contains a wide variety of species, and we used this table to obtain more than 70% of genes in P. vulgaris and M. truncatula and 58% in G. max. An analysis of legumes using Ensembl Plants provided more than 70% of ATGs in the legumes under study. Other studies were performed through a HMMER analysis using Ensembl databases and the InParanoid tools in Phytozome. The obtained sequences were verified using Pfam to acquire the positions of the families, domains and repeats, and the protein motifs were determined with MEME. Additional studies were performed using EggNOG, which provided a list of orthologs, particularly in P. vulgaris (Supp. S3). C h a p te r II. id e n tifica tio n o f a u top ha g y g en e s in P . v u lg a ris a n d le g u m e s 4 9 T a b le 2 . Id e n tifica tio n o f 1 7 g e n e fa m ilie s in A . th a lia n a , P . v u lg a ris, M . tru n ca tu la a n d G . m a x Chapter II. identification of autophagy genes in P. vulgaris and legumes 50 Phylogenetic relationships, chromosome localization of ATG families To understand the evolutionary relationships among ATGs, we generated 17 phylogenetic trees, one for each ATG family in A. thaliana, P. vulgaris, M. truncatula and G. max as per the classification in A. thaliana. The primary protein sequences of A. thaliana, P. vulgaris, M. truncatula and G. max were aligned using Clustal Omega with the Hidden Markov Model, and phylogenetic trees were obtained with the neighbor-joining method. Each of the ATG sequences was also subjected to a motif analysis, which revealed that the sequences and motifs in all the studied legumes showed a high identity to their homologs in Arabidopsis. The phylogenetic trees also revealed that the majority of the ATG families are predominantly composed of Medicago sequences that were more closely related to those in Arabidopsis. Among all the phylogenetic trees of ATGs developed, 11 contained only one clade (ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG10, ATG11, ATG12, ATG14 and ATG101), even if there was more than one isoform, and most of the motif P-values were greater than 1e-100. ATG8 and ATG18 were the families with the highest number of members: ATG18, eight each in Arabidopsis, Medicago and Phaseolus and 19 in G. max; ATG8, nine in Arabidopsis, eight in Medicago, six in P. vulgaris and 10 in G. max. The phylogenetic analysis of ATG8 and ATG18 was divided into three clades with motif P-values between 1 x1013 and 1 x1090 (Fig. 5). The close association of the homologs in all the species studied depicts the conservation of sequences and hence implies biological function conservation. Chapter II. identification of autophagy genes in P. vulgaris and legumes 51 Figure 5. Phylogenetic tree and protein motifs of 17 ATG families in A. thaliana, P. vulgaris, M. truncatula and G. max. Conserved motifs are identified using the MEME search tool. The phylogenetic tree was constructed using the neighbor-joining method in ClustalW2 and visualized using evolview. The chromosome localization of ATGs in the A. thaliana and legume genomes was mapped using OmicCircos (Hu et al., 2014) (Fig. 6). The distribution of ATG homologs among the chromosomes was uneven in all the species compared. Among all 17 families, the maximal number of homologs was located on chromosome 3 in A. thaliana (8) and P. vulgaris (6), chromosome 4 in M. truncatula (6) and chromosomes 4 and 17 in G. Chapter II. identification of autophagy genes in P. vulgaris and legumes 52 max (6). The chromosome localization is accompanied with macrosynteny analysis to compare genomes and reveal the genomic evolution. Figure 6 The chromosomal localization, synteny relationship and gene expression of autophagy genes were integrated into the Circos plot designed using OmicCircos. The outermost circle shows the A. thaliana (blue), P. vulgaris (green), M. truncatula (pink) and G. max (brown) chromosomes. The inner circle is a heatmap that shows the log2 RPKM values of gene expression in leaves and roots under ammonia, nit rate and urea treatments. The innermost line is the synteny of autophagy genes, but the yellow, purple and red lines represent ATG18b subfamilies I, II and III, respectively. Identification of ATG18 family in plants ATG18 families as largest family required extensive study to identify, classify and determine subfamilies of each ATG18 member and reveal a possible function. Here, we selected 27 plant species starting from the early plant lineage Chlorophyta, Charophyta, Chapter II. identification of autophagy genes in P. vulgaris and legumes 53 liverworts, mosses and higher plants such as monocots and dicots. As with other ATGs, the ATG18 family is also well conserved in all the studied plant species; herein, a total of 280 genes and amino acid sequences were identified and retrieved from various databases. Initially, we identified the ATG18 homologs through a BLAST search of NCBI, and we then used the Pfam database to ensure the presence of WD40 repeats in the characteristic ATG18 members. The identified members were named using the aliases registered in the legume information system, NCBI, Phytozome, InParanoid, EGGNOG and Ensembl (Supp. S5 & S6). The genes with the same names were distinguished by adding a Roman numeral: The number I indicated the closest sequence to that in NCBI. For the primitive plants Physcomitrella patens, Chara braunii, Chlamydomonas reinhardtii, Dunaliella salina, Volvox carteri, Klebsormidium nitens, Micromonas pusilla, Ostreococcus lucimarinus, Ostreococcus tauri and Coccomyxa subellipsoidea, we retained the same names that were reported by Norizuki and colleagues (Norizuki et al., 2019). Principal components analysis for ATG18 family Multidimensional scaling analysis using Bios2mds demonstrates the similarity between 280 ATG18 protein sequences from 27 different species. The plot clearly shows that orthologs (genes with closely related sequences and having the same function in different species) are more similar than paralogs (genes that have similar sequences but have different functions in the same species). The plots show that all ATG18 sequences were grouped into three clusters (Fig. 7). The PRINCIPAL COMPONENTS (PCs) allowed us to construct graphs with PC1, PC2 and PC3, and we then applied the K-means method. Cluster I formed a subfamily with ATG18a, c, d and e members from all the higher plant species studied. Cluster II contained only ATG18b homologs, and cluster III contained ATG18f, g and h members. Cluster III consisted of 3 groups: Lower plants formed a distant group, the second group contained the monocot-derived proteins, and the third group harbored all dicots except Arabidopsis, which was more similar to monocots than dicots. Lower plant species were found to be distributed mostly in clusters I and II with the exception of K. nitens, C. subellipsoidea, M. polymorpha and P. patens, which were Chapter II. identification of autophagy genes in P. vulgaris and legumes 54 also grouped in cluster III but exhibited more similarities among themselves than with higher plants. These clusters were named subfamilies I, II and III for convenience. Figure 7 3D representation of 280 ATG18 proteins from a different plant species analyzed by Multidimensional scaling using Bios2mds. ATG18 subfamilies colors code is subfamily I (Yellow), subfamily II (Purple), subfamily III (Red). PC principal component. Axis are principal components (PC): the x-axis (PC1); y-axis (PC2); z-axis (PC3). Phylogenetic relationship of ATG 18 family in plants To understand the evolutionary relationship among primitive and advanced dicot plant species, a multiple sequence alignment of 280 ATG18 amino acid sequences was performed. The aligned sequences were used to generate phylogenetic trees based on the maximum likelihood using MEGA (Fig.8). The largest clade was subfamily III followed by subfamily I, which was mainly composed of ATG18 a, c, d and e. Subfamily II harbored ATG18b. Subfamilies II and III consisted of the Bryopsida, Charophyceae, Klebsormidiophyceae, Mamiellophyceae and Trebouxiophyceae plants, which is important for understanding the divergence of ATG18 homologs. Chapter II. identification of autophagy genes in P. vulgaris and legumes 55 Figure 8 Phylogenetic tree of ATG18 proteins in plants. Protein sequences were aligned using Clustal Omega and the phylogenetic tree was constructed using the ML method in MEGA X software. 280 sequences of ATG18 are distinguished by subfamilies: subfamily I (Yellow), subfamily II (Purple), subfamily III(Red). The plant species are differentiated by letters. A. thaliana (At), M.polymorpha (Mpo), O.sativa (Os), T. aestivum (Ta), Zea mays (Zm), A. duranensis (Ad), A. ipaensis (Ai), C. cajan (Cc), L. Japonicus (Lj), C. arietinum (Ca), L. angustifolius (La), P. sativum (Ps), V. angularis (Va), V. radiata (Vr) and Trifolium pratense (Tp), P. Patens, C. braunii (Cb), C. reinhardtii (Cr), D. salina (Ds), V. carteri (Vc), K. nitens (Kn) , M. pusilla (Mpu), O. lucimarinus (Ol), O. tauri (Ot) and C. subellipsoidea ( Cs). The branch lengths are labeled. Chapter II. identification of autophagy genes in P. vulgaris and legumes 56 Conserved protein motif analysis of ATG18 family. For the detection of motifs in 280 aa sequences, we identified four main motifs using MEME software. Motif 1 (SGVHLYKLRRGATNAVIQDIAFSHDSQWJAISSSKGTVHIF) contained 41 aa, and the motif sequence matched that of the WD40 family (PF00400) and propeller clan 186 (CL0186) in the Pfam database. The InterProScan results also showed that motif 1 belongs to the superfamily WD40 (IPR036322), WD40 repeat-like (SSF50978) and breast carcinoma amplified sequence 3 (PTHR13268). Motif 2 (VIAQFRAHTSPISALCFDPSGTLLVTASVHGHNINVFRIMP) contained 41 aa and the motif sequences were further analyzed with PfamScan to identify the repeats, domains and families. Subfamily I was characterized by motifs 1 and 4, which consisted of WD40 and ANAPC4_WD40 repeats. These motifs also had two domains and eight families, although these Pfam family results are not representative of the subfamily. Subfamily II had motifs 1, 2 and 4, and we detected WD40 and ANAPC4_WD40 repeats in all the members. Only the green alga O. tauri contained leucine‐rich repeats (LRR9 and LRR4). A total of four domains were identified: Gel_WD40, which was the largest, a defensin domain and PQQ and SecA preprotein crosslinking domains. Subfamily II also consisted of three families in six plants (Fig.9). It was similar to motif 1 but contained an additional domain (WD40/YVTN repeat-like domain, IPR015943). Moreover, motifs 3 (VRCSRDRVAVVLATQIYCYBA) and 4 (GYGPMAVGPRWLAYASNPPLLSNT GRLSPQN) did not belong to any protein family. Subfamily III had all four motifs, and we found PD40 repeats along with WD40 and ANAPC4_WD40 repeats. Among the 27 plant species analyzed, nine of them had 12 domains and ATP synthase was specific Z. mays. Breast carcinoma amplified sequence 3 (BCAS3) is a characteristic domain found in most members. Chapter II. identification of autophagy genes in P. vulgaris and legumes 57 Figure 9 Protein motif of ATG18 family from different plant species. Conserved motifs are identified by MEME. The amino acid sequence of the ATG18 family is represented by lines and motifs by boxes using Tbtools. Motif 1 (green), motif 2 (yellow), motif 3 (dark green), and motif 4 (pink). Chapter II. identification of autophagy genes in P. vulgaris and legumes 58 Promoter analysis, Expression profiling and Transcriptome of ATGs families Promoter analysis is an important method for understanding the regulatory mechanisms governing ATGs in response to growth and developmental issues and to environmental cues. The analysis of cis-acting elements in the promoters of all 17 ATG families resulted in 44 different transcription factors. The most abundant transcription factors identified were B-Proto-Oncogene-MYB involved in the ABA response and C-Proto-Oncogene- MYC related to jasmonate signaling, and the transcription factors with the motifs ethylene response elements (ERE), TATA box, CAATT-box and G-box were found for all ATGs in A. thaliana, P. vulgaris, M. truncatula and G. max (Fig. 10). Our results also showed that the ATG8 and ATG18 families contained the highest numbers of MYB, MYC, ERE and Box 4 (ATTAAT) transcription factor-binding sites. Most of the promoters contained MeJA-, SA-, GA- and ABA-responsive elements. Furthermore, light-responsive transcription factors such as BOX-4, G-box, GT1 motif, MRE and ACE were also detected abundantly in most of the families. Figure 10 Transcription factor binding sites in ATG promoters (2000pb) using PlatCare. Interestingly, we elucidated the influence of nitrogen sources on ATG expression in the legume members P. vulgaris, M. truncatula and G. max due to their ability to establish symbiotic associations with nitrogen-fixing Rhizobia. Gene expression data from the Chapter II. identification of autophagy genes in P. vulgaris and legumes 59 Phytozome database were retrieved for leaf and root tissues under urea as the organic source and nitrate and ammonia as inorganic sources, as depicted in Figure 6. The highest expression of ATGs was recorded in roots treated with ammonia and leaves treated with urea. ATG8i and ATG3 showed the highest abundance in all the treatments, and the lowest expression levels were recorded for ATG18b, e, c and h, ATG2 and ATG2.II in G. max and ATG3 and ATG8c in M. truncatula. The ATG18 family homologs ATG18a.II, ATG18g and ATG18h showed induced expression in all tissues under all treatments. Also, in Phytozome database, we obtained the ATGs genes expression in whole plant without treatment. The ATG gene expression at large are low expressed in organs without abiotic and biotic stress. However, there are six genes which are expressed in all tissues, most of them are three ATG8, one ATG18 and ATG3. But only ATG8 and ATG3 are reported in nodules. The flower expression is much higher than other part of the plant. In nodules, only ATG8 and ATG3 have more expression that other ATG genes (Fig. 11). Figure 11 Expression profiles of ATGs in P. vulgaris. Expression profile in different tissues and organs obtained in Phytozome database. The heatmap was built with the log2 of FPKM value and ordered by distances between samples (represented by dendrograms) Furthermore, the differential expression analysis of ATGs in P. vulgaris tissues showed very low expression in young pods collected 1 to 4 days post floral senescence, whereas the fix-(inefficient) nodules collected at 21 days showed the most abundant expression of Chapter II. identification of autophagy genes in P. vulgaris and legumes 60 all ATGs. Interestingly, inefficient fixation increased the expression levels compared with those found with efficient fixation. Among all PvATGs, the ATG1, ATG10, ATG13b, ATG18c and ATG18g.I genes showed the lowest expression in all the analyzed tissues and a total of 16 ATGs were found to be expressed in most of the tissues (Fig. 12). Figure 12 Expression profiles of ATGs in P. vulgaris. Heat map of differential expression of ATGs in tissues and organs during different stages of development and during rhizobia infections obtained in PvGEA database. Expression values are FPKM normalized with Log2. To extend expression findings, we performed RNA-seq analysis and RT-qPCR on our candidate gene using P. vulgaris roots inoculated with Rhizobium (21dpi) and wild type roots as a control. RNAseq comprise RPKM 27,083 values for control and inoculated roots. We calculated the Fold Change (FC) values compared inoculated roots with control and here we found 239 was upregulated (FC>2) and 334 was downregulated (FC<2). Then, we extracted fold change values of autophagy genes identified for P. vulgaris and we detected 12 ATG genes expressed within PvATG9b showing high expression. PvATG9b expression was corroborated with the RT-qPCR (Fig. 13). Chapter II. identification of autophagy genes in P. vulgaris and legumes 61 A B C Figure 13 Trasncriptomic data and Expression patterns of P.vulgaris noludated roots (A) Log2 RPKM and Fold change of Control and nodulated roots. Red represent the FC>2 and Blue FC<2. (B) Fold change of Autophagy core in nodulated root of P. vulgaris. (C) Expression of PvATG9 control by RT-qPCR analysis. Transcript accumulation was normalized to the expression of metalloproteinase as reference gene. DISCUSSION Autophagy is recognized as a highly selective cellular clearance pathway that helps maintain homeostasis in eukaryotic cells. The genes involved in autophagy are highly conserved from yeast to humans, and the process is the result of the interaction of these ATGs and other associated genes. The number of identified ATGs shows a marked variation among different species. In yeast, a total of 41 genes have been identified to date, and several studies on plant ATGs have also identified a varied number of genes. In the present investigation, we attempted to perform a comprehensive study for identifying ATG families in three important legume species, namely, P. vulgaris, M. truncatula and G. max. Furthermore, we focused on the ATG18 gene family, the largest Chapter II. identification of autophagy genes in P. vulgaris and legumes 62 of all the families, to identify and phylogenetically compare 27 plant species starting from early plant lineages, chlorophytes to higher plants including legumes. Using Arabidopsis ATGs as a reference, we retrieved ATG homologs in all the species listed in various databases, including Phytozome, and the sequences were confirmed to be affiliated with ATG-like homologs by analyzing their Pfam matches in the Pfam database. We identified a total of 32, 28 and 61 ATG homologs in P. vulgaris, M. truncatula and G. max, respectively. The identified homologs could be classified into 17 families based on their phylogenetic relationships and motifs. The phylogenetic analysis revealed that homologs in Medicago were located closer to Arabidopsis than those in other species. Unlike in yeast, which contains a single copy of each family, many of the gene families have multiple copies. ATG1 has 4, 3, 2 and 6 homologs in Arabidopsis, Medicago, Phaseolus and Glycine, respectively, ATG13 has 2 homologs in Arabidopsis, Medicago and Phaseolus (2 in each) and 4 homologs in G. max, ATG9 has 2 or 4 homologs in Medicago, Phaseolus and G. max and ATG14 and ATG4 have 2 homologs in Arabidopsis and 2 homologs in G. max. The analysis of larger families revealed that ATG8 has 9, 6, 7 and 10 homologs in Arabidopsis, Medicago, Phaseolus and G. max, respectively, and that ATG18 has 8 homologs in Arabidopsis, Medicago and Phaseolus (8 in each) and a maximum of 19 homologs in G. max. Similar results were also obtained with O. sativa (Xia et al. 2011), Nicotiana tabacum (Zhou et al. 2015), Vitis vinifera (Shangguan et al. 2018), Musa acuminate (Wei et al. 2017) and Setaria italic (Li et al. 2016). However, in most of the families, the homologs were placed in one clade, which clearly showed sequence similarity and the derivation of statistically reliable pairs of possible orthologous proteins sharing similar functions from a common ancestor, consistent with the results from a previous study conducted by Kellogg (2001). ATG18 was the family with the highest number of homologs; hence, we chose this family for a comprehensive analysis of the family from the early plant lineage to legumes. The multiple sequence alignment and phylogeny of ATG18 homologs resulted in separation of the homologs into three clades. Each of the clades had subfamily members, as determined by the multidimensional scaling projection of 280 ATG18 homologs in 27 photosynthetic organisms. Unlike previous studies by Norizuki and colleagues, the Chapter II. identification of autophagy genes in P. vulgaris and legumes 63 classification of the ATG18 family was not based on the BCAS3 domain alone. Knockout of the BCAS3 gene in Dictyostelium resulted in a reduction in early autophagosomes compared with that found in wild-type cells (Yamada et al. 2021). In the present study, due to the multidimensional scaling projection of the retrieved sequences, we classified the ATG18 sequences into three subfamilies. Subfamily I contained ATG18a, ATG18c, ATG18d and ATG18e homologs, subfamily II had only ATG18b, and subfamily III had ATG18f, ATG18g and ATG18h members. All homologs with BCAS3 were found to be clustered within subfamily III. Subfamily II, which contained only ATG18b homologs, had few members but was detected in all the plant species investigated in this study, which suggested the sequence and functional conservation of these proteins. Among the early photosynthetic organisms, we identified at least one homolog in subfamilies I and II, but significant divergence was detected, particularly within subfamily III. Among monocots, O. sativa had 8 homologs, whereas 32 and 21 homologs were found in Z. mays and T. aestivum, respectively. The analysis of dicots revealed 8 homologs in each of Arabidopsis, L. japonicus, M. truncatula and P. vulgaris, whereas Arachis sp. had 9 and 10. The maximum number of homologs was recorded in C. cajan (18), G. max (18), C. arietinum (20), Vigna sp. and L. angustifolius (27). The legume family includes one of the most agroeconomically important plant crops after Poaceae (Lewis et al., 2005). Of the three subfamilies within Fabaceae, Papilionoideae is the largest, the most recently evolved and monophyletic. Because Papilionoideae includes the most important cultivated legumes, we sought to determine the members of this subfamily in different clades. In the present study, the maximum number of homologs (27) was identified in L. angustifolius, which belongs to the genistoid clade and exhibited an early divergence at approximately 56.4 2 million years ago(mya). Furthermore, in Arachis species, we found less than half of the ATG18 homologs, indicating possible deletions. Among the members of the next recent (45 mya) clade, which consisted of milletoids, an increase in the number of homologs (18) was detected, which might be due to whole-genome duplication in G. max. However, P. vulgaris had only eight members of ATG18, indicating possible divergence prior to wholegenome duplications, whereas Vigna sp. was found to have high numbers of homologs. Furthermore, more recent robinioid (48.3 ± 1.0 mya) and IRLC (39.0 ± 2.4 mya) clade members had fewer members with the exception of the tribe Chapter II. identification of autophagy genes in P. vulgaris and legumes 64 Vicieae, whose gene numbers were due to genome expansion and related genomic events. In contrast, syntenic relations were not disrupted due to differences in genome sizes (Choi et al., 2004; Lee et al. 2017). A phylogenetic analysis revealed that the ATG18 homologs of Chlorophyta, Charophyta, Marchantiophyta and Bryophyta were always grouped together, and similar results were obtained for monocots and dicots. However, in a comparison of a broad class of species, it is often not simple to precisely define orthologous genes or genomic loci in a straightforward manner, and this analysis is complicated due to gene duplication, recurring polyploidy and extensive genome rearrangement (Tang et al., 2008). Furthermore, the ATG families identified constituted a relatively complete autophagic machinery in forming the complexes, namely, the ATG1 kinase complex, class III PI3K complex, ATG9 recycling complex, Atg8-lipidation system and Atg12-conjugation system. ATG17 is an important accessory protein along with ATG31-ATG29, which acts as a scaffold/modulator in linking the ATG1-ATG13 complex to the phagophore assembly site in yeast. Homologs of the ATG17-ATG31-ATG29 subcomplex were not detected in Arabidopsis. However, single orthologs of ATG11 and ATG101 were identified, and ATG11 reportedly contains a short cryptic ATG17-like domain with weak identity to yeast ATG17 (Li et al.,2014). The identification of ATG homologs in the present study revealed one homolog of ATG11 and one homolog of ATG101 in all the legumes analyzed. Our study of ATGs we detected hypothetical transcription factors binding sites and revealed that several light-responsive transcription factors, such as BOX-4, G-box, GT1- motif, MRE and ACE, were abundant in most of the ATGs. Furthermore, cis-acting elements related to circadian control were also identified. Phytohormones play key roles in different plant processes, including stress responses. The ATGs analyzed exhibited TF-binding sites for EREs, ABA-responsive ABREs, MeJA-responsive CGTCA motifs, auxin-responsive TGA elements and gibberellin-responsive GARE motifs. Ethylene is considered a key regulator of autophagy in petal senescence in petunia, and ERF5 is also shown to induce autophagy by binding to ATG8 and ATG18h under drought stress in tomato. Upregulation of autophagy by low concentrations of salicylic acid is found to Chapter II. identification of autophagy genes in P. vulgaris and legumes 65 delay methyl jasmonate-induced leaf senescence in Arabidopsis (Yin et al. 2020; Shibuya et al. 2013; Zhu et al. 2018). In addition, several wound-responsive, pathogen responsive, flavonoid biosynthetic gene regulation-related and meristem-specific elements were also detected. Based on all the results, the involvement of autophagy in the regulation of plant responses to hormones is undeniable. To assess the differential expression pattern and responsive nature of ATGs to the presence of different nitrate sources, we developed heatmaps using the data retrieved from databases and from a previous RNA-seq analysis performed by our research group. The differential expression pattern in Phaseolus tissues showed that most of the ATGs were expressed in all tested tissues. Nitrogen is an essential component of life that is needed for building proteins and DNA, and despite its abundance in the atmosphere, only limited reserves of soil inorganic nitrogen are accessible to plants, and this nitrogen is primarily in the forms of nitrate and ammonium. Legumes have a unique ability to establish a symbiotic association with nitrogen-fixing Rhizobia. Due to our understanding of the evolution of ATGs in legumes, we opted to understand the response of both aerial and root tissues of these legumes to different nitrate sources. The expression patterns showed that the highest expression was found in roots treated with ammonia and leaves treated with urea. ATG18 homologs a, g and h were specifically induced in all tissues and by all treatments, indicating the nitrate-responsive nature of these genes. Furthermore, an analysis of the differential expression patterns of ATGs in Phaseolus tissues revealed that the highest expression level was noted in 21-day fix (-) nodules, which could be due to the involvement of the autophagic process in providing the necessary amino acids for the synthesis of nitrogen in the absence of the symbiont. In yeast and other eukaryotes, it has been proven that nitrogen deficiency induces autophagy. A recent study using yeast cells also suggested that autophagy sustains glutamate and aspartate synthesis during nitrogen starvation (Liu et al. 2021). RNA-seq data from early symbiosis with Rhizobia and Mycorrhizae showed differential ATG expression, and more ATGs were upregulated in Rhizobia-inoculated roots than in Mycorrhizae-inoculated roots. This analysis provided candidate genes that could play pivotal roles in symbiosis. The involvement of Chapter II. identification of autophagy genes in P. vulgaris and legumes 66 ATG6/beclin has previously been reported in P. vulgaris during Rhizobial infection progression and arbuscule maturation (Estrada- Navarrete et al. 2016). MATERIAL AND METHODS Identification of ATG families in legumes. Arabidopsis (taxid:3702) ATG family gene sequences were retrieved from Araport (https://www.araport.org) and TAIR (https://www.arabidopsis.org) databases through Phytozome v.13(Gou et al., 2019; J. Wang et al., 2019). Using these sequences, a BLAST(Altschul et al., 1997) (http://www.ncbi.nlm.nih.gov; search was conducted to identify the homologs of ATG genes in Phaseolus vulgaris v2.1 (taxid:3885), Medicago truncatula Mt4.0v1 (taxid:3880) and Glycine max Wm82.a2.v1(taxid: 3847). The stringency of search was maintained by keeping a mean BLAST result within a query coverage of 93.85% and 67.78% identity. The detection of homologs was further optimized by using other programs such as, KEGG (www.genome.jp/kegg/;Feng et al., 2012), EnsemblPlants (https://plants.ensembl.org; Bolser et al., 2017), HMMer suite server (http://hmmer.org;Potter et al., 2018), Inparanoid 4.1 (Remm et al., 2001) Additional we examined the ontology IDs for all ATG families using KOG (EuKaryotic Orthologous subfamilies) in eggNOGv5.0 database (Huerta- Cepas et al., 2019) (http://eggnog.embl.de), the ID for Protein ANalysis THrough Evolutionary Relationships in PANTHER (PANTHER v.14.0, http://www.pantherdb.org) and recognition of Pfam ID in the portal version 33.1 version (http://pfam.xfam.org/about). The ATG18 protein family was studied in 27 photosynthetic organisms, 13 dicot – (Legumes), 3 monocot crops and 10 plants through evolution of land plants from an algal ancestor. We obtain the ATG18 proteins sequences of liliopsida crops such as Zea mays (taxid:4577), Triticum aestivum (taxid:4565), Oryza sativa (Rice; taxid:4530) and legumes such as Arachis duranensis (Peanut; taxid:130453), Arachis ipaensis (taxid:130454), Cajanus cajan (taxid:3821), Lotus Japonicus (taxid:34305), Cicer arietinum (taxid:3827), Lupinus angustifolius (taxid:3871), Pisum sativum (Pea;taxid:3888),Vigna angularis (taxid:3914), Vigna radiata (taxid:157791) and Trifolium pratense (red clover; taxid: 57577) after using BLAST analysis in NCBI and analysis in Phytozome and Legumeinfo Chapter II. identification of autophagy genes in P. vulgaris and legumes 67 (https://legumeinfo.org), KEGG, Inparanoid, Ensembl, Eggnog and PFam. Additionally, we used the Norizuki report of early-divergent plant lineages to extract the ATG18 proteins sequences in Bryopsida (Physcomitrella patens-taxid:3218), Charophyceae (Chara braunii-taxid:69332), Chlorophyceae (Chlamydomonas reinhardtii-taxid:3055, Dunaliella salina-taxid:3046),Volvox carteri-taxid:3067), Klebsormidiophyceae ( Klebsormidium nitens-taxid:105231), Mamiellophyceae (Micromonas pusilla-taxid:38833, Ostreococcus lucimarinus-taxid:242159, Ostreococcus tauri -taxid:70448) and Trebouxiophyceae (Coccomyxa subellipsoidea-taxid: 248742)(Norizuki et al., 2019). Alignment and Phylogenetic tree analysis The proteins sequences of ATGs families were aligned with Clustal Omega (Sievers & Higgins, 2018; www.clustal.org & www.ebi.ac.uk) using default parameters., the phylogenetic tree is a Neighbour-joining without distance corrections. From there we extracted the outputs, and we generated the circular phylogram and cladogram tree image in evolview. The different phylogenetic trees were combined with the MEME results for all sequences, the final details were using inkscape software (Subramanian et al., 2019; https://www.evolgenius.info/evolview/). Multiple sequence alignment of 280 intraspecies protein sequences of ATG18 family members was performed using Clustal Omega. The phylogenetic analysis was performed using MEGA X with the maximum likelihood method and Bayes analyses with 1000 bootstrap replicates and the default parameters (Subramanian et al., 2019). Phangorn and APE packages in R were used to build the phylogenetic trees (Kumar et al.,2018; Akaike,1974). In Phangorn, we used the Akaike information criterion and the Whelan and Goldman matrix (WAG) as the substitution model. Chromosome localization. The chromosomal localization of ATG family homologs in A. thaliana, P. vulgaris, M. truncatula and G. max was verified using NCBI. Furthermore, Ensembl Plants was used to compare and explore the gene alignments and generate a segment to link the genomes. The synteny relation of ATG genes was drawn using OmicCircos in R (Bolser et al., 2017; Hu et al., 2014). Chapter II. identification of autophagy genes in P. vulgaris and legumes 68 Promoter analysis, Expression profiling and Transcriptome of ATGs families The 2000-bp upstream sequences of ATG genes were retrieved from Phytozome, and these sequences were used as query sequences in PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) the results were analyzed and the most abundant transcription factors were identified using ggplot2 in R. ATG gene expression data for A. thaliana, M. truncatula and G. max were extracted from Phytozome to determine the differential expression of the genes under different nitrogen treatments (Cleary et al., 2018). Data on the differential expression of genes in P. vulgaris under nitrogen treatments and after fixation and inoculation with Rhizobium tropici (CIAT899) were obtained from the PvGEA website (https://plantgrn.noble.org/PvGEA/). we calculated the Log2 values of the RPKM of A. thaliana, M. truncatula and G. max, we used the OmicCircos package and constructed subfamilies using the synteny graph. However, for P. vulgaris, we constructed an independent heatmap of ggplot2 because the amounts of treatments and tissues were higher. to be able to make the comparison. The expression data for ATG family genes under Rhizobia symbiotic conditions are taken from global transcriptomic analysis. For transcriptome analysis we isolated the RNA from roots of P.vulgaris by RNeasy Plant mini kit (Quiagen) and cleaned with RNase-free DNase followed by Dynabeads (spherical superparamagnetic polymer particles with a uniform size), RNaDIRECT micro kit (Life technologies). For the cDNA library, the fragmented RNA (100ng of mRNA fragmented with RNAse II) was hybridized with ion adapters and mixed with reverse transcriptase. The template preparation consisted in use 10pM of barcoded cDNA libraries in Ion PI template OT2 solutions 200 Kit and amplified using IonTouch2 instrument (Life technologies). Each beads had many copies and then was sequence on the chip into Ion Proton sequencer. Then the results were aligned to the P. vulgaris references v2.1 and analyzed with strand NGS software and plotted in R. Dot plot which compared the RPKM of nodulated roots with control and the histogram using the fold change values was constructed using ggplot2 package. Quantitative Real Time PCR Analysis PVATG9 gen were selected for RT-qPCR analysis, which was performed to validate the RNA-seq data. High-quality total RNA was isolated from frozen P. vulgaris root inoculated Chapter II. identification of autophagy genes in P. vulgaris and legumes 69 with Rhizobium (21dpi) using TRIzol reagent (Sigma) according to the manufacturer’s instructions. RNA integrity was verified by gel electrophoresis and RNA concentration was assessed using a NanoDrop spectrophotometer (Thermo Scientific). RNA was treated with DNase to eliminate DNA contamination (1 u/μL; Roche, USA) according to the manufacturer’s instructions. Reverse-transcription quantitative PCR (RT-qPCR) analysis was performed using a DNA-free RNA and iScriptTM One-Step RT-PCR Kit with SYBR® Green (Bio-Rad) according to the manufacturer’s instructions. To confirm the absence of DNA contamination, a sample lacking reverse transcriptase was included. Relative expression values were calculated using the 2-ΔCt method, where the quantification cycle (Cq) value equals the Cq value of the gene of interest minus the Cq value of the reference gene (Nanjareddy et al., 2017). Gene-specific primers were used for RT-qPCR analysis (Supp.S9). The values presented are averages of three biological replicates, and each data set was recorded using triplicate samples. Principal components analysis for ATG18 family Based on multiple alignments of ATG18 protein sequences, we converted the information into a distance matrix calculated by bios2mds packages (https://CRAN.R- project.org/package=bios2mds) in R. The matrix used was BLOSUM62 (BLOcks of Amino Acid SUbstitution Matrix), and sequences 62% identity were obtained into sequences. Using the same packages we obtain the K-means and principal components to generate the Multidimensional scaling projection to define the subfamilies into the protein family. 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Mitochondrial alternative oxidase-dependent autophagy involved in ethylene-mediated drought tolerance in Solanum lycopersicum. Plant Biotechnol. J. 2018, 16, 2063–2076 Zhuang, X., Chung, K. P., Cui, Y., Lin, W., Gao, C., Kang, B. -H., & Jiang, L. (2017). ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis. Proceedings of the National Academy of Sciences , 114(3), E426–E435. https://doi.org/10.1073/pnas.1616299114 Zhuang, X., Cui, Y., Gao, C., & Jiang, L. (2015). Endocytic and autophagic pathways crosstalk in plants. Current Opinion in Plant Biology, 28, 39–47. https://doi.org/10.1016/j.pbi.2015.08.010 CHAPTER III UNDERSTANDING THE ROLE OF ATG9 DURING SYMBIOSIS BETWEEN Phaseolus vulgaris AND Rhizobium tropici Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici ABSTRACT The autophagy is a conserved degradation process leaded by AuTophaGy genes (ATG). The sequence of steps implies function of ATG9 which participate in the membrane recruitment to form the autophagosomes. In P. vulgaris, ATG9 showed abundant expression compared with other autophagy genes during symbiosis with R. tropici. In this regard, we performed hairy root transformation mediated by Agrobacterium rhizogenesis to characterization of ATG9 in expression, silencing, overexpression, and localization studies. Our results showed high expression of PvATG9b detected by GUS in root tip, root vascular tissue, lateral root primordia and in vascular tissue of young and mature nodules. Silencing of PvATG9b showed least staining of GUS in nodules, short roots, yellowish leaves. Contrary, PvATG9b overexpression contain abundant staining in nodules, long roots and green leaves. These phenotypes suggest a role of PvATG9b in intricate symbiosis relation particularly in P. vulgaris and R. tropici. Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 74 INTRODUCTION Autophagy is an ancient degradation process that mediate recycling to maintain the homeostasis in eukaryotic cells. The intracellular and intercellular recycling is essential for controlling the metabolism and nutrient management (Zientara-Rytter & Sirko, 2016). Autophagosome formation imply a sequence of steps that include the ATG genes. The essential ATGs are known as autophagy core. Briefly, the steps in the autophagy process are divided as, autophagy initiation complex (complex ATG1; ATG1, ATG11, ATG13 and, ATG101), membrane recruitment to autophagosome (complex ATG2-ATG18; ATG2, ATG9 and, ATG18), autophagosome formation (complex PI3K; ATG6 and, ATG14) and ubiquitin-like protein conjugation systems (ubiquitin-like conjugation ATG8; ATG3, ATG5, ATG7 and ATG8 and ubiquitin-like conjugation ATG12; ATG10, ATG12 and ATG16) (Tang & Bassham, 2018). ATG9 is the unique transmembrane protein in the autophagy core and it is essential to generate the autophagosome from ER membrane in plants, yeast and mammals provides lipids for the autophagosome at the beginning and have able to form vesicles from Golgi- endosomal system (Masclaux-Daubresse et al., 2020). Several studies of ATG9 were performed in different organism mainly in mammals and yeast, some of which I should like to mention to complement. During early steps of autophagy ATG1 phosphorylate an ATG9 at multiple serine residues required to recruit ATG18 and ATG8. ATG9 allows the position of ATG2 in autophagosome and forms the complex ATG9-ATG2-ATG18 participating in the lipid transport from ER into phagophore in yeast (Gómez-Sánchez et al., 2018; Papinski & Kraft, 2014). In Drosophila midgut, ATG9 acts as a negative regulator of TOR-mediated cell growth independent of ATG1. Thus, ATG1 and ATG9 might be negatively regulated by TOR under different conditions and, ATG1 might acts independent of ATG9 (Wen et al.,2017). In mammals, mATG9 studies suggested that it is involved in mitochondrial integrity, fundamental in initiation and vesicular trafficking through multiple organelles including endosome recycling (Orsi et al., 2012; Tang et al, 2019). Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 75 During nitrogen starvation AtATG5, AtATG9 and AtATG10 have shown high expression (Bedu et al., 2020). It is already known that ATG5 is considering important in nitrogen storage in seed and under low phosphate in Arabidopsis (Guiboileau et al., 2012; Sakhonwasee & Abel, 2009). In apple, MdATG10 and MdATG9 promote nitrogen uptake and tolerance to nitrogen starvation respectively (Huo et al., 2020). ATG8 participates in nitrogen remobilization in rice and Camelia sinensis (Huang et al., 2020). Besides, ATG5, ATG18a and ATG9 result essential in nitrogen use efficiency (Masclaux-Daubresse & Chardon, 2011). Autophagy sustains glutamate and aspartate synthesis during nitrogen starvation (Liu et al., 2021). To analyze this interesting gen in plants, some experiments were performed with atg9 mutants. For instance, atg9-3 defective mutant has shown abnormal autophagosomal tubular structure which is the membrane continuity with ribosome-free ER membrane that suggest the importance of ATG9 at initial steps of autophagosome (Fig.14) (Zhuang et al., 2018). In atg9 mutant have an early leaf senescence and when this mutants are under treatments with inhibitor of vacuolar degradation generated less autophagy bodies (Guiboileau et al., 2012; Hanaoka et al., 2002; Inoue et al., 2006; Shin et al., 2014). Moreover, atg9 knockout mutant under nitrogen starvation accumulate amino acids such as glutamate and aspartate. Also, transcriptomic profiles of atg9 mutant under low nitrogen at days after sowing (DAS) shows plant immunity affected and malfunction at ROS detoxifying (Masclaux-Daubresse et al., 2014; Yoshimoto et al., 2009). Figure 14 Autophagosome morphology in wild type and atg9 mutant obtained in electron microscopy (EM) or fluorescence microscopy (FM)(Zhuang et al., 2018). Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 76 These interesting reports about ATG9 in yeast, mammals and plants showed us the complex of the protein but it has not been reported during nodulation in P. vulgaris yet. Until now, reports mention increasing expression levels of ATG1, ATG13 and ATG8 in TOR-RNAi transgenic roots and Pv-Beclin1/ ATG6 loss-of function in PvPI3K-RNAi roots. Now, we present our results based on transcriptome data where we recognized the PvATG9b expression. Our studies consider expression, silencing, and overexpression analysis and also our preliminary studies to determinate the subcelullar localization to understand the role of protein during the nodulation of P. vulgaris RESULTS Structure and Phylogenetic analysis of PvATG9 As discussed in the previous chapter, our analysis of ATG genes in P. vulgaris showed the presence of two ATG9 genes, PvATG9a (Phvul.001G159900) and (Phvul.007G194300). We found two sequences of ATG9 in Phaseolus vulgaris. PvATG9a (Phvul.001G159900) is in chromosome 1: 41311908- 41319321 with 10 exons and 9 introns and the primary transcript has 2573pb and protein 858 a. a. (Supp. S7). The other is PvATG9b (Phvul.007G194300) gene is located on chromosome 7: 31618092- 31623866 and count with 5775pb, where it has exons 9 and 8 introns (Fig.15). The CDS comprise 2622pb and the protein 875a.a. (Supp. S8). Figure 15 Schematic representation of PvATG9b (Phvul.007G194300). PvATG9b contains 5.775Kb with nine exons and eight introns. Blue boxes: exons; Black line: introns. We analyzed the sequences, using BLAST results we compared At2g31260 with ATG9a and resulted in 99% of query cover with 65.2% of identity while ATG9b resulted in 97% and 59.77% respectively. Furthermore, we generated a phylogenetic analysis where we considered our previous data, identifying the autophagy core homologs in M. truncatula and G. max but here we added A. thaliana and two more organisms there are the humans Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 77 and yeast. The phylogenetic tree shows the PvATG9a (Phvul.001G159900) is near to Arabidopsis protein (sp|Q8Rus5|ATG9 ARATH) (Fig.16). Figure 16 Phylogenetic tree of ATG9. Neighbor-joining tree using protein sequences of A. thaliana, P. vulgaris, M. truncatula, G. max, yeast, and human in Clustal omega and designed in evolview. Expression of PvAtg9b gene in roots and nodules Based on the transcriptomic data, PvATG9b had a significant expression in Phaseolus roots and nodules. Hence, we considered to analyze the spatio-temporal expression pattern of PvATG9b by taking 1080-bp region upstream of the translation initiation codon. The promoter fragment was fused to the chimeric reporter GUS and enhanced GFP (pPvAtg9b::GUS-GFP). The pPvATG9b::GUS-GFP reporter construct was transfected into bean via hairy root transformation. Transcriptional activation of the reporter gene in the transgenic hairy roots of bean was monitored with and without Rhizobia inoculation. In the uninoculated roots 6 days post inoculation (dpi), notable GUS expression was observed at the root tip. Rhizobium inoculation increased the GUS expression in the meristematic zone, columella cells and in the elongation zone. At 10 dpi, the same zones both in uninoculated and inoculated roots showed stronger promoter expression (Fig. 17). Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 78 A B C D Uninoculated Inoculated Uninoculated Inoculated Figure 17 Root expression of PvATG9b gene promoter. Promotor activity was detected by GUS staining (Blue) during P. vulgaris roots of 6 and 10 days using optical microscopy. (A) Uninoculated root ,6days (B) Inoculated root ,6 days.(C)Uninoculated root, 10 days. (D)Inoculated root, 10 days. Inoculated root showed more GUS staining tha uninoculated roots. Elongated zone (EZ), Transition zone (TZ), Meristematic zone (MZ),Lateral root cap (LRC) and Vascular tissue(V). Scale Bar:1mm (A and B), 2mm (C and D). Promoter activity also was found at the site of the lateral root primordium. At 13 dpi, lateral root primordium uninoculated showed expression in central of primordia vasculature. In primordia and lateral root of inoculated roots showed the promoter activity in peripheral cells (Fig. 18). Figure 18 PvATG9b expression patterns during lateral root formation. Promotor activity was detected by GUS staining (Blue) during P. vulgaris lateral root development using optical microscopy. (A) and (B) GUS staining was detected in central cells of lateral root primordium in stage VII (uninoculated, 13days). (C) Lateral root primordium in stage VII (Inoculated, 13 dpi) (D) Emergence of lateral root (inoculated, 13dpi). (C) and (D) showed the GUS staining in peripherial cells. Epidermis (E);Vasculature (V) Scale barr: 1mm To analyze the promoter activation upon inoculation with Rhizobium, hairy roots were inoculated with Rhizobium tropici, and GUS activity was observed at periodic intervals post inoculation. At the early stages of infection, promoter expression could be recorded in the root hairs infected with Rhizobium. The figure 19A shows PvATG9b promoter Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 79 expression in the outer cortex of the nodule primordium and at 10 dpi, the expression was mostly restricted to the outer cortex and root vascular tissue (Fig. 19B). A B Figure 19 Expression patterns in early stages of nodule development. Promotor activity was detected by GUS staining (Blue) during P. vulgaris nodule development using optical microscopy (A) Nodulated Root and curly hairy root (HR), which expression was detected in two nodule primordium and vasculature. (B) Nodule primordia of 13 dpi has expression in vasculature and around the infection zone. Scale Barr:1.25mm Further, 15 dpi the promoter activity was recorded in the nodule vascular elements and inner cortex (Fig. 20A-F). In the mature nodules, no promoter activity was noticeable in the infected or uninfected cells of the nodule. The promoter expression was mostly restricted to the vascular tissues, inner and outer cortex (Fig. 20 F). At 30 dpi, when the nodule senescence started, the PvATG9b expression continued to be seen in the vascular tissues but in the cortex, it was only seen the inner cortex (Fig. 21A-C). Figure 20 Expression of PvATG9b during nodulation. Promotor activity was detected by GUS staining (Blue) during P. vulgaris nodule development using optical microscopy. (A) and (B) Rhizobia invasion into nodule primordia. (C) and (D) Young nodule. (E) Nodule transition to maturation. (F) Mature N-fixation nodule. The expression was maintained in vascular tissue. Nodule primordium(P); Provascular bundle (PVB); Vasculature tissue (V); Bacteria (B); Nitrogen fixing Zone (NFZ) Scale Bars: 1.25mm(A);2mm(B,C,D);1mm(D) Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 80 A B Figure 21, PvATG9b expression in mature nodules. Promotor activity was detected by GUS staining (Blue) during P. vulgaris nodule development using optical microscopy. (A) Mature nodule, Longitudinal view(B) Mature nodule- Trasnversal view. The expression apperead in vasculature. Nodule Cortex (NC);Vascular bundle (VB);Developmental zone (DZ); infected zone (IZ); Nodule Parenchyma (NP);Nodule meristem (NM). Scale Barrs: 2mm Transcript downregulation of ATG9b in P. vulgaris hairy roots To functionally characterize PvATG9b during symbiosis, we took advantage of the bean root transformation system that uses Agrobacterium rhizogenes. An RNAi construct harboring a non-conserved region of the C terminus and 3´ UTR of PvATG9b (pTdT-35S- PvAtg9b-RNAi) and an empty vector (pTdT-35S-RNAi) were expressed individually in hairy roots of the composite plants. An RT-qPCR analysis of hairy roots isolated at 10 d post emergence (dpi) confirmed the reduction of PvATG9b mRNA levels, with levels ranging from 70% to 80% in transgenic roots expressing pTdT-35S-PvATG9b -RNAi (henceforth 35S-PvATG9b-RNAi) compared with transgenic control roots containing the empty vector (henceforth control roots). Our results indicated that the 35S-PvATG9b- RNAi constructs specifically down-regulated ATG9b transcript levels in transgenic roots (Fig.22). Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 81 Figure 22 Transcript levels of 35S-PvATG9b-RNAi by RT-PCR in hairy roots (dpi). Relative transcript levels were normalized with metalloprotease. We compared transformed hairy roots of silencing (35S-PvATG9b-RNAi) with empty vector (EV). Transcriptional downregulation of PvATG9b affected the root hair length in the root elongation zone (Fig. 23). The root hairs in 35S-PvATG9b-RNAi were found to be shorter than the empty vector control root hairs. A B Figure 23 Hairy roots of silencing of PvAtg9b. Roots observed under optical microscopy without staining. (A)Empty vector (Control-EV) and (B) silencing of PvAtg9b (PvATG9b-RNAi) We observed the reduced size of hairy roots in PvATG9b-RNAi transformed roots compared with Control-EV. Scale barr:2mm Composite plants expressing 35S-PvATG9b-RNAi construct exhibited some changes in the overall plant growth. The shoots and roots of the RNAi plants were typically shorter when compared to the vector controls (Fig. 24). To quantify the growth parameters, we measured the internodal length, root length, root numbers and weight in 35S-PvATG9b- RNAi and control plants of 35dpi. Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 82 A B Figure 24 Silencing of PvATG9b phenotype. (A) Pots and (B) Roots of P. vulgaris plants at 35 dpi grown with nitrogen- limited B&D solution (KNO3 2nM) to promote nodulation. PvATG9b-iRNA size is reduced compared with the Control- EV Scale bar: 7cm As discussed earlier, the root length of RNAi plants grew shorter and similarly, internodal length was also reduced (Fig. 25A). Root weight remained the same though the whole plant weight was higher in control plants (Fig. 25B). The number of primary, secondary, and tertiary roots remained to be the same in 35S-PvATG9b-RNAi and control plants (Fig. 25C). A B C Figure 25 Root architecture of PvATG9b silencing plants. Bar plots of P. vulgaris roots at 35 dpi grown with nitrogen- limited B&D solution (KNO3 2nM) to promote nodulation. (A)Root and internode length.(B)Root and plant weight.(C) Primary, secondary and tertiary roots. Red boxes:Control empty vector; Blue boxes:PvATG9b-iRNA. Significative difference values at P < 0.05 Student’s t test (***). Another important observation in the aerial parts of the plant is the leaf phenotype. The leaves in 35S-PvATG9b-RNAi plants were smaller and yellowish when compared to controls (Fig. 26). Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 83 A B C Figure 26 Leaves phenotype of PvATG9b-RNAi. Plants of P. vulgaris plants at 35 dpi grown with nitrogen-limited B&D solution (KNO3 2nM) to promote nodulation. (A)Leaves of Control-EV and PvATG9b.(B) Length and (C) Width.PvATG9b-RNAi showed smaller and yellowish leaves compared with control. Red boxes:Control empty vector; Blue boxes:PvATG9b-RNAi. Significative difference values at P < 0.05 Student’s t test (***). To assess the role of PvAtg9b in nodulation, transgenic hairy roots expressing 35S- PvATG9b-RNAi were inoculated with R. tropici expressing a GUS marker (Vinuesa et al., 2003). Light microscopic observations revealed that the Rhizobium-infected root hair cells of both control and 35S-PvATG9b-RNAi plants show the typical root hair curling and Rhizobial microcolonies of wild-type roots (Fig. 27). The infection events of the Rhizobium were accompanied by the cortical cell divisions in the root cortex. A B Figure 27. Infection threat of PvATG9b silencing roots. Roots observed under optical microscopy with GUS staining. (A) control and (B) Infection threat PvATg9b-iRNA in transgenic root. Both showed typical curly hairy roots Roots of P. vulgaris plants at 30 dpi grown with nitrogen-limited B&D solution (KNO3 2nM) to promote nodulation. The colonization of the Rhizobium bacteria in the nodules of 30 dpi was found to be normal when compared to the controls (Fig.28). The nodule morphology was also found to be normal indicating less impact of PvATG9b silencing in P. vulgaris and Rhizobium Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 84 interaction. The silencing nodules show less GUS staining in vasculature bundle and infection zone compared with control. Figure 28. Mature nodules of PvATG9b-iRNA at 30 dpi. Roots observed under optical microscopy with GUS staining. (A)Control -Empty vector (EV). (B) Silencing of PvATG9b with iRNA(PvATG9-iRNA). P. vulgaris plants at 35 dpi grown with nitrogen-limited B&D solution (KNO3 2mM) to promote nodulation. PvATG9b-iRNA showed less expression in infection zone. Infection zone (IZ),Vascular bundle (VB), Nodule cortex(NC). Scale barrs: 2mm Overexpression of PvATG To analyze the impact of overexpression of PvATG9b transcript on P. vulgaris and Rhizobium interaction, the PvAtg9b cDNA was isolated and the along with the 3′- untranslated region, was inserted into the pH7WG2D.1 binary vector under the control of the constitutive 35S promoter (Karimiet al., 2002). Empty pH7WG2D.1 vector was used as the control. The Agrobacterium rhizogenes /K599 strain carrying the construct was used to generate hairy root formation on P. vulgaris tissues and form composite plants after transformation transgenic hairy roots expressing pH7WG2D-PvATG9b-OE vector were selected under an epifluorescence stereomicroscope using the green fluorescent protein (GFP) filter with an excitation of 488 nm and emission fluorescence from 510 to 540 nm. Fifteen-day-old non inoculated composite plants grown in vermiculite were utilized to determine growth parameters such as root length, lateral root density, and tertiary and quaternary root numbers. The PvATG9b-OE plants were larger than the control plants (Fig. 29) both in root and shoot length. The root volume was higher in PvATG9b-OE plants due to an increase in primary and secondary root numbers. Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 85 A B Figure 29 Overexpression of PvATG9b phenotype. (A) Pots and (B) Roots of P. vulgaris plants at 35 dpi grown with nitrogen-limited B&D solution (KNO3 2nM) to promote nodulation. PvATG9b-OE size is greater compared with the Control-EV Scale bar: 7cm On the other hand, the shoot weight increased without showing any significant increase in internodal length (Fig. 30). Figure 30 Root architecture of PvATG9b-OE plants. Bar plots of P. vulgaris roots at 35 dpi grown with nitrogen-limited B&D solution (KNO3 2nM) to promote nodulation. (A)Root and internodes length.(B)Root and total plant weight (C) Primary, secondary and tertiary roots. Green boxes:Overexpression control; Purple boxes:PvATG9b Overexpression. Significative difference values at P < 0.05) Student’s t test(***). While comparing the foliage phenotype, PvATG9b-OE plants had larger and greener leaves when compared to controls (Fig. 31). Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 86 A B Figure 31 Leaves phenotype of PvAtg9b overexpression Plants of P. vulgaris plants at 35 dpi grown with nitrogen- limited B&D solution (KNO3 2nM) to promote nodulation. (A)Leaves of Control-EV and PvATG9b-OE (B)Boxplot. Scale Bar. 3cm. Green boxes:Overexpression control; Purple boxes:PvAtg9b Overexpression. Significative difference values at P < 0.05 Student’s t test (***). The P. vulgaris roots inoculated with R. tropici expressing a GUS marker (Vinuesa et al., 2003). Light microscopic studies revealed that the Rhizobium-infected root hair cells of both control and PvATG9b-OE plants show the typical root hair curling. In nodules, GUS staining of nodules is strong in infection zone when we compared with control (Fig.32). Most of the nodules in PvATG9b-OE roots were remained white even at 30 dpi when, the controls were pink until 28 dpi and reached senescence by 30-35 dpi. A B Figure 32 Nodules of PvATG9b overexpression roots at 30dpi . Roots observed under optical microscopy with GUS magenta staining (A)Control-OE, (B)Over expression of PvATG9b (PvAtg9-OE). P. vulgaris plants at 30 dpi grown with limited B&D solution (KNO3 2mM)to promote nodulation (Nodule Cortex(NC); Vascular Bundle(VB); Infection zone(IZ); Vascular tissue (V). Scale 2mm. Molecular analysis of the PvATG9b-OE roots showed the expression of common symbiotic gene expression NODULE INCEPTION (NIN), EARLY-NODULINE 40 (ENOD40) and ENDOPLASTIC RETICULUM TO NUCLEOUS SIGNALLING 1 (ERN1) in Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 87 PvATG9b-OE roots. NIN, ENOD40 and ERN1 had higher when compared to control (Fig.33). Figure 33 Fold Change of relative expression of NIN, ENOD40 and ERN1 in PvATG9b overexpression roots and the control. Relative transcript levels were normalized with metalloprotease. The expression of NIN, ENOD40 and ERN1 showed higher expression than control. Green boxes:Overexpression control; Purple boxes:PvATG9b Overexpression. Subcellular localization of ATG9b-YFP protein in P. vulgaris Coding region of PvATG9b was fused to the YFP in the N-terminus under 35S promoter. For these studies, we used the P. vulgaris hairy roots infected by A. rhizogenes. The hairy roots expressing non-fused YFP served as controls. Yellow fluorescence (YFP- PvATG9b) was detected in the plasma membrane and nucleus of primary roots and lateral roots. We could detect the fluorescence particularly in vascular tissue (Fig. 34). A B C Figure 34 Analysis of subcellular localization of PvATG9b for YFP fusion protein in P. vulgaris. Roots at 30d observed in confocal microscopy. (A)Primary root and lateral root. (B)and (C)Primary root. Vascular tissue (V) Scale bar: 1mm Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 88 DISCUSSION In autophagy, ATG9 is the unique transmembrane protein. The Arabidopsis core of autophagy genes contains only one ATG9 which is ATG231260. However, P. vulgaris and M. truncatula contain two ATG9 genes identified and four in G. max. PvATG9a (Phvul.001G159900) and PvATG9b (Phvul.007G194300) are in P. vulgaris. PvATG9a has a higher query cover and percentage of identity with the ATG9 of Arabidopsis, but PvATG9a has seven transcripts and PvATG9b only one. ATG9a has been studied more than ATG9b. Many of the studies of ATG9 under nitrogen starvation focused on ATG9a and are accompanied by ATG10, ATG5 and ATG1 to maintain the glutamine and aspartate levels in Arabidopsis (Bedu et al, 2020; Masclaux- Daubresse et al., 2014). Our studies PvATG9b has showed higher expression in our previous transcriptome and in our real time PCR using P. vulgaris but also MtATG9b showed high expression pattern in 10 and 28dpi of M. truncatula with Sinorhizobium meliloti (establishes nitrogen-fixing symbiosis). Both results support the hypothesis that ATG9b is involved in symbiosis with nitrogen-fixing bacteria. Subsequently, we generated promoter analysis of PvATG9b which showed higher GUS activity in vascular tissue in inoculated roots. This expression increased while day post inoculation increased. During the lateral root formation, the GUS staining appear in center cells of uninoculated roots changed to lateral sides of primordia of inoculated roots (13dpi) showed a change from the center cell to the lateral sides of the primordia. The expression of autophagy genes has been reported during vascular tissue differentiation and root senescence, but nothing during symbiosis (Escamez et al., 2016; Wojciechowska et al., 2018, 2021). PvATG9 could be viewed from the perspective that this tissue can transport nutrients between the plant and symbiosome. In nodules, the vascular bundles connect the nodule with the root generating an interchange of metabolism material and plant supply water to nodules (Livingston et al., 2019; Turgeon & Wolf, 2009). Interestingly, the promoter findings of Pv-PI3K that form a complex during autophagy confirmed the expression in nodule vascular bundles like PvATG9 suggesting the function in nutrient mobilization from the formed sink organ (Estrada-Navarrete et al., 2016). Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 89 The phenotype of PvATG9b-RNAi during R. tropici nodulation are short hairy roots and fewer tertiary roots. At first glance, we could observe the few and short hairy roots, but we used RT-qPCR to demonstrate low expression. The short hairy roots impact directly in the establishment of bacteria interaction. The tertiary roots of PvATG9b-RNAi showed significant reduction and that imply an incomplete root system to interact with bateria and to capture resources such as nutrients and water. In the shoot system, the plants exhibited yellow color and small leaves that could have been affected by not being able to obtain soil resources. Nodules of PvATG9b-RNAi seemed to mature faster but this needs to be studied with more detail, but we found less GUS staining when we compared with control. The lack of hairy roots and tertiary roots in plant structure and possible problems in nutrients transport could be the reason that PvATG9b-RNAi plants are affected. To complete the studies, we examined the overexpression of PvATG9b (PvATG9b-OE) on the nodulation. The results were larger root and shoot length compared with control and PVATG9b-iRNA. The root volume was abundant due to the increase of primary and secondary roots that we quantified. Only, secondary roots had a significant change compared with control. The foliage phenotype in PvATG9b-OE has larger and greener leaves. The size was supported by length and width data analysis and statics carried out a significant change when we compared it with control and silencing construction. The infection treated was typical curling and the number of infection events increased significantly. Also, the GUS staining in nodules evidenced the postponed maturation in nodules. This led us to examine the expression of NIN, ENOD40 and ENR1 which are regulators of Nod factor perception in common symbiotic pathway. NIN is a transcription factor that plays an important role in nodule initiation, ENOD40 is a marker gene for nodule primordium initiation, and ENR1 is a transcription factor that is activated in response to calcium spiking in root hairs (Nanjareddy et al., 2017; Liu et al., 2021; Schauser et al., 1999; Crespi, 1994). In nodulated roots of 28 to 30 dpi, the expression of NIN, ENOD40 and ENR1 presented more expression than the control which indicates the function of the molecular machinery or common symbiotic pathway in early stages. Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 90 Moreover, we visualized PvATG9b subcellular localization in plasmatic membrane and nucleus, but there is preliminary result. The fact that PvATG9b is in the plasmatic membrane could be due to this protein having transmembrane domains, but in the nucleus is an expected result that we need to analyze. These studies are an effort that requires more microscopy efforts to identify more valuable information in plants and during nodulation. The experiments performed in this chapter give evidence of the important role of PvATG9b in nodulation. The comparison of silencing and overexpression plants of PvATG9b are clearly dissimilar in shoot and root system. We suggested that size differences are related to the transport and resources interchange between bacteria and plants. MATERIAL AND METHODS Nomenclatures: we used the capital letters and italics to genes and the capital letter to refer to proteins. Plant Material We use P. vulgaris commonly named "common bean" variety Negro Jamapa obtained by Biotechnology Institute, UNAM. Seeds were sterilized (sterile distilled water for 1min, 10% of sodium hypochlorite for 5min, sterile distilled water for 1min) and germinated on sterile wet paper with B&D at 25°C for 2-4 days in the dark (Supp.S1). After 2 days, we transferred to pots (50% Vermiculite, 50% Peatmoss) or glass tubes with B&D for hairy root transformation (Broughton & Dilworth, 1971). The plants growth in chambers (16h/8h light-dark cycle) and 65% relative humidity at 28 ºC. Bacteria Material For our studies, we used Rhizobium tropici CIAT899 (CIAT, Centro Internacional de Agricultura Tropical) for nodule induction. R. tropici growth in PY medium (0.5g peptone, 0.3g yeast extract and 7 mM ml–1 CaCl2 and 20 mg ml–1 nalidixic acid and specific Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 91 antibiotic) incubated 30ºC for 24h and 200rpm shaking (Supp.S16; Nanjareddy et al., 2017). Structure and Phylogenetic analysis Arabidopsis sequence of Atg9 was the reference to find the sequence in P. vulgaris. We used NCBI BLAST searching using (Phvul.007194300) and we chose the highly similar sequences; also, we used orthologs software. The phylogenetic tree was performed in Simple phylogenetic (https://www.ebi.ac.uk/Tools/phylogeny/simple_phylogeny/) and here we include the sequence of Phaseolus vulgaris, Medicago truncatula, Glycine max, Arabidopsis thaliana, human and yeast ATG9 sequences. Plasmid construction and transformation Cloning of PvATG9 Promoter We used primer-specific oligonucleotides for the promoter designed 1, 080pb upstream of the translation start site of PvATG9b gen (Phvul.007G194300) from P. vulgaris DNA (Supp.S9). The fragment was amplified and cloned using Invitrogen Gateway system. The entry vector was pENTR/D-TOPO in the BP reaction (Supp. S11). We used E. coli Top 10 for transformation (kanamycin 50µg/ml-LB medium). We extracted the DNA plasmid Mini prep Kit GenElute and we corroborated the cloning with PCR reaction. Then, we amplify the promoter using M13 oligonucleotides and put fragments into the destination binary vector pBGWSF7.0 using the LR clonase (Supp.S12-S14 & S18). The recombinant plasmids were introduced by electroporation into K599 strain of Agrobacterium rhizogenesis which induces the hairy roots. GFP was used to select the transformed roots in Leica epifluorescence stereo microscope and then transformed into P. vulgaris roots, which were inoculated with R. tropici-GUS grown in PY (Supp.S16) (Karimi et al., 2002). Silencing PvATG9b gene iRNA construction was designed with the fragment of PvATG9b (369pb) from the region 3’ of the cDNA sequence using specific oligonucleotides and then was cloned with GATEWAY system (Supp. S9). After BP reaction pENTR/SD/D-TOPO vector we continue with transformation using Db3.1 competent cells (1 ml of kanamycin stock 50mg/ml-LB medium) (Supp.S15). We grew selected colonies with white color, and we extracted the Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 92 DNA plasmid (Mini prep Kit GenElute) and corroborate the cloning with PCR reaction. Then, with 1–7 µl (50-150 ng), we prepared the LR reaction with the pTdT-DC-RNAi vector (Supp. S10-14& S19). The vector contains the NOSpro:ptdT cassette to express the red fluorescent protein as molecular marker (tdTomato; excitation/emission max, 554 nm/582 nm) that help us to corroborate the iRNA forms a loop under 35SCaMV promoter. The empty vector pTdT-DC-RNAi (EV) was used as control. Both recombinant plasmids were introduced by electroporation into K599 strain of Agrobacterium rhizogenesis and then were transformed into P. vulgaris roots to later inoculate with R. tropici-GFP and GUS. Overexpression of PvATG9 Overexpression was performed with the DNA fragment of complete PvATG9b gene (2613pb). The fragment was cloned into pENTR/SD/D-TOPO intermediate vector which was transformed using E. coli Top10 competent cells (kanamycin μg/mL -LB medium (Supp.S15)). Selected white colonies were grown that after we used the Mini prep Kit GenElute to extract the DNA plasmid and corroborate the cloning with PCR reaction (Supp.S20). The next cloning was into pEarleyGATE plasmid (Earley et al., 2006). This plasmid includes a 35SCaMV promoter (Supp. S11). The recombinant plasmids were introduced by electroporation into K599 strain of Agrobacterium. Consequently, we transformed P. vulgaris roots to later inoculate with R. tropici-RFP and GUS. Subcellular localization of PvATG9b To obtain the subcellular localization, we used our cloned strain that contains the pENTR/SD/D-TOPO vector with the DNA fragment of complete PvATG9b which was transformed in E. coli Top10 competent cells (kanamycin 50 μg/mL -LB medium (Supp.15S & S20)). Then, We cloned into pEarleyGATE 104 plasmid (Earley et al., 2006). This plasmid includes a Yellow Fluoresce Protein (YFP) (Supp. S12). The recombinant plasmids were introduced by electroporation into K599 strain of A. rhizogenesis. The roots of P. vulgaris roots were transformed to later inoculate with R. tropici. Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 93 Plant transformation Sterilized seeds grew for 3 days at 28°C in the dark condition. After the three days, we injured in the hypocotyl of P. vulgaris with a syringe, which contain the binary vector. The binary vectors were prepared 2 days before the transformation. For these experiments, the binary vector pPvATG9b::GUS-GFP, EV-Control, PvATG9b::iRNA, OE-Control, PvATG9b::OE, PvATG9b::YFP was grown in independent plates covering all surface of medium in LB and Spe100 in the dark at 28°C. The bacteria were scraped to be collected and resuspend in a tube of 1.5ml, thus put the resuspend bacteria in syringe. Then, the plants were moved to sterile glass tube (22cm) which was previously prepared with dH2O but into these tubes, we put a falcon tube (15ml) with B&D (Supp.S17), covered with aluminum foil. We drilled above the aluminum foils to put the plants, to give support and to always keep the B&D handy to moisten the roots. This system was maintained with enough water and medium to maintain the humify during all experiments. Histochemical GUS staining The transgenic roots and nodules were cut and placed in small plates containing 5ml of β-Glucuronidase (GUS) solution and incubated at 37°C in dark for 12hrs (Supp. S21) (Jefferson, 1987). The GUS solution contained X-Gluc or Magenta-Gluc (diluted in dimethyl formaldehyde). To clarify and remove the excess of GUS solution, we used 2% of chlorine. Finally, we used the optic and stereo microscope. Phenotype analysis We measured the root size, root height, total height, distance between nodes, primary roots, secondary and tertiary roots, and nodules of our transgenic plants. We had independent biological replicates and we use analysis of variance (ANOVA) and T-test. Our statistical results and boxplot were performed by gglot2 package in R language (Supp. S22-S27). Quantitative real time- RT-PCR analysis We collected roots and nodules that was pulverized with liquid nitrogen. The RNA was extracted using TRizol reagent, according to the manufacturer’s recommendations (Thermo Scientific, Waltham, USA). DNA contamination from RNA samples was eliminated by incubating the samples with RNase-free DNase (1 U µl–1) at 37 °C for Chapter III. Understanding the role of ATG9 during symbiosis between Phaseolus vulgaris and Rhizobium tropici 94 15 min and then at 65 °C for 10 min. RNA integrity and concentration were determined by electrophoresis and NanoDrop ND-2000 (Thermo Scientific, Wilmington, USA) spectrophotometry. Quantitative real-time PCR was performed using an iScript One-step RT-PCR Kit with SYBR Green (Bio-Rad, Hercules, California, USA), following the manufacturer’s instructions, in a Real-time PCR Detection System (Bio-Rad, Hercules, California, USA). For the reaction, we used 40 ng of RNA as template. A control sample, which lacked reverse transcriptase (RT), was included to confirm the absence of contaminant DNA. Relative gene expression levels were calculated using the formula 2– ΔCT, where cycle threshold value (ΔCT) is the CT of the gene of interest minus the CT of the reference gene. Transcript accumulation was normalized to the expression of metalloproteinase as reference gene. The data are averages of three biological replicates and each sample was assessed in triplicate. 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To deliver or to degrade – an interplay of the ubiquitin–proteasome system, autophagy and vesicular transport in plants. The FEBS Journal, 283(19), 3534–3555. https://doi.org/10.1111/febs.13712 CHAPTER IV. DECIPHERING THE PVATG9B INTERACTION NETWORK DURING SYMBIOSIS BETWEEN Phaseolus vulgaris AND Rhizobium tropici Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 97 ABSTRACT ATG9 is the unique transmembrane protein in autophagy and recently it was revealed as an important element in phospholipids translocation during autophagy together with ATG2 and ATG18. Also, ATG9 was found in the cytoplasmic membrane and in vesicular trafficking. These results extended possibilities and increased the function of PvATG9b during Rhizobium symbiosis in P. vulgaris. For this reason, we constructed the PvATG9b network based on our studies of yeast two-hybrid (Y2H) during symbiosis with Rhizobium where we found 24 proteins that do not include autophagy proteins. Then, we expanded the protein-protein interaction network, overlapping our transcriptome data and results showed the expected enrichment in the endoplasmic reticulum, ribosome, ubiquitination, and endocytosis. We found that PLANT CYSTEINE OXYGENASE (PCO2) is the PvATG9-interacting partner with more expression in our transcriptome. In the PCO2 network, the up regulation of HRA1 and HRE2 revealed the hypoxia response that is critical for the function of Nitrogenase during nitrogen fixation. PvATG9b could interact with PCO2 modulating degradation via N-end rule pathway derived by hypoxia and autophagy. Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 98 INTRODUCTION ATG9 is an autophagy protein which plays a pivotal role in autophagosome formation. This protein is the unique transmembrane protein in the autophagy core. ATG9 appears in early stages of autophagosome formation where ATG1 phosphorylate ATG9 protein to recruit ATG2 and ATG18 vesicles and even studies suggest the also ATG8 (Papinski & Kraft, 2014). ATG9 has been related to the autophagosome membrane, and is related with vesicle trafficking in the endomembrane system (Søreng et al., 2018; Yang et al., 2021). Understanding the context of the ATG9, its structure and interaction of the protein is possible to understand more about this protein. Using cryoelectronic microscopy, Guardia et al. 2020 described the structure of the human ATG9 with four transmembrane helices, which contains homotrimer domain- swapped architecture that contributes to forming the central pore, multiple membrane spans and a network of branched cavities. In contrast, Arabidopsis ATG9 has six transmembrane alpha helices. AtATG9 is located between the cytoplasmic and membrane-embedded regions, forming the trimer that creates a central cavity of ~20 Å in diameter (Lai et al., 2020). Recent studies of ATG9 in yeast describe the lipid scramblase activity of this protein to expand the autophagosome. ATG9 would translocate phospholipids from cytoplasmic leaflet of the ER to the cytoplasmic leaflet of INITIAL ISOLATION MEMBRANES (IM; also named as phagophore in yeast). ATG9 does not work by itself. The system needs ATG2 and now we know more elements such LONG-CHAIN-FATTY-ACID-COA LIGASE 1 (Faa1) which at the IM produces acyl-CoA from free fatty acid and CoA utilizing ATP and the connection with a lipid synthetases localized at the ER (Matoba et al., 2020; Noda, 2021).In Yeast and Arabidospsis, ATG9 has only one gene, however in P. vulgaris and in mammals are two genes. In mammals, mATG9a and mATG9b are localized in different places but generally appear in growing autophagosomal membrane through the ubiquitin- Interacting motives (UIMs) and mATG9a has only one UIMs, while ATG9b has the double, that means the different genes could have different functions (Zhang et al., 2020). Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 99 A B Figure 35 ATG9 protein structure and ATG2-ATG18 complex. A) ATG9 contain transmembrane helices and forms a pore. B)ATG9- mediate lipid transfer from ER to the isolation membrane for expansion together with ATG2 and ATG18.Figure based on Matoba & Noda,2020; Lai et al., 2020. Furthermore, ATG9 has been found on cytoplasmic vesicles of 32-35.6nm that are generated from Golgi apparatus, these vesicles contain around 30 ATG9 proteins in yeast (Reggiori et al. 2012). The amount of these ATG9 vesicles is increased during starvation or rapamycin treatment that contribute to forming autophagosomes. Cytoplasmic ATG9 vesicles (at least 3 vesicles in yeast) are assembled individually at PRE AUTOPHAGOSOME-STRUCTURE (PAS) and the outer autophagosome membrane that finally are recycled as new ATG9 vesicles (Yamamoto et al., 2012). Despite the important role of ATG9 is not present in the whole autophagic flux in Arabidopsis and Drosophila (Wen et al., 2017; Zhuang et al., 2017). In addition, ATG9’s role is related with vesicular trafficking machinery during autophagosome biogenesis. This connection appeared during the studies that explain the origin of the autophagosome (Yang et al., 2021). Vesicular trafficking is the transportation of materials between different cellular compartments, between cells and its environment, regulating various intra and extracellular signals to respond to different cellular stressors and metabolic states such as degradation (Søreng et al., 2018; Tokarev et al., 2013). ENDOPLASMIC RETICULUM (ER) is part of the vesicular trafficking and endomembrane precursor of the autophagosome and probably the major origin site particularly the rough ER where no ribosomes are positioned. Approximately 70% of autophagosome content is derived from ER. (Hayashi-Nishino et al., 2009). Also, the ER-GOLGI INTERMEDIATE Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 100 COMPARTMENT (ERGIC), ER-EXIT SITES (ERES), mitochondrion, ER-mitochondria contact, nuclear membrane, plasma membrane and recycling endosomes may be a source of autophagosome membrane (Figure. 39) (Rubinsztein et al., 2012; Yang et al., 2021). On the other hand, some ATG proteins in yeast and mammals participate in the remodeling of the ERES-ERGIC-COPII system. COPII vesicles (COPII; COAT ASSEMBLY PROTEIN) during nitrogen starvation stops the vesicle trafficking and diverted to macroautophagy where ATG1 tethers ATG9-containing vesicles with COPII vesicles (Ge et al., 2014; Jia et al., 2019; Wang et al., 2014). ATG9 vesicles (generated by TRANS-GOLGI NETWORK (TGN) and endocytic recycling system) define the number of the autophagosome (Feng & Klionsky, 2017; Ge et al., 2013; Jin & Klionsky, 2014). Figure 36. ATG9 in vesicular trafficking and autophagosome formation. ATG9 is internalized from the plasma membrane, VAMP3-mediated fusion between the ATG16L1 and ATG9 vesicles. ATG9 cycles between the TGN and a peripheral pool, in recycling endosomes that is mediated by TRAPPII-like complex and RAB1. Atg9 vesicles form the autophagosome (Søreng et al., 2018). In mammals, ATG9a vesicles are localized by clatherin-coated structures, internalized by endocytosis pathway, and are fused with ATG16L1 vesicles. Also, ATG16L1 vesicles are internalized by clatherin during endocytosis from the plasma membrane but by different pathways. During starvation, studies show that membrane recycling is reduced but the fusion of mATG9a-ATG16L vesicles that depend on VAMP3 are increased during starvation (Puri et al., 2013, 2014). Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 101 Other proteins related to ATG9 and membrane are proteins of multisubunit tethering complex (MTC) which are essential for transport and transmembrane lipid scramblase. Some of them are COG, GARP, TRAPPII and TRAPIII which are studied in yeast. For example, Recent results show that TRAPIII requires Drs2 to stabilize in ATG9 vesicles under cold environment (Pazos et al., 2021; Shima et al., 2019). This means that ATG9 vesicles associations impact in regulating transport and autophagosome formation. One of the most extensive studies to date found that ATG9 is interacting with 42 proteins of membrane transports, RNA regulation, TOR signaling, vacuole fusion and as they expected autophagy genes. They determine the interaction of Glo3 with ATG9 during retrograde transport (Peng et al., 2021). As we can appreciate, the ATG9 interactions goes far beyond the autophagy interaction in the process. The canonical function of autophagy genes has been described but different authors have been considering the alternatives functions of autophagy genes. The no-canonical functions of ATGs are mainly reported in Homo sapiens and other mammals (Jülg et al.,2020; Dopont et al., 2013). In ATG9, we present some interaction examples in Table 3 that include ATGs. Our aim in this chapter is identify PvATG9 interactors creating an expanded network and contrasting with our transcriptome. The results allowed us to decipher large possibility of PvATG9 functions and find the most related with nodulation in P. vulgaris. Table 3 ATG9 interactions reported in Yeast, Mammals and Plants. ATG9 interactors Name Organism Title of publication References ATG1/ULK1 AuTophaGy 1 Yeast The Atg1–kinase complex tethers Atg9-vesicles to initiate autophagy Rao et al., 2016; Atg1 kinase organizes autophagosome formation by phosphorylating Atg9 Papinski et al.,2014 Mammal Regulation of mATG9 trafficking by Src- and ULK1- mediated phosphorylation in basal and starvation- induced autophagy Zhou et al.2017 Ap1/Ap2 complex Adaptor protein-1 Adaptor protein-2 Mammal Mammalian Atg9 contributes to the post‐Golgi transport of lysosomal hydrolases by interacting with adaptor protein‐1. Jia et al., 2017 ATG11 AuTophaGy 11 Yeast Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast. He et al. 2006 Atg11 tethers Atg9 vesicles to initiate selective autophagy Matscheko et al., 2019 Mammal Regulation of mATG9 trafficking by Src- and ULK1- mediated phosphorylation in basal and starvation- induced autophagy Zhou et al.2017 Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 102 ATG2 AuTophaGy 2 Yeast Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores. Gomez-Sanchez et al., 2018 Plant Autophagy-related (ATG) 11, ATG9 and the phosphatidylinositol 3-kinase control ATG2- mediated formation of autophagosomes in Arabidopsis. Kang et al., 2018 ATG5 AuTophaGy 5 Plant ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis. Zhuang et al.,2017 ATG17 AuTophaGy 17 Yeast The Atg1–kinase complex tethers Atg9-vesicles to initiate autophagy Rao et al., 2016 ATG9 AuTophaGy 9 Yeast Self-interaction is critical for Atg9 transport and function at the phagophore assembly site during autophagy He et al., 2008 GLO3 GLyOxalase 3 Yeast Atg9-centered multi-omics integration reveals new autophagy regulators in Saccharomyces cerevisiae. Peng et al., 2021 Rab1B Ras-related protein Rab-1B Mammal Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation Kakuta et al., 2017 SCS7 Ceramide very long chain fatty acid hydroxylase Yeast Atg9-centered multi-omics integration reveals new autophagy regulators in Saccharomyces cerevisiae. Peng et al., 2021 OPTN Optineurin Mammal Critical role of mitochondrial ubiquitination and the OPTN–ATG9A axis in mitophagy Yamano et al.,2020 PATJ PALS1-associated tight junction protein Drosophila Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila Wen et al., 2017 TRS85 Trafficking protein particle complex III- specific subunit 85 Yeast Atg9 vesicles recruit vesicle-tethering proteins Trs85 and Ypt1 to the autophagosome formation site Kakuta et al., 2012 dTRAF2/TRAF6 Drosophila tumor necrosis factor receptor-associated factor 2 Drosophila Atg9 interacts with dTRAF2/TRAF6 to regulate oxidative stress induced JNK activation and autophagy induction Tang et al., 2013 tumor necrosis factor receptor- associated factors 6 Mammal TBC1D5 TBC1 Domain Family Member 5 Mammal TBC 1 D 5 and the AP 2 complex regulate ATG 9 trafficking and initiation of autophagy. Popovic et al., 2014 TFR Transferrin receptor (recycling endosome marker) Mammal Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy Orsi et al., 2012 TMEM74 Transmembrane pro tein 7 Mammal TMEM74 promotes tumor cell survival by inducing autophagy via interactions with ATG16L1 and ATG9A. Sun et al., 2017 VAMP7 Vesicle Associated Membrane Proteins 3 Mammal VAMP7 regulates autophagosome formation by supporting Atg9a functions in pancreatic β-cells from male mice. Aoyagi et al., 2018 p38IP p38- interacting protein Mammal Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP Webber et al.,2010 PI(4)KIIα Phosphatidylinositol -4-kinase type II alpha The Golgi as an Assembly Line to the Autophagosome De Tito et al., 2020 SUI2,KSP1,TOR2,VTI1,PHO80, YPT7,VPS9,VPS21, CDC48,PHO23,SSA1,COG3,CCZ ,SCS7,SEC22,SEC4,SEC23,GLO3 ,YPT31,SEC17,ARP2,TLG2,SEC 18,VPS34,ATG27,TRS85,ATG2 3,ATG11, ATG18,ATG2,ATG12, ATG14,ATG7,ATG14, ATG10,ATG2,ATG17,ATG8, ATG6,ATG1, ATG5 *Abbreviations Table Page 10 Yeast Atg9-centered multi-omics integration reveals new autophagy regulators in Saccharomyces cerevisiae. Peng et al., 2021 Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 103 RESULTS PvATG9b protein interactions during nodulation in P. vulgaris To test the interactions of PvATG9b, we used Yeast Two-Hybrid (Y2H). The bait and the pray interactions were screened on plates lacking Leucine, Tryptophane, Histindine, Adenine (SC-LTHA) with 0.25mM 3-aminotriazol (3-AT) and with 2.5mM 3-AT, respectively. Approximately 6.6 x 10^6 diploids were screened per library. Positive colonies were picked from the screening plates and regrown in a grid on Quadruple dropput (QDO) media with the corresponding 3-AT concentration (Fig. 37). Inserts were amplified by PCR, sequenced, and used to probe both NCBI and JGI databases by both blastn and blastx. We identified 24 putative interactors which are listed in Table 4. Most of the interacting proteins have never been reported as the interacting proteins of PvATG9b in any plants previously. Table 4. 24 interacting partners of PvATG9b Interactors of PvATg9b 1 Phvul.001g009100 2 Phvul.001g103600 3 Phvul.001g108101 4 Phvul.002g249800 -CUPIN1 5 Phvul.002g282500 6 Phvul.002g324300 7 Phvul.003g054600 8 Phvul.004g026900 - PCO2 9 Phvul.004g102800 10 Phvul.005g096700 11 Phvul.005g172400 12 Phvul.006g125700 13 Phvul.006g203200 14 Phvul.007g053500 15 Phvul.007g150800 16 Phvul.007g162300 17 Phvul.008g290800 18 Phvul.009g042900 19 Phvul.009g210564 20 Phvul.009g236600 21 Phvul.010g095300 22 Phvul.011g033650 23 Phvul.011g048200 24 Phvul.011g065900 Figure 37 Y2H of ATG9 and Cupin, CDO proteins grown for 3-5 days on the selective medium synthetic (SD) DDO(−Leu/−Trp) and QDO (SD/-Ade/-Trp/-Leu/-His). Identification of PvATG9b-interacting partners during nodulation in P. vulgaris. In our results of Y2H screening, PvATG9b interacts with 24 proteins. The variety of proteins is wide and uses annotations of diverse databases. We recognize the descriptions and annotations of the genes with three different identifiers (Supp. S28). The Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 104 descriptions have shown that the genes are different. In figure 38, we can properly observe the function of the proteins assigned so far. Using the enrichment of gene ontology associated with the primary proteins of the 24 genes gave us a broader vision. Figure 38 PvATG9b-interacting partners. 24 proteins interact directly with PvATG9b in Y2H screening (Blue points; represent the protines), The list of them contain a briefly description. The cellular components related to five PvATG9b interacting partners are the nucleus and endoplasmic reticulum membrane as the most abundant. Biological processes in interactors are related to the oxidation- reduction process. Molecular function enrichment carried out 33 different predicted functions (Table.5; Supp. S29). The smallest protein is Phvul.006G203200.1. p (No. 13) with 37 a.a., in contrast with the protein Phvul.002G282500.1.p (No. 5) with 923 a.a. . Of all proteins, only six proteins have high probability of containg transmembrane domains: Phvul.001G108101.p.1 (No.3), Phvul.004G102800.1.p (No.8), Phvul.006G125700.1.p (No. 12), Phvul.006G203200.1.p (No.13), Phvul.007G053500.1.p (No.14), ´Phvul.008G290800.1.p (No.17) (Supp. S30). These initial results helped us to understand the type of proteins that are interacting with PvATG9b during nodulation Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 105 . Table 5. Ontology enrichment of PvATG9b-interacting partners by PANTHER. Name/Description ID Accession Term PvATG9b Network Node * - Histone H3 and H4 Phvul.001G009100.1 Cellular Component GO:0000786 nucleosome 1 Phvul.001G009100.1 Cellular Component GO:0005634 nucleus RPS16C 40S Ribosomal protein S16 Phvul.002G324300.1 Cellular Component GO:0055114 oxidation-reduction process 6 - Protein glycosyltrasferase subunit 4A Phvul.006G203200.1 Cellular Component GO:0005789 endoplasmic reticulum membrane 13 Phvul.006G203200.1 Cellular Component GO:0016020 membrane Phvul.006G203200.1 Cellular Component GO:0016021 integral component of membrane TFIIS Trasncription elongation factor Phvul.009G042900.1 Cellular Component GO:0005634 nucleus 18 . Inorganic diphospho pyrophosphate Phvul.011G065900.1 Cellular Component GO:0005634 nucleus 24 Phvul.011G065900.1 Cellular Component GO:0005783 endoplasmic reticulum CLP1 ATPdepent CLP protease Phvul.002G282500.1 Biological Process GO:0019538 protein metabolic process 5 PCO2 Plant cysteine oxidase 2 Phvul.004G026900.1 Biological Process GO:0055114 oxidation-reduction process 8 SEC1A Proteins containging the FAD bindin domain Phvul.006G125700.1 Biological Process GO:0055114 oxidation-reduction process 12 - Dehydrogenases with different specificities Phvul.010G095300.1 Biological Process GO:0055114 oxidation-reduction process 21 - Predicted E3 ubiquitin ligase Phvul.011G048200.1 Biological Process GO:0016567 protein ubiquitination 23 - Histone H3 and H4 Phvul.001G009100.1 Molecular function GO:0003677 DNA binding 1 Phvul.001G009100.1 Molecular function GO:0046982 protein heterodimerization activity DTX22 DTX22- Protein DETOXIFICATION 22 Phvul.001G103600.1 Molecular function GO:0015238 xenobiotic transmembrane transporter activity 2 Phvul.001G103600.1 Molecular function GO:0015297 antiporter activity Phvul.001G103600.1 Molecular function GO:0042910 xenobiotic transmembrane transporter activity Cupin1 Phvul.002G249800.1 Molecular function GO:0045735 nutrient reservoir activity 4 CLP1 ATPdepent CLP protease Phvul.002G282500.1 Molecular function GO:0000166 nucleotide binding 5 Phvul.002G282500.1 Molecular function GO:0005515 protein binding Phvul.002G282500.1 Molecular function GO:0005524 ATP binding Phvul.002G282500.1 Molecular function GO:0016887 ATPase activity RPS16C 40S Ribosomal protein S16 Phvul.002G324300.1 Molecular function GO:0003735 structural constituent of ribosome 6 EIF3 Transcription initiation factor 3 subunit EIF-3G Phvul.003G054600.1 Molecular function GO:0003676 nucleic acid binding 7 Phvul.003G054600.1 Molecular function GO:0003723 RNA binding Phvul.003G054600.1 Molecular function GO:0003743 translation initiation factor activity PCO2 Plant cysteine oxidase 2 Phvul.004G026900.1 Molecular function GO:0016702 oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen 8 Phvul.004G026900.1 Molecular function GO:0017172 cysteine dioxygenase activity Phvul.004G026900.1 Molecular function GO:0046872 metal ion binding SLAH3 S-type union channel Phvul.004G102800.1 Molecular function GO:0008308 voltage-gated anion channel activity 9 GTE4 Transcription initiation factor TFII, subunit BDF1 Phvul.005G096700.2 Molecular function GO:0005515 protein binding 10 . F-box domainKelch motif Phvul.005G172400.1 Molecular function GO:0005515 protein binding 11 SEC1A proteins containging the FAD bindin domain Phvul.006G125700.1 Molecular function GO:0016491 oxidoreductase activity 12 Phvul.006G125700.1 Molecular function GO:0016614 oxidoreductase activity, acting on CH-OH group of donors Phvul.006G125700.1 Molecular function GO:0050660 flavin adenine dinucleotide binding Phvul.006G125700.1 Molecular function GO:0071949 FAD binding PBL7 Serine/ threonine Phvul.007G162300.1 Molecular function GO:0000166 nucleotide binding 16 Phvul.007G162300.1 Molecular function GO:0004672 protein kinase activity Phvul.007G162300.1 Molecular function GO:0004674 protein serine/threonine kinase activity Phvul.007G162300.1 Molecular function GO:0005524 ATP binding Phvul.007G162300.1 Molecular function GO:0016301 kinase activity Phvul.007G162300.1 Molecular function GO:0016740 transferase activity Phvul.008G290800.1 Molecular function GO:0008270 zinc ion binding TFIIS -Trasncription elongation factor Phvul.009G042900.1 Molecular function GO:0003676 nucleic acid binding Phvul.009G042900.1 Molecular function GO:0008270 zinc ion binding 18 Phvul.009G042900.1 Molecular function GO:0046872 metal ion binding - Predicted E3 ubiquitin ligase Phvul.011G048200.1 Molecular function GO:0004842 ubiquitin-protein transferase activity 23 Phvul.011G048200.1 Molecular function GO:0016740 transferase activity Phvul.011G048200.1 Molecular function GO:0046872 metal ion binding * PvATG9b Network Node (Figure 41) Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 106 Interaction Network of PvATG9b in P. vulgaris We built the interaction network of PvATG9, beginning with the predicted functional partners related to the co-expression, experiments, databases and textmining. Our network contains 11 nodes that represent the proteins, with 55 edges (Fig. 39). The functional enrichment using STRING, KEGG Pathway, Uniprot, Pfam and Interpro databases converge in autophagy. The strongest protein-protein interaction is with ATG18 supported by six experiments (Papinsky et al., 2014; Nagy et al., 2014; Sun et al., 2017; Reggiori et al. 2004;Gomez-Sanchez et al.,2018). There are the techniques (affinity chromatography technology assay, coimmunoprecipitation assay, biochemical assay and two hybrid assays) in yeast, Drosophila melanogaster and Homo sapiens (Supp.S31). All proteins are coexpressing, ATG13 has the highest coexpression score 0.743 and ATG7 with 0.601 (Supp. S32). Until now the data did not have experimental information on P. vulgaris. Figure 39 PvATG9 network. PvATG9 interact with 10 protein results based on cooexpression and texmining from STRING databases. (Purple square: nodes; Blue square: PvATG9b) We expanded the network by adding 24 PvATG9 interacting-partner proteins that we detected in our previous screening and their own interaction (Figure 40). Therefore, the topological parameter for our network has 241 nodes, and 734 edges (Supp. S33). The nodes form six edges on average, forming a network density of 0.27 (Number 1 is the value of the most density network). In this network the heterogeneity increased compared with the first PvATG9 network reflecting the tendency to contain new hubs of nodes additional to autophagy. Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 107 Figure 40. Expanded Network of PvATG9b. PvATG9 expanded network contain 241 nodes that include the STRING results and Y2H screening. (Blue point:nodes) Functional enrichment analysis of PvATG9b network formed 10 hubs. The highest number of nodes are ribosome, protein procession in endoplasmic reticulum and ubiquitin mediated proteolysis hubs. In MAPK signaling pathway, endocytosis and RNA polymerase hubs have 6 to 9 nodes that are linked with only one node which interacts with PvATG9b. The least number of nodes are in Circadian rhythm, folates and basal transcription factor. Several nodes are associated with two hubs, for example, five nodes in MAPK signaling pathway, two in ribosome and folate biosynthesis, and three in ubiquitin mediated proteolysis. To focus on specific hubs, we integrated our transcriptome data that we shall hereafter show. Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 108 Expression profile of PvATG9b-interacting partners The co-expression of 24 interacting-partners with PvATG9b are 9 genes that increase, and 8 genes decrease, their expression was obtained from our transcriptome during P. vulgaris nodulation (21dpi). The most abundant expression is PLANT CYSTEINE OXIDASE 2 (PCO2; Phvul.004G026900; No. 8) followed by Phvul.008G290800 (No.17), SECRETORY 1A (SEC1A; Phvul.006G125700; No.12) and Phvul.011G065900 (No.24). On the opposite side, Phvul.009G236600 (No. 19), Phvul.010G095300 (No.20) and Phvul.009G042900 (No.21) have the lowest expression (Fig. 41). These data were contrasted with PvGEA database to have more information about expression of these genes in roots and nodules. The lack of nodes is because they do not have quantitative value in our transcriptome data. Figure 41 PvATG9b-interacting partners coexpression during nodulation. Nine PvATG9b-interacting partners increased their expresion while eight decreased the expression during R. tropici symbiosis in P. vulgaris The expression of the 24 genes is compared in roots and nodules (Fig. 42). In roots, we compare the pre-fixing nodules (5 d) with roots separated from nodules nitrogen fixers (Fix +) and not nitrogen fixers (Fix -). In nodules, the samples are pre-fixing nodules (5 days), effectively and ineffectively fixing nodules (21 days after inoculation). The expression of the PCO2 (Phvul.004G026900; No. 8) and Phvul.011G065900 (No.24) have the highest coexpression in our transcriptome and in PvGEA database. The expression increases in roots and nodules with effective fixing compared with the other Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 109 samples. The results bring us the reason to examine mainly the node PCO2 and Phvul.011G065900 (No.24). Figure 42 Expression profile of PvATG9b-interacting partners in P. vulgaris roots and nodules. The network of PCO2 is composed of 11 nodes (Fig. 43). Firstly, we examined this gene PCO2 (Phvul.004G026900) and we found that the gene is a cysteamine dioxygenase/persulfurase which involved in processes such amino acid biosynthesis, nitrogen metabolism, carbohydrate metabolism, membrane transport and sulfur assimilation. PCO2 increases the expression during symbiosis together with the coexpression of HYPOXIA REPSONSE ATTENUATOR1 (HRA1), VACUOLAR SORTING PROTEIN 39 (VPS39) and ETHYLENE-ESPONSIVE FACTOR 71 (ERF71). Meanwhile, the nodes ARGINYL TRANSFERASE (ATE1) and PROTEOLYSIS 6 (PRT6) decrease the expression. These genes play a role during the normoxia and hypoxia. Hypoxic conditions lead to an increase in Nitrogen Oxide levels, that allow the NO2/O2 balance. In accordance with the expression of genes, the decrease of ATE1 and PRT6 suggested that normoxia stopped and PCO2 is participating in the NO2/O2 balance during symbiosis and at the same time ATG9 is interacting. Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 110 A B Figure 43 Plant Cysteine Oxigenase 2 (PCO2) Network. (A) 10 nodes are interacting with PCO2 of which HRA1, ERF71, VPS39 and HRA1 increased their expression during symbiosis between R. tropici and P. vulgaris.(B) Normoxia pathway that involves the Plant Cysteine Oxigenase 2 (PCO2) network (Taylor‐Kearney et al. 2022). DISCUSSION In this chapter, we present 24 interacting partners of PvATG9b obtained by Y2H screening. The 24 interacting partners had not previously been reported and were included in PvATG9b network, generating 241 nodes and 734 edges. The expanded network exposed a various biological process of which ribosome, in endoplasmic reticulum and ubiquitin mediated proteolysis are the most abundant. Additionally, we contrasted expression data of the PvATG9b-interacting partners between our Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 111 transcriptome and PvGEA data. In this way, we could recognize PCO2 as a candidate due to their highest expression during nitrogen fixation. Several studies of proteins interacting with ATG9 in diverse organisms reported different proteins and we did not detect the same proteins. Neither have ATG proteins in our results that could imply ATG9 is playing a role in non-canonical autophagy function. It might be for the ATG9 structure because the reports mention the vary lengths ranging from 700 to 1,000 a. a. residues where N- and C-terminal are significant different structures among organisms (Maeda et al.,2020). As well as ATG9 of S. cerevisiae and H. sapiens contain the same transmembrane domains but the amino acid sequences exposed to the cytosol are different. That means that ATG9 interacts with ATG13, ATG23, ATG27 and ATG17 in yeast, while ATG9a in humans interacts with AP complex in the same exposed sequences as ATG9 in yeast (Nishimura et al., 2020). Also, ATG9a structures of cryogenic electron microscopy (Cryo-EM) in Arabidopsis and humans exhibit the self-interaction of ATG9 as a trimer forming a pore embedded in nano disks of the membrane scaffold protein 2N2 (MSP2N2) that can participate in lipid scrambling activity (Maeda et al., 2020; Guardia et al.,2020). The range of exposes of the protein could have few specific interactions or give a greater capacity to interact with at proteins, but it has not been probed yet. During P. vulgaris nodulation, we did not register the self-interacts, but ATG9 might be as a vesicle. The ATG9 vesicles originated from the Golgi apparatus (Yamamoto et al., 2012). These vesicles participate in membrane-trafficking processes, such as budding and fusion (Noda et al.,2017). The interactome of ATG9 performed by Peng found proteins related to membrane trafficking, protein transport and RNA regulation in yeast (Peng et al., 2021). Also, we presented proteins of membrane trafficking in our interaction results such as SEC1 and YPT/RAB Specific GTPase-activating protein GYP. SEC1 has not been reported interacting with ATG9 in any organism, but in yeast Peng reported SEC4, SEC17, SEC18 and SEC22 that are secretory proteins. SEC1 contributes during membrane fusion, interacting with SNARE complex (Carr et al.,1999). In Arabidopsis, Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 112 SEC1/MUNC18 (SM) was reported in pollen fertility by membrane trafficking disruption (Beuder et al., 2022). On the other hand, YPT/RAB Specific GTPase-activating proteins allow the reaction to associate targeting molecules located on the surfaces of transport vesicles (Pfeffer et al.,1994). RAB1 was described in proteomic of immunoisolation of mATG9A-containing membranes of human cells. Rab1 is indispensable in endoplasmic reticulum-to-Golgi vesicle trafficking and mutants suppress autophagy. Rab5 and Rab7 form part of a complex with Vps34 and Beclin1 necessary for autophagosome formation in mammals while Rab11 facilitates the cross talk between autophagy and the endosomal pathway in Drosophila (Stein et al 2005; Ravikumar et al., 2008). In legumes, other small GTPase of the Rab family was studied during symbiosis between P. vulgaris with Rhizobium etli and results mention that Rab2 acts in polar growth of root hairs and is required for reorientation of the root hairs growth during infection (Blanco et al., 2009). By this we mean that Rab family is related to autophagy and membrane trafficking. We contrasted all 24 interacting partners of PvATG9b with PvGEA data base that includes many stages of nodulation and with our transcriptome using P. vulgaris nodulated with 21 dpi. These studies were based on yeast two hybrid that require another experiment to corroborate the interaction. For now, we found 17 interaction partners in our transcriptome and only 9 have up regulation. Meanwhile, 4 interacting partners maintain high expression in efficient and inefficient fixation. SEC1, eIF3 and PCO2 showed high expression in both analyses. SEC1 as I mentioned earlier, is a protein involved in membrane fusion. eIF3 is a scaffold protein that forms a complex to scan, precise the start codon selection, and can mediate the translational mechanism controlling energy metabolism (Shah et al. 2016). eIF3 is participates in translational control that plays an important role in novo protein biosynthesis since early association with Arbuscular mycorrhizae (Van Buuren et al., 1999). Until now, there are not reports in nodulation. These two interesting proteins need to be explored in nodulation we only could think that membrane trafficking and the translational mechanism are active. PCO2 has the highest expression in our transcriptome and remarkable expression in nodules with efficient fixation in databases. PCO2 as a plant cysteine oxidase is classified Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 113 as oxygen-sensing enzymes in plants, controlling hypoxia-dependent processes (White et al., 2018). Regulation of oxygen in Rhizobium symbiosis is required to induce mechanisms of nitrogen fixation. The nitrogen fixation is performed under low oxygen because nitrogenase enzyme is intolerant to oxygen. This is a reason that the plant host a Rhizobia in nodules, to maintain the anoxic environment but also provide demand for resources. PCO2 as a node in network is related to 10 proteins and 4 of them presented high expression in our transcriptome. The network contains the amino-end rule pathway that mediates the oxygen sensing in plants. In our results, we found two HRA1 with the highest expression, ERF71 and VPS39. The vacuolar sorting proteins VPS39 in our transcriptome appear two transcripts with same name and opposite expression but is interesting because was analyzed during symbiosis revealed the dynamic of vacuole consist in contract the vacuoles to allow the expansion of symbiosome (Gavrin et al.,2014). The expression of one of them has sense with the fusion and membrane dynamics that we consider during symbiosis. In yeast, VPS39 is required phospholipids transport in contact sites among mitochondria, endoplasmic reticulum, and vacuole (Iadarola et al., 2020). Probably, if the membrane of the vacuole is shrinking for the symbiosome the ATG9 vesicles could be more abundant. ERF71/HRE2 is induced during hypoxia and is recognized in direct role in ROS perception (Yao et al., 2017). Results of ERF71 studies in Lotus japonicus confirmed an important function in successful infection by Mesorhizobium loti (Asamizu et al., 2008). Also, HRA1 is a transcription factor that can act on RAP2.12. The upregulation of HRA1 was detected in low oxygen and promoted the expression of anaerobic gene by RAP2.12 (Giuntoli et al., 2014). ERF71, HRA1 and PCO2 expression is induced by the barrier generated by the bacteria to maintain low oxygen. PvATG9b might interact with PCO2 to transport the protein in cytosol. However, this interaction required future analysis to understand this fascinating process. Finally, under this context, PvATG9b is not in the autophagy process but is interacting with diverse proteins related to membrane trafficking and co expressed with proteins that Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 114 response to hypoxia. The main interaction is with the plant cysteine oxygenase PCO2. we suggest that PvATG9b is involved in membrane trafficking and hypoxia mechanisms. MATERIAL AND METHODS Yeast two-hybrid screening Plant material and Rhizobium inoculation Seeds of Phaseolus vulgaris L. cv. Negro Jamapa were surface-sterilized, germinated in the dark on wet filter paper for two days at 28 °C, transferred to sterile vermiculite, and grown under a 16-hphotoperiod at 28 ± 1 °C. Five-day-old plants were inoculated with R. tropici and irrigated twice weekly with no nitrogen. At 7 dpi roots samples were collected and were immediately frozen in liquid nitrogen and stored at -80º C for RNA extraction. RNA Isolation Total RNA was isolated from the harvested root tissues using TRIzol (Invitrogen, USA) reagent according to the manufacturer's protocol. Quality of all the samples was assessed on 1.2% formaldehyde agarose gel, while quantification was done by measuring A260/A280 ratio in Nanodrop. First strand cDNA was synthesized from the total RNA (2.5 μg), using cDNA synthesis kit (Superscript® III, Invitrogen, USA) following manufacturer's instructions. Cloning of the Atg9 CDS into Yeast Bait Plasmid First strand cDNA was synthesized using RNA extracted from the P. vulgaris roots (7 dpi) according to the “First-Strand cDNA Synthesis” protocol (Invitrogen, USA) using 2 μg of DNAase free RNA. The coding sequence (CDS) region encoding the PvATG9b protein with restriction sites attached, was amplified (primer pair sequences provided in Supp.S9 from single stranded cDNA. For ligation, the pGBKT7 vector (2.5 μg) was double digested (EcoR1 and BamH1) and gel purified. The purified PCR product (150 ng) was ligated with 50 ng of pGBKT7 vector using the 5 × In-Fusion® HD Enzyme Premix, containing the “In- Fusion Enzyme.” Five microliters of the ligated product were transformed into 100 μl of Stellar™ Competent Cells (Clontech, USA) and selected on LB plates with Kanamycin (Kan; 50 μg/ml). Colonies were picked and inoculated into 5 ml LB/Kan broth and grown overnight with shaking at 37°C. Plasmids were extracted from these cultures using a Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 115 purification kit (NucleoSpin® Plasmid, Macherey-Nagel, Germany) and screened for the presence of inserts with restriction digestion. To confirm the successful cloning of the PvAtg9 CDS, the pGBKT7 vectors containing inserts were sequenced using CDS specific primers. The confirmed PvATG9b clone was selected and transformed into competent S. cereviceae Y2HGold using a high-efficiency polyethylene glycol (PEG)/LiAc-based method (Yeastmaker™ Yeast Transformation System 2 User Manual, Clontech, USA). Transformed yeast cells were selected on the minimal YSD medium deficient in TRP (SD/-W). Generation of rhizobium inoculated Root cDNA Library The cDNA library was constructed from the roots of the rhizobium inoculated P. vulgaris roots, in S. cereviceae Y187α using Make Your Own “Mate and Plate™” Library System (Clontech, USA) following the manufacturers' instructions. Equal amounts of double stranded cDNA (3 μg) and “prey” library vector (3 μg; pGADT7-Rec) were mixed for the homologous recombination-mediated cloning using the library-scale transformation protocol (Yeast Transformation System 2 Manual, Clontech, USA). After 4 days of incubation, all the colonies were harvested in freezing medium (YPDA in 25% glycerol) and stored in aliquots at –80°C. Y2H assay An aliquot (1 ml) containing >2 × 107 cells of the harvested S. cereviceae 187α strain (harboring library constructs in pGADT7-Rec) was mated with 4–5 ml (>1 × 108 cells per ml in SD/-W) of S. cereviceae Y2HGold (containing the PvATG9 constructs in pGBKT7) based on the Matchmaker™ Gold Y2H (Clontech, USA) manual. The re-suspended cells in YPDA/Kan were spread on the selective media [double dropouts (SD/−Leu/−Trp) and incubated at 30°C for 3–5 days. Positive and negative control matings were then carried out as per the Matchmaker™ Gold Y2H manual and plated on DDO media. Single colonies were patched on QDO (SD/-Ade/-Trp/-Leu/-His), followed by incubation at 30°C for 3–5 days. Yeast colony PCR using 5′ and 3′ PCR primers (Supplementary Table 1), were performed on the blue colonies identified on the QDO media to determine the presence of inserts in the prey, pGADT7-Rec clones. Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 116 Following this, plasmids were isolated from yeast colonies picked from the QDO selective media using the Easy Yeast Plasmid Isolation kit (Clontech, USA), and the “prey” vectors containing inserts of candidate interactors, were isolated by transforming into Stellar™ Competent Cells and plating on LB with ampicillin (Amp), (selective for only pGADT7-Rec clones). Colonies were picked, cultured in LB/Amp (overnight) and the plasmids were purified. The PPIs were confirmed by co-transforming S. cerevicieae Y2HGold with the “bait” (ATG9 in pGBKT7) clone together with the interactor “prey” clone (in pGADT7-Rec) and plated on QDO To check for any false positive interactions, the empty “bait” vector was co-transformed with the interactor prey clone and plated as above. The pGADT7- Rec clones were sequenced in the forward and reverse directions using T7 and 3′AD primers. The sequences of the identified interactors were subjected to BLASTN and BLASTX (NCBI, http://www.ncbi.nlm.nih.gov/; JGI, https://phytozome-next.jgi.doe.gov/ ) Analyses for identification and confirming the correct orientation of the interactor sequences and to rule out any false-positive or large ORFs in the wrong reading frame. Identification and characteristics of proteins The sequenced proteins were aligned with Joint Genome JGI institute data bases in the first instance. As well, we collected the diverse names corresponding to Phytomine (https://phytozome-next.jgi.doe.gov/phytomine), Ensembl (https://www.ensembl.org/ ) and National Center for Biotechnology information (NCBI, https://www.ncbi.nlm.nih.gov ) to develop the networks and get the gen and protein features. Then, we obtained the homologs in A. thaliana to assign the names (Supp. S34). The association of annotations of Gene ontology (GO) was performed in Panther (http://pantherdb.org/webservices/go/overrep.jsp). The protein physical and chemistry parameters were carried out using the sequence in PROTOPARAM software (https://web.expasy.org/protparam/). Interaction network construction The interaction network was constructed as a full STRING network where the edges indicate the functional and physical protein association (https://string-db.org/). The interaction sources considered are textmining, experiments, databases, co-expression, Chapter IV. Deciphering the PvATG9b interaction network during symbiosis between Phaseolus vulgaris and Rhizobium tropici 117 neighborhood, gene fusion, co-occurrence data and we incorporated our interaction and expression data. The final image was drawn using Cytoscape software which maintains the minimum of interaction score of 0.04 (https://cytoscape.org/) for complete network. Expression profiling and transcriptome Expression data from transcriptome was obtained from roots of P. vulgaris (wild type and nodulated). The RNA was isolated using RNeasy Plant mini kit (Quiagen) and cleaned with RNase-free DNase followed by Dynabeads, RNaDIRECT micro kit (Life technologies). The cDNA was hybridized with ion adapters and mixed with reverse transcriptase. The technology for the transcriptome was prepared to introduce the chip into Ion Proton sequencer. Then the results were aligned to the P. vulgaris references v2.1 and analyzed with strand NGS software. Then we plotted the Log2 of RPKM of comparing the wild type and nodulated roots. To enhance the studies, we used the Log2 of RPKM of Whole roots separated from 5 days old pre-fixing nodules, Whole roots separated from fix+ and fix- nodules collected 21 days after inoculation, Pre-fixing (effective) nodules collected 5 days after inoculation, Effective and Ineffective fixing nodules collected 21 days after inoculation obtained by PvGEA (https://www.zhaolab.org/PvGEA/page/download). Chapter IV. 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General Discussion and Conclusion 121 General Discussion The greatly expanding knowledge of autophagy has shown its relevant role in growth, development, and enduring responses to abiotic and biotic stresses mainly in model plants. However, it is necessary to understand this process in other plants, such as legumes. An initial objective of the thesis was to identify the autophagy genes in legumes. As a result, we found 17 families, from whom ATG14 family was recently identified by Tang & Bassham (2018). We defined the families based on homologs analysis in various six databases and were contrasted using a phylogenetic tree, synteny and motif identification analysis. The lack of autophagy genes in plants compared with yeast, made necessary to study the detail of the ATGs function because they could be supplementing for the function of other genes. That was the case of ATG11 that in its protein sequence contains ATG17 domain (Li & Vierstra, 2014). In our study, we focused in ATG18 family, one of the biggest families in autophagy genes. To have a better understanding of this family, we started reviewing and analyzing each dataset, because all of them had different annotation schemes. These differences resulted on inconsistent gene naming, obscuring the associations with the correct gene. As a result of our analysis, we proposed to divide the family in three subfamilies. We expect that this new family subdivision helps to understand the different functions of ATG18 genes. Promotor analysis and gene expression where our main tools to increase our understanding about the ATG families. Promotor studies showed several light response, circadian control, ethylene and ABA transcription factors that are also abundant in autophagy genes promotors. This is consistent with other plant studies. For example, ethylene is considered a key regulator in petunia petal senescent (Shibuya et al., 2013). Moreover, our transcriptome data of P. vulgaris (21 dpi with Rhizobium) reveled a PvATG9b, PvATG12 and PvAT18g.II. But in the databases, such as PvGEA and Phytozome, the expression of PvATG9b was not reported to date. So, understanding PvATG9b became our priority in this thesis. Particularly, ATG9 is the unique transmembrane protein that appears in vesicles, and it is essential in autophagy to generate the autophagosome in plants, yeast, drosophila, and mammals but does not appear in the whole process. ATG9 was reported to have Chapter V. General Discussion and Conclusion 122 high expression levels in starvation and nitrogen use efficiency as well as early senescence in plants (Bedu et al., 2020; Buchanan-Wollaston et al., 2005; Masclaux- Daubresse et al., 2014; Nishimura & Tooze, 2020). So, we began to study the PvATG9b using two methods, on one hand we used the cloning technology (expression, silencing, overexpression, and localization) and, on the other hand, we created the PvATG9b network using yeast two hybrid (Y2H). Y2H gave us a different perspective about the role of ATG9, since we expected to find a PvATG9 interaction with PvATG18 and PvATG2, but we could not find it. To explain our findings, one possibility is PvATG9b is transitory, which means it is not required in the whole autophagy process. Other, it is possible that ATG9 vesicles could have other functions. Previous reports suggested the ATG9 participate in autophagy during starvation, but as vesicle in normal level of nutrients (Søreng et al., 2018). Before to stablish the symbiotic relation. With that in mind, our results of GUS staining analysis were used to analyze the expression pattern of PvATG9b promoter, and we found expression in vascular tissue. Our results coincided with other plants, like PvATG9b in Lotus japonicus. Researchers found that, in Lotus japonicus, the LjSYP71 protein is located at the plasma membrane and participates in vesicle trafficking. Transcripts were also detected in vascular tissue, revealing its participation in the transport of substances produced from nitrogen fixation. Nodule products are exported by the xylem, and shoot products are secreted by the phloem and transported to the nodules (Hakoyama et al., 2012). These observations may support the hypothesis of the same mechanism occurring on our research when we see PvATG9b expression in vascular tissue. The results in RNAi of PvATG9b showed a deformed hairy root and lack of expression in nodule vascular tissues, especially concentrated in the cortex of the nodule. Also, the phenotype in secondary roots and leaves were affected showing small yellowish colors, which may be the failure in vesicular trafficking that did not allow the transport between the nodules and the whole plant. The phenotype in overexpression of PvATG9b was opposite in the case of RNAi. In this case, we found expression in the whole nodules, secondary roots, and big greenish Chapter V. General Discussion and Conclusion 123 leaves. It seems possible that these results were due to a problem when mobilizing material between the nodules and the plant. Our last point regarding PvATG9b network is that we contrasted the 24 interacting- partners with our transcriptome and PCO2 had larger expression than any interactant- partners. This finding was unexpected, and one possibility was that the interaction of these proteins could regulate the sensing of oxygen and nitric oxide to maintain the function of the nitrogenase enzyme during nodulation (Pucciariello et al., 2019; Pucciariello & Perata, 2017). Further research is required to establish this possibility and to understand the function of PvATG9b when other proteins interactions occur. There is abundant room for further progress. Therefore, we propose the study of PvATG9a in order to understand the whole function of the gene, by performing finer microscopy studies using the localization construct and also, we propose to look if this protein is also involved in mycorrhizal symbiosis. Chapter V. General Discussion and Conclusion 124 Conclusions This current thesis gives valuable data to increase the knowledge of autophagy in plants. Overall, we made considerable contributions to identify the autophagy core in legumes and, divided the subfamilies in ATG18. Our findings allowed to understand the function of the members of ATG family. Moreover, we detected the high expression of PvATG9b in P. vulgaris, which was examined using novel cloning tools to suggest the function and finally we constructed the network of PvATG9b to complement. Here, we listed below our conclusions of each mentioned part. • 32 genes were identified in P. vulgaris, 39 genes in M. truncatula, and 61 genes in G. max. • The 17 gene families in autophagy of A. thaliana were conserved in P. vulgaris, M. truncatula and G. max • ATG18 family was divided into 3 subfamilies. Subfamily I has a high proportion of proteins named ATG18a, ATG18c, ATG18d, Subfamily II are ATG18b and Subfamily III are ATG18f, ATG18g, ATG18f. • PvATG9b autophagy gene is the highest expressed in P. vulgaris during symbiosis with Rhizobium • PvATG9b expression is concentrated in the vascular tissue of whole plant including the nodule. • The silenced PvATG9b phenotype shows deformed infection threads, short secondary roots, short yellowish leaves. • PvATG9b overexpression phenotype is the huge secondary roots, huge greenish leaves, and an increased number of nodules. • Preliminary localization studies of PvATG9b were found the protein in vascular tissue, tip of lateral root and hairy roots. • 24 unreported proteins that interact with PvATG9b were found. • The expanded PvATG9b network has 241 nodes based on STRING data and yeast two-hybrid analysis. • Plant Cysteine oxidase 2 (PCO2) interacting with PvATG9b was found to show high expression during 21 days of symbiosis between P. vulgaris and Rhizobium. • In the PCO2 network, HRA1, VPS39 and ERF71 we found to increase the expression. Chapter V. General Discussion and Conclusion 125 Discusión General Con los recientes estudios sobre autofagia se ha demostrado que este proceso juega un papel relevante en el crecimiento, el desarrollo y durante las respuestas al estrés abiótico y biótico principalmente en plantas modelo. Sin embargo, se deben hacer más estudios en otras plantas como en las leguminosas. Así que el objetivo inicial de esta tesis fue identificar los genes de autofagia en leguminosas, y obtuvimos 17 familias, de las cuales la familia de ATG14 fue la última añadida por haber sido recientemente añadida por Tang (Tang & Bassham, 2018). Definimos las familias con base a los análisis de homólogos de seis bases de datos y se contrastó con el árbol filogenético, la sintenia y el análisis de identificación de motivos. La falta de genes de autofagia en plantas comparada con levadura hace necesario estudiar el detalle de los ATGs porque podrían estar supliendo la función de otros genes como se reportó con ATG11 que en su secuencia de proteína contiene el dominio ATG17 (Li & Vierstra, 2014). En nuestros estudios en la familia ATG18, una de las familias más grandes en autofagia, requerimos un análisis profundo para dar los nombres porque las anotaciones cambian en las diferentes bases de datos y dificulta la asociación con el gen correcto. Nuestro esfuerzo clasificamos a la familia en tres subfamilias que podrían ayudar a comprender las diferentes funciones de los genes ATG18. Nos esforzamos por conocer y comprender mejor las familias ATG con análisis de promotores y expresión génica. Los estudios de promotores muestran varios factores de transcripción sensibles a la luz, control circadiano, etileno y ABA que son abundantes en los promotores de genes de autofagia. Algunos esfuerzos han estado apareciendo, por ejemplo, el etileno se considera un regulador clave en la senescencia de los pétalos de petunia (Shibuya et al., 2013). Además, nuestros datos de transcriptoma de P. vulgaris (21 dpi con Rhizobium) revelaron a PvATG9b, PvATG12 y PvAT18g.II. Pero en las bases de datos como PvGEA y Phytozome no se reporta la expresión de PvATG9b por lo que entender este gene también se convirtió en nuestra prioridad en esta tesis. Chapter V. General Discussion and Conclusion 126 Particularmente, ATG9 es la única proteína transmembranal que aparece en vesículas, y es esencial en la autofagia para generar el autofagosoma en plantas, levaduras, Drosophila y mamíferos, pero no aparece en todo el proceso. ATG9 fue reportada con alta expresión durante la inanición y la eficiencia del uso de nitrógeno, así como la senescencia temprana en las plantas (Bedu et al., 2020; Buchanan- Wollaston et al., 2005; Masclaux-Daubresse et al., 2014; Nishimura & Tooze, 2020). Para entender a PvATG9b, por un lado, usamos la tecnología de clonación (expresión, silenciamiento, sobreexpresión y localización) y por otro creamos la red PvATG9b usando Y2H. El análisis de interacción de doble hibrido nos dio una visión diferente de ATG9 ya que esperábamos la interacción de PvATG9 con PvATG18 y PvATG2, pero no la encontramos. Una posibilidad por la que tal vez no encontramos las interacciones con PvATG9b es porque es una proteína transitoria, lo que significa que no es requerida durante todo el proceso de autofagia, pero otra razón podría ser que está cumpliendo una función como vesículas ATG9. Estudios previos si han sugerido que ATG9 funciona en la autofagia durante la inanición pero como una vesícula en el nivel normal de nutrientes (Søreng et al., 2018). Con eso en mente, nuestro resultado del análisis de tinción GUS se utilizó para analizar el patrón de expresión del promotor PvATG9b, y encontramos la expresión en el tejido vascular. En Lotus japonicus, LjSYP71 está localizado en la membrana plasmática e involucrado en el tráfico de vesículas. Esta proteína también se expresa en el tejido vascular y participa en el transporte de sustancias producidas por la fijación de nitrógeno. Las sustancias generadas en los nódulos son exportadas por xilema y las sustancias del brote se secretan al floema para ser transportadas a los nódulos de la misma manera. Podrías estar pasando lo mismo con PvATG9b y por eso encontramos expresión en el tejido vascular (Hakoyama et al., 2012). Los resultados en RNA interferente de PvATG9b mostraron un pelo radicular deformado y la falta de expresión en tejido vascular en el nódulo, pero se encontró alta concentración de expresión en la corteza del nódulo. En cuanto al fenotipo las raíces secundarias reducidas y las hojas son pequeñas y amarillentas, lo que puede Chapter V. General Discussion and Conclusion 127 ser la falla de transporte entre el nódulo con toda la planta. El fenotipo en la sobreexpresión de PvATG9b es opuesto al del RNA interferente, encontramos la expresión en nódulo completo mientras que en el fenotipo encontramos las raíces secundarias y en hojas grandes y estas últimas verdosas, es posible que estos resultados se deban a un flujo adecuado en el transporte en la planta. Nuestro último punto es la red de PvATG9b que, como se mencionó antes, nos ayuda a tener otra perspectiva de la función de la proteína. Así que, con las 24 proteínas que encontramos que interactúan con PvATG9b las contrastamos con nuestros datos de transcriptoma y encontramos que PCO2 que interactúan directamente con PvATG9b es altamente expresado. Este hallazgo fue inesperado, esta interacción podría estar regulando que la planta detecte el oxígeno y el óxido nítrico para mantener la función de la enzima nitrogenasa durante la nodulación (Pucciariello et al., 2019; Pucciariello & Perata, 2017). Se requiere más trabajo para comprender la función de PvATG9b al interactuar con otras proteínas. Para continuar se propone estudiar a PvATG9a para poder comprender la función del gen, además de estudios finos de microscopia usando las construcciones de localización y además de entender si esta proteína también está involucrada en la simbiosis con micorrizas. Chapter V. General Discussion and Conclusion 128 Conclusiones Esta tesis presenta datos que permiten entender con más detalle la autofagia en las plantas. En general, hacemos una contribución considerable para identificar las familias de autofagia, así como dividir la familia ATG18 que permite comprender la función de los miembros de la familia. Además, detectamos la alta expresión de PvATG9b en P. vulgaris que examinamos usando herramientas de clonación para sugerir la función y finalmente construimos la red PvATG9b. A continuación, enumeramos nuestras conclusiones. • Se identificaron 32 genes en P. vulgaris, 39 genes en M. truncatula y 61 genes en G. max. • Las 17 familias de autofagia A. thaliana se conservaron en P. vulgaris, M. truncatula y G. max. • la familia de ATG18 se dividió en 3 subfamilias: La subfamilia I tiene una alta proporción de proteínas nombradas como ATG18a, ATG18c, ATG18d, Subfamilia II con ATG18b y Subfamilia III con ATG18f, ATG18g, ATG18f. • El gen de autofagia PvATG9b es el más expresado en P. vulgaris durante la simbiosis con Rhizobium. • La expresión de PvATG9b se concentra en el tejido vascular de toda la planta, incluido el nódulo. • El fenotipo de PvATG9b silenciado muestra hilos de infección deformados, raíces secundarias cortas, hojas amarillentas y cortas. • El fenotipo de sobreexpresión de PvATG9b son las raíces secundarias y hojas verdosas de gran tamaño y además un mayor número de nódulos comparado con las plantas silvestres. • Los estudios preliminares de localización de PvATG9b se encontraron en el tejido vascular, punta de raíz lateral y pelos radiculares. • Se encontraron 24 proteínas no reportadas que interactúan con PvATG9b. • La red expandida de PvATG9b tiene 241 nodos que se basaron en los datos de STRING y análisis de doble híbrido. • Se encontró que la Oxidasa de cisteína de plantas (PCO2) interactúa con PvATG9b muestra alta expresión durante 21 días de simbiosis entre P. vulgaris y R. tropici. • Se encontró que en la red de PCO2, HRA1, VPS39 y ERF71 aumentan la expresión. 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We obtained the Query cover and Percentage of identity value compared A. thaliana protein sequence with legumes: (A) P. vulgaris; (B) M.truncatula; (C) G.max. .(A) Arabidopsis Protein accession numbers Phaseolus vulgaris Protein accession numbers Query Cover Per. Ident At1g49180.1 Phvul.010G120500 74 39,22 At1g49180.2 Phvul.010G120500 81 38,96 At2g37840.1 Phvul.010G015100 86 57,32 At2g37840.2 Phvul.010G015100 93 53,38 At2g37840.3 Phvul.010G015100 95 48,13 At3g53930.1 Phvul.010G015100 86 55,3 At3g53930.2 Phvul.010G015100 86 55,45 At3g53930.3 Phvul.010G015100 98 50 At3g53930.4 Phvul.010G015100 98 50,18 At3g53930.5 Phvul.010G015100 98 50 At3g61960.1 Phvul.010G120500 99 48,16 At3g61960.2 Phvul.010G120500 99 45,55 At3g19190.1 Phvul.003G295800 99 44,68 At3g19190.2 Phvul.003G295800 99 46,62 At3g19190.3 Phvul.003G295800 99 46,62 At5g61500.1 Phvul.011G006500 99 85,67 At5g61500.2 Phvul.011G006500 81 77,82 At2g44140.1 Phvul.008G048900 99 58,61 At2g44140.2 Phvul.008G048900 95 61,43 At2g44140.3 Phvul.008G048900 99 58,61 At2g44140.4 Phvul.008G048900 99 62,44 At2g44140.5 Phvul.008G048900 99 59,14 At3g59950.1 Phvul.008G048900 100 55,98 At3g59950.2 Phvul.008G048900 91 54,85 At3g59950.3 Phvul.008G048900 91 65,45 At3g59950.4 Phvul.008G048900 99 62,41 At3g59950.5 Phvul.008G048900 99 57,61 At5g17290.1 Phvul.008G241000 97 62,46 At3g61710.1 Phvul.005G029900 94 74,9 At3g61710.2 Phvul.005G029900 94 71,7 At3g61710.3 Phvul.005G029900 92 73,99 At3g61710.4 Phvul.005G029900 93 73,23 At5g45900.1 Phvul.011G010700 96 70,18 At4g21980.1 Phvul.011G103300 95 84,72 At4g21980.2 Phvul.011G103300 84 84,82 At4g04620.1 Phvul.003G079300 95 80,51 At4g04620.2 Phvul.003G079300 95 80,51 At4g04620.3 Phvul.003G079300 95 80,51 At1g62040.1 Phvul.011G103300 99 90,76 At1g62040.2 Phvul.011G103300 88 90,75 At2g05630.1 Phvul.011G103300 99 91,67 At2g05630.2 Phvul.011G103300 66 91,74 At2g45170.1 Phvul.003G219600 95 81,36 Supplemental Material 131 At2g45170.2 Phvul.003G219600 95 81,36 At4g16520.1 Phvul.003G219600 96 91,38 At4g16520.1 Phvul.002G062200 96 91,38 At4g16520.2 Phvul.003G219600 95 91,38 At4g16520.3 Phvul.003G219600 95 91,38 At3g60640.1 Phvul.003G219600 91 86,61 At3g06420.1 Phvul.007G210800 94 68,14 At3g15580.1 Phvul.007G210800 98 71,68 At2g31260.1 Phvul.001G159900 99 65,2 At2g31260.1 Phvul.007G194300 97 59,77 At3g07525.1 Phvul010G036300 96 52,73 At3g07525.2 Phvul.010G036300 96 52,49 At4g30790.1 Phvul.003G153800 99 60,79 At1g54210.1 Phvul.010G130300 94 89,13 At1g54210.2 Phvul.010G130300 90 47,83 At1g54210.3 Phvul.010G130300 94 89,13 At3g13970.1 Phvul.010G130300 100 82,98 At3g13970.2 Phvul.010G130300 83 71,43 At3g13970.3 Phvul.010G130300 83 71,43 At3g13970.4 Phvul.010G130300 85 78,67 At3g49590.1 Phvul.008G187800 98 49,11 At3g49590.2 Phvul.008G187800 98 49,11 At3g49590.3 Phvul.008G187800 99 47,69 At3g18770.1 Phvul.002G269600 96 54,19 AT1G77890.1 Phvul.008G169200 96 51,21 AT1G77890.2 Phvul.008G169200 96 49,67 AT1G77890.3 Phvul.008G169200 96 51,21 AT4G08540.1 Phvul.008G169200 99 71,49 At5g50230.1 Phvul.003G207100 99 72,98 At3g62770.1 Phvul.007G196400 88 74,41 At3g62770.1 Phvul.001G205000 98 66,36 At3g62770.3 Phvul.007G196400 86 74,79 At4g30510.1 Phvul.003G152800 97 68,95 At4g30510.2 Phvul.003G152800 99 72,06 At2g40810.1 Phvul.009G041700 98 69,82 At2g40810.2 Phvul.009G041700 98 69,82 At2g40810.3 Phvul.009G041700 98 67,24 At3g56440.1 Phvul.009G041700 97 68,11 At3g56440.2 Phvul.009G041700 98 68,12 At3g56440.3 Phvul.009G041700 95 69,72 At5g05150.1 Phvul.009G041700 97 48,05 At5g54730.1 Phvul.005G091300 88 42,8 At5g54730.2 Phvul.011G140900 89 40,66 At1g03380.1 Phvul.001G146700 86 57,35 At1g54710.1 Phvul.007G183100 98 55,02 At1g54710.2 Phvul.007G183100 100 53,1 At5g66930.1 Phvul.003G248000 87 80,58 At5g66930.2 Phvul.003G248000 100 75,8 At5g66930.3 Phvul.003G248000 81 75 (B) Arabidopsis Protein accession numbers Medicago truncatula Protein accession numbers Query Cover Per. Ident At1g49180.1 MTR_3g095620 62 57,65 At1g49180.2 MTR_3g095620 67 57,65 At2g37840.1 MTR_4g019410 97 63,5 At2g37840.2 MTR_4g019410 93 58,51 At2g37840.3 MTR_4g019410 95 53,47 At3g53930.1 MTR_4g019410 98 59,89 At3g53930.2 MTR_4g019410 98 59,94 At3g53930.3 MTR_4g019410 98 53,79 At3g53930.4 MTR_4g019410 98 53,87 At3g53930.5 MTR_4g019410 98 53,79 At3g61960.1 MTR_8g024100 98 49,71 At3g61960.2 MTR_8g024100 98 46,76 At3g19190.1 MTR_4g086370 99 43,74 At3g19190.2 MTR_4g086370 98 46,76 At3g19190.3 MTR_4g086370 99 45,9 At5g61500.1 MTR_4g036265 99 84,98 At5g61500.2 MTR_4g036265 80 76,65 At2g44140.1 MTR_7g081230 99 58,85 At2g44140.2 MTR_7g081230 96 60,38 At2g44140.3 MTR_7g081230 99 58,85 At2g44140.4 MTR_7g081230 99 62 At2g44140.5 MTR_7g081230 99 59,54 At3g59950.1 MTR_7g081230 99 56,26 At3g59950.2 MTR_7g081230 90 55,05 At3g59950.3 MTR_7g081230 91 63,01 At3g59950.4 MTR_7g081230 99 61,75 At3g59950.5 MTR_7g081230 99 57,36 At5g17290.1 MTR_5g076920 99 59,44 At3g61710.1 MTR_3g018770 99 74,27 At3g61710.2 MTR_3g018770 94 73,9 At3g61710.3 MTR_3g018770 92 77,36 At3g61710.4 MTR_3g018770 93 74,37 At5g45900.1 MTR_0003s0540 97 68,98 At4g21980.1 MTR_2g023430 95 84,75 At4g21980.2 MTR_2g023430 85 84,75 At4g04620.1 MTR_2g023430 96 82,35 At4g04620.2 MTR_2g023430 96 82,35 At4g04620.3 MTR_2g023430 96 82,35 At1g62040.1 MTR_4g048510 96 82,35 At1g62040.1 MTR_4g037225 95 60,87 At1g62040.2 MTR_2g023430 96 82,35 At2g05630.1 MTR_4g048510 98 90,76 At2g05630.2 MTR_4g048510 65 90,74 At2g05630.1 MTR_2g088230 98 72,88 At2g45170.1 MTR_4g101090 92 83,33 At2g45170.2 MTR_4g101090 92 83,33 At4g16520.1 MTR_4g101090 96 92,31 At4g16520.2 MTR_4g101090 96 92,31 At4g16520.3 MTR_4g101090 76 86,96 At4g16520.1 MTR_1g086310 96 53,85 At3g60640.1 MTR_4g101090 99 80,99 At3g06420.1 MTR_4g123760 96 73,04 At3g15580.1 MTR_4g123760 99 71,3 At3g15580.1 MTR_7g096540 98 79,03 At2g31260.1 MTR_7g096680 99 65,31 Supplemental Material 132 At2g31260.1 MTR_1g070160 98 65,22 At3g07525.1 MTR_8g010140 96 55,25 At3g07525.2 MTR_8g010140 96 54,09 AT4G30790.1 MTR_4g130370 99 60,69 At1g54210.1 MTR_8g020500 94 91,3 At1g54210.2 MTR_8g020500 90 47,83 At1g54210.3 MTR_8g020500 94 91,3 At3g13970.1 MTR_8g020500 97 87,1 At3g13970.2 MTR_8g020500 80 76 At3g13970.3 MTR_8g020500 80 76,36 At3g13970.4 MTR_8g020500 80 76,36 At3g49590.1 MTR_5g068710 98 48,78 At3g49590.2 MTR_5g068710 98 48,78 At3g49590.3 MTR_5g068710 98 47,45 At3g18770.1 MTR_3g095570 95 50,16 AT1G77890.1 MTR_5g061040 96 55,88 AT1G77890.2 MTR_5g061040 96 49,34 AT1G77890.3 MTR_5g061040 96 50,88 AT4G08540.1 MTR_5g061040 99 71,42 At5g50230.1 MTR_4g104380 99 67,19 At5g50230.1 MTR_4g007500 88 56,28 At3g62770.1 MTR_1g083230 88 73,49 At3g62770.3 MTR_1g083230 87 73,5 At4g30510.1 MTR_4g130190 99 68,71 At4g30510.2 MTR_4g130190 99 72,87 At2g40810.1 MTR_7g108520 88 62 At2g40810.1 MTR_3g093590 98 71,92 At2g40810.2 MTR_3g093590 98 71,92 At2g40810.3 MTR_3g093590 98 73,53 At3g56440.1 MTR_3g093590 96 74,23 At3g56440.2 MTR_3g093590 96 74,59 At3g56440.3 MTR_3g093590 97 74,66 At3g56440.1 MTR_1g088855 90 53,8 At5g05150.1 MTR_3g093590 96 48,16 At5g54730.1 MTR_3g093590 88 44,62 At5g54730.2 MTR_3g093590 89 43,11 At3g56440.1 MTR_2g082770 21 29,9 At1g03380.1 MTR_1g089110 86 58 At1g54710.1 MTR_1g082300 99 53,68 At1g54710.2 MTR_1g082300 100 52,44 AT5G66930.1 MTR_8g079240 75 83,33 AT5G66930.2 MTR_8g079240 100 75,34 AT5G66930.3 MTR_8g079240 81 74,52 (C) Arabidopsis Protein accession numbers Glycine max Protein accession numbers Query Cover Per. Ident At1g49180.1 GLYMA_04G215500 61 61,81 At1g49180.2 GLYMA_04G215500 61 61,81 At2g37840.1 GLYMA_03G069800 97 64,49 At2g37840.2 GLYMA_03G069800 93 58,89 At2g37840.3 GLYMA_03G069800 95 53,94 At2g37840.1 GLYMA_01G099600 97 63,99 At2g37840.1 GLYMA_06G150700 34 42,86 At2g37840.1 GLYMA_02G220700 98 49,84 At3g53930.1 GLYMA_03G069800 98 61,41 At3g53930.2 GLYMA_03G069800 98 61,83 At3g53930.3 GLYMA_03G069800 98 55,15 At3g53930.4 GLYMA_03G069800 98 55,72 At3g53930.5 GLYMA_03G069800 98 55,15 At3g61960.1 GLYMA_07G048400 98 52,71 At3g61960.2 GLYMA_07G048400 98 49,77 At3g61960.1 GLYMA_16G017300 98 50,82 At3g19190.1 GLYMA_02G133400 99 45,07 At3g19190.2 GLYMA_02G133400 99 47,13 At3g19190.3 GLYMA_02G133400 99 47,13 At5g61500.1 GLYMA_12G005700 99 87,22 At5g61500.2 GLYMA_12G005700 80 79,38 At5g61500.1 GLYMA_09G231000 99 78,21 At2g44140.1 GLYMA_09G244800 99 59,02 At2g44140.2 GLYMA_09G244800 96 61,12 At2g44140.3 GLYMA_09G244800 99 59,02 At2g44140.4 GLYMA_18G248400 99 62,84 At2g44140.5 GLYMA_09G244800 99 59,9 At3g59950.1 GLYMA_09G244800 99 56,22 At3g59950.2 GLYMA_09G244800 91 55,59 At3g59950.3 GLYMA_09G244800 91 65,04 At3g59950.4 GLYMA_09G244800 99 61,8 At3g59950.5 GLYMA_09G244800 99 58,23 At5g17290.1 GLYMA_14G210200 98 62,57 At5g17290.1 GLYMA_02G240700 98 62,68 At3g61710.1 GLYMA_11G153900 99 74,07 At3g61710.2 GLYMA_11G153900 94 72,8 At3g61710.3 GLYMA_11G153900 92 75,34 At3g61710.4 GLYMA_11G153900 93 74,14 At3g61710.1 GLYMA_04g141000 99 73,68 At5g45900.1 GLYMA_12G010000 98 70,52 At4g21980.1 GLYMA_15G108200 95 86,44 At4g21980.2 GLYMA_17G013000 95 79,55 At4g04620.1 GLYMA_15G108200 95 82,2 At4g04620.2 GLYMA_15G108200 95 82,2 At4g04620.3 GLYMA_15G108200 95 82,2 At4g04620.1 GLYMA_15G188600 56 74,29 At1g62040.1 GLYMA_12G098400 99 91,6 At1g62040.2 GLYMA_12G098400 88 91,6 At1g62040.1 GLYMA_06G306300 99 90,76 At1g62040.1 GLYMA_09G003900 97 88,89 At1g62040.1 GLYMA_07G261000 99 88,03 At2g05630.1 GLYMA_12G098400 99 90,83 At2g05630.2 GLYMA_12G098400 66 90,83 At2g45170.1 GLYMA_17G140700 92 84,21 At2g45170.2 GLYMA_17G140700 92 84,21 At4g16520.1 GLYMA_17G140700 95 93,16 At4g16520.2 GLYMA_17G140700 95 93,16 At4g16520.3 GLYMA_17G140700 75 88,04 At3g60640.1 GLYMA_17G140700 95 83,76 At3g06420.1 GLYMA_02G008800 94 68,14 Supplemental Material 133 At3g15580.1 GLYMA_02G008800 97 90,83 At2g31260.1 GLYMA_13G122200 99 84,21 At2g31260.1 GLYMA_03G162100 99 64,88 At2g31260.1 GLYMA_19G163500 99 64,88 At3g07525.1 GLYMA_03G097000 96 84,21 At3g07525.2 GLYMA_03G097000 96 93,16 At4g30790.1 GLYMA_17G071400 99 93,16 At1g54210.1 GLYMA_07G038100 94 88,04 At1g54210.2 GLYMA_16G007300 90 83,76 At1g54210.3 GLYMA_07G038100 94 68,14 At3g13970.1 GLYMA_07G038100 98 90,83 At3g13970.2 GLYMA_16G007300 79 71,68 At3g13970.3 GLYMA_16G007300 82 65,87 At3g13970.4 GLYMA_07G038100 85 65,89 At3g49590.1 GLYMA_14G187000 98 54,55 At3g49590.2 GLYMA_14G187000 98 53,64 At3g49590.3 GLYMA_14G187000 98 62,23 At3g18770.1 GLYMA_05G189000 96 90,22 AT1G77890.1 GLYMA_13G085400 96 50,22 AT1G77890.2 GLYMA_13G085400 96 49,12 AT1G77890.3 GLYMA_13G085400 96 50,22 AT4G08540.1 GLYMA_14G167200 99 70,53 At5g50230.1 GLYMA_05G043700 99 61,97 At5g50230.1 GLYMA_17G126200 99 73,43 At3g62770.1 GLYMA_20G235800 88 90,22 At3g62770.3 GLYMA_20G235800 87 82,98 At3g62770.1 GLYMA_10G152500 89 72,94 At3g62770.1 GLYMA_03G212100 99 68,94 At3g62770.1 GLYMA_19G209200 79 73,76 At4g30510.1 GLYMA_17G070200 99 74,07 At4g30510.2 GLYMA_17G070200 99 71,43 At2g40810.1 GLYMA_06G140400 99 78,67 At2g40810.2 GLYMA_06G140400 99 50,08 At2g40810.3 GLYMA_06G140400 99 50,08 At2g40810.1 GLYMA_10g126200 89 68,94 At2g40810.1 GLYMA_04g224300 96 73,76 At2g40810.1 GLYMA_07g203900 18 65,22 At3g56440.1 GLYMA_06G140400 97 48,42 At3g56440.2 GLYMA_06G140400 96 56,26 At3g56440.3 GLYMA_06G140400 93 73,57 At5g05150.1 GLYMA_06G140400 97 73,82 At5g05150.1 GLYMA_16g109400 60 25,43 At5g54730.1 GLYMA_13G287000 88 74,43 At5g54730.2 GLYMA_13G287000 89 70,65 At5g54730.1 GLYMA_12g214600 91 44,23 At5g54730.1 GLYMA_12g136000 88 43,33 At5g54730.1 GLYMA_06g267000 88 42,5 At1g03380.1 GLYMA_03G148700 86 75 At1g03380.1 GLYMA_19g152000 86 58,47 At1g03380.1 GLYMA_20g230900 75 56,93 At1g54710.1 GLYMA_10G157700 98 71,71 At1g54710.2 GLYMA_10G157700 99 71,71 AT5G66930.1 GLYMA_17G180900 87 82,01 AT5G66930.2 GLYMA_17G180900 100 76,71 AT5G66930.3 GLYMA_17G180900 81 75,96 Supplemental Material S3. Percentage of legume ATG homologs in different softwares. Bar graph showing the P. vulgaris (Red bar), M. truncatula (Orange bar), G. max (Pink bar) results using BLAST, EGGNOG, ENSEMBL, HMMER, INPARANOID,and KEGG. Supplemental Material 134 Supplemental Material S4 List of accession numbers of ATG (A) genes, (B) transcripts and (C)proteins in P. vulgaris (A) Gene accession numbers Gene Length Gene Chromosome Primary Identifier Gene location Gene location Phvul.007G210800 1251 Chr07 33282212 33283462 Phvul.006G149640 1933 Chr06 25471813 25473745 Phvul.011G103300 2352 Chr11 11510662 11513013 Phvul.010G130300 2465 Chr10 41141057 41143521 Phvul.003G079300 2947 Chr03 12725555 12728501 Phvul.001G205000 3005 Chr01 46312575 46315579 Phvul.007G196400 3036 Chr07 31976845 31979880 Phvul.010G036300 3199 Chr10 5365166 5368364 Phvul.003G219600 3412 Chr03 44794445 44797856 Phvul.002G062200 3611 Chr02 7317826 7321436 Phvul.003G207100 3740 Chr03 43273987 43277726 Phvul.006G173700 4372 Chr06 27671872 27676243 Phvul.010G120500 4567 Chr10 40105482 40110048 Phvul.011G006500 4613 Chr11 472854 477466 Phvul.008G187800 4725 Chr08 52535454 52540178 Phvul.009G041700 4840 Chr09 8502323 8507162 Phvul.002G269600 5044 Chr02 44011076 44016119 Phvul.011G010700 5130 Chr11 817643 822772 Phvul.008G048900 5411 Chr08 4270236 4275646 Phvul.007G194300 5775 Chr07 31618092 31623866 Phvul.011G140900 6185 Chr11 36027897 36034081 Phvul.003G152800 6378 Chr03 36768348 36774725 Phvul.005G091300 6464 Chr05 23812386 23818849 Phvul.001G146700 6471 Chr01 39328927 39335397 Phvul.003G153800 6786 Chr03 36865951 36872736 Phvul.005G029900 7212 Chr05 2766598 2773809 Phvul.007G183100 7327 Chr07 30276041 30283367 Phvul.001G159900 7414 Chr01 41311908 41319321 Phvul.003G248000 7507 Chr03 48513916 48521422 Phvul.008G241000 7959 Chr08 58970503 58978461 Phvul.008G169200 9652 Chr08 47325764 47335415 Phvul.008G088100 10302 Chr08 8689853 8700154 Phvul.003G295800 11590 Chr03 53263303 53274892 Phvul.010G015100 14023 Chr10 2253323 2267345 Phvul.008G087800 15146 Chr08 8649887 8665032 Phvul.002G049900 31655 Chr02 4626523 4658177 (B) Gene accession numbers Transcript accession numbers Transcript Gene Length Phvul.001G146700 Phvul.001G146700.1 3938 Phvul.001G146700 Phvul.001G146700.2 3459 Phvul.001G146700 Phvul.001G146700.3 3989 Phvul.001G159900 Phvul.001G159900.1 3211 Phvul.001G159900 Phvul.001G159900.2 3108 Phvul.001G159900 Phvul.001G159900.3 3744 Phvul.001G159900 Phvul.001G159900.4 2972 Phvul.001G159900 Phvul.001G159900.5 3173 Phvul.001G159900 Phvul.001G159900.6 3457 Phvul.001G159900 Phvul.001G159900.7 2658 Phvul.001G205000 Phvul.001G205000.1 1718 Phvul.002G062200 Phvul.002G062200.1 762 Phvul.002G062200 Phvul.002G062200.2 888 Phvul.002G269600 Phvul.002G269600.1 2262 Phvul.003G079300 Phvul.003G079300.1 777 Phvul.003G079300 Phvul.003G079300.2 629 Phvul.003G152800 Phvul.003G152800.1 1735 Phvul.003G152800 Phvul.003G152800.2 1732 Phvul.003G153800 Phvul.003G153800.1 4725 Phvul.003G207100 Phvul.003G207100.1 2169 Phvul.003G207100 Phvul.003G207100.2 1997 Phvul.003G219600 Phvul.003G219600.1 743 Phvul.003G248000 Phvul.003G248000.1 1198 Phvul.003G295800 Phvul.003G295800.2 6545 Phvul.003G295800 Phvul.003G295800.3 6535 Phvul.003G295800 Phvul.003G295800.4 6413 Phvul.005G029900 Phvul.005G029900.1 1810 Phvul.005G091300 Phvul.005G091300.1 3077 Phvul.005G091300 Phvul.005G091300.2 3159 Phvul.005G091300 Phvul.005G091300.3 2997 Phvul.006G149640 Phvul.006G149640.1 944 Phvul.006G149640 Phvul.006G149640.2 837 Phvul.006G173700 Phvul.006G173700.1 1534 Phvul.007G183100 Phvul.007G183100.1 3428 Phvul.007G194300 Phvul.007G194300.1 2728 Phvul.007G196400 Phvul.007G196400.1 1651 Phvul.007G210800 Phvul.007G210800.1 654 Phvul.008G048900 Phvul.008G048900.1 2194 Phvul.008G048900 Phvul.008G048900.2 2140 Phvul.008G048900 Phvul.008G048900.3 2014 Phvul.008G087800 Phvul.008G087800.1 5007 Phvul.008G088100 Phvul.008G088100.1 4269 Supplemental Material 135 Phvul.008G088100 Phvul.008G088100.2 2714 Phvul.008G088100 Phvul.008G088100.3 3401 Phvul.008G169200 Phvul.008G169200.1 1930 Phvul.008G169200 Phvul.008G169200.2 2489 Phvul.008G187800 Phvul.008G187800.1 2667 Phvul.008G187800 Phvul.008G187800.3 2743 Phvul.008G187800 Phvul.008G187800.4 2619 Phvul.008G187800 Phvul.008G187800.5 2470 Phvul.008G241000 Phvul.008G241000.1 1336 Phvul.009G041700 Phvul.009G041700.1 2026 Phvul.010G015100 Phvul.010G015100.2 2578 Phvul.010G015100 Phvul.010G015100.3 2530 Phvul.010G015100 Phvul.010G015100.4 2576 Phvul.010G036300 Phvul.010G036300.1 1173 Phvul.010G120500 Phvul.010G120500.1 2185 Phvul.010G120500 Phvul.010G120500.2 2127 Phvul.010G130300 Phvul.010G130300.1 602 Phvul.011G006500 Phvul.011G006500.1 1341 Phvul.011G006500 Phvul.011G006500.2 1329 Phvul.011G010700 Phvul.011G010700.1 2459 Phvul.011G103300 Phvul.011G103300.1 848 Phvul.011G103300 Phvul.011G103300.2 863 Phvul.011G140900 Phvul.011G140900.1 3507 (C) Protein accession numbers Protein accession numbers Protein length Isoelectric point Proteins Molecular Weight Phvul.010G120500.1 627 6.34 70430.22 Phvul.010G120500 Phvul.010G120500.2 477 5.59 53617.62 Phvul.010G015100 Phvul.010G015100.2 733 6.16 81386.04 Phvul.010G015100.3 717 6.49 79717.22 Phvul.010G015100.4 655 6.25 72675.94 Phvul.003G295800 Phvul.003G295800.2 1977 5.43 217499.88 Phvul.003G295800.3 1977 5.43 217499.88 Phvul.003G295800.4 1933 5.32 212448.85 Phvul.011G006500 Phvul.011G006500.1 314 4.73 35345.69 Phvul.011G006500.2 310 4.73 34932.21 Phvul.008G048900 Phvul.008G048900.1 489 5.45 53395.03 Phvul.008G048900.2 489 5.45 53395.03 Phvul.008G048900.3 397 4.98 43725.09 Phvul.008G241000 Phvul.008G241000.1 349 4.79 39237.55 Phvul.005G029900 Phvul.005G029900.1 489 5.91 55623.84 Phvul.011G010700 Phvul.011G010700.1 700 5.67 77256.47 Phvul.003G079300 Phvul.003G079300.1 119 7.92 13755.77 Phvul.003G079300.2 119 7.92 13755.77 Phvul.011G103300 Phvul.010G103300.1 120 8.78 13891.19 Phvul.010G103300.2 120 8.78 13891.19 Phvul.003G219600 Phvul.003G219600.1 123 7.85 14165.28 Phvul.002G062200 Phvul.002G062200.1 131 7.85 15086.34 Phvul.002G062200.2 131 7.85 14973.18 Phvul.007G210800 Phvul.007G210800.1 122 6.73 14135.17 Phvul.001G159900 Phvul.001G159900.1 857 6.35 98197.83 Phvul.001G159900.2 857 6.35 98197.83 Phvul.001G159900.3 857 6.35 98197.83 Phvul.001G159900.4 857 6.35 98197.83 Phvul.001G159900.5 857 6.35 98197.83 Phvul.001G159900.6 857 6.35 98197.83 Phvul.001G159900.7 733 6.64 84490.12 Phvul.007G194300 Phvul.007G194300.1 873 6.24 101816.55 Phvul.010G036300 Phvul.010G036300.1 239 5.75 27706.34 Phvul.003G153800 Phvul.003G153800.1 1153 5.69 130516.48 Phvul.010G130300 Phvul.010G130300.1 94 9.25 10536.18 Phvul.008G187800 Phvul.008G187800.1 593 8.71 20572.81 Phvul.008G187800.3 593 8.71 20572.81 Phvul.008G187800.4 593 8.71 20572.81 Phvul.008G187800.5 590 8.89 65397.9 Phvul.002G269600 Phvul.002G269600.1 625 8.83 69262.82 Phvul.003G207100 Phvul.003G207100.1 514 6.1 56511.95 Phvul.003G207100.2 514 6.1 56511.95 Phvul.007G196400 Phvul.007G196400.1 380 8.09 42023.77 Phvul.003G152800 Phvul.003G152800.1 359 8.86 38887.62 Phvul.003G152800.2 358 8.86 38800.54 Phvul.009G041700 Phvul.009G041700.1 422 8.6 46963.54 Phvul.005G091300 Phvul.005G091300.1 889 6.53 97215.94 Phvul.005G091300.2 889 6.53 97215.94 Phvul.005G091300.3 865 6.79 94521.68 Phvul.001G146700 Phvul.001G146700.1 975 5.39 106417 Phvul.001G146700.2 978 5.36 106730.35 Phvul.001G146700.3 758 6.7 82592.35 Phvul.011G140900 Phvul.011G140900.1 925 646 100644.54 Phvul.007G183100 Phvul.007G183100.1 907 5.68 98293.52 Supplementary Material 136 Supplemental Material S5. Identification of ATG8 in 13 legumes Supplemental Material S6. Identification of ATG18 proteins in 15 plants Supplementary Material 137 Supplemental Material S7. Sequence and structure of ATG9a (Phvul.001G159900) (A) DNA sequence obtained in Phytozome. Green Highlight:5’UTR, GreenBlue; Highlight:5’UTR , Green Exons; Pink Highlight:5’UTR 3’UTR (B)CDS structure of 7 t structures of ATG9a designed in GSDS.v2: Dark blue boxes: CDS; Lines: Introns; Dark green boxes: upstream /downstream. (C) Protein sequence features carried out by HMMER. Green boxes: Pfam domain; Purple boxes: disorder regions obtained by IUPred; Red boxes: Transmembranal region and signal peptide obtained by Phoibus. Supplementary Material 138 Supplemental Material S8. Sequence and structure of ATG9a (Phvul.007G194300) (A) DNA sequence obtained in Phytozome. Green Highlight:5’UTR, Blue; Highlight: Exons; Pink Highlight: 3’UTR (B)CDS structure of 7 t structures of ATG9a designed in GSDS.v2: Dark blue boxes: CDS; Lines: Introns; Dark green boxes: upstream /downstream. (C) Protein sequence features carried out by HMMER. Green boxes: Pfam domain; Purple boxes: disorder regions obtained by IUPred; Red boxes: Transmembranal region and signal peptide obtained by Phobius. Supplemental Material S9. Oligonucleotides sequences pb Forward Reverse ATG9 promotor 1080pb 5´-CAC CAG TTT CCT TAT CTG TTG TTG ATG-3´ 5´-GTT AAA CAT TTT TCA GAC AGA AGA CAA TTG G -3´ ATG9 - iRNA 369pb 5´-CAC CAT AGA AGT CAA CCC CGG ATT G-3´ 5´-CAG TCA GTG CTT GAA TTT ACA GTG GGA- 3´ ATG9 localization 2613pb 5´-CAC CAT GTT TAA CTG GCC AAG AGA-3´ 5´-CTA GGGGGGGCTGCAATAAACA-3´ M13 300pb 5´-GTA AAA CGA CGG CCA G-3´ 5´-CAG GAA ACA GCT ATG AC-3´ Forward Reverse ATG9 qRT-PCR 5´-CCA GGA CCC TTG AGT TGG CTT TA-3´ 5´-TCA GAA AGA GAT GTC CCA GCA TG-3´ ATG2 qRT-PCR 5´-CAA CAC AAT GCT TGC ACG GTG A-3´ 5´-GTG CTA CCA TTG TTC AAA GGT GA-3´ ATG8i qRT-PCR 5´-GCG ATC TGC CTG AGT TGG AG-3´ 5´-CAG TTT GAG GCA AGG TAT TCT TCA-3 ATG10 qRT-PCR 5´-TGG GCA ACT ATT GCC GCT CAA-3´ 5´-CAT CCA TTC ACT CGT ACC ACA TGG-3´ ATG18g.II qRT-PCR 5´-TGAGCATGACACCCCCACCTCC-3´ 5´-ACA GCA GAA ACA GCA CCA GAT GG-3´ PvNIN qRT-PCR 5′-GGGGATTCAGAGATTTGCAG-3′ 5′-AACCCACTCTTGAGCATCGT-3′ PvENOD40 qRT-PCR 5′-AGTTTTGTTGGCAAGCATCC-3′ 5′-TAAGCACAAGCAAACTGTTG-3′ PvERN1 qRT-PCR 5′-GGAGCTGTCTTTGATCGTTTTCC-3′ 5′-CAAATTCAGAAAGCTCCAAGTCAGC-3′ Aquaporina 5´-CGC CGC TGT TTG AGC CCT CG-3´ 5´-TTG CGC ATC GTT TGG CAT CG-3´ Metalloprotease 5´-TGA CCC GTC CTA CAC ATG AGC T-3´ 5´- CCC CAA CCT CGG TGG GAA CAC-3´ Supplementary Material 139 Supplemental Material S10. Thermocycling program for PCR and qRT-PCR ATG9 promotor Initial denaturation of 4 minute, 94 °C hot start to activate the Taq, followed by 31 cycles: 45 seconds at 94 °C (denaturation), 45 seconds at 55 °C (annealing) and 2 min at 72 °C (elongation); followed by 2 min at 72 °C (extension) PvATG9 localization Initialization of 4 minute, 94 °C followed by 31 cycles: 45 seconds at 94 °C (denaturation), 45 seconds at 55 °C (annealing) and 2 min at 72 °C (elongation); followed by 2 min at 72 °C (extension) SiATG9 Initialization of 3 minute, 95 °C followed by 34 cycles: 30 seconds at 95 °C (denaturation), 45 seconds at 56 °C (annealing) and 50 seconds at 72 °C (elongation); followed by 10 min at 72 °C (extension) Aquaporin Initialization of 3 minute, 94 °C followed by 35 cycles: 30 seconds at 94 °C (denaturation), 30 seconds at 60 °C (annealing) and 1 min at 72 °C (elongation); followed by 3 min at 72 °C (extension) qPCR Initialization of 3 minute, 95 °C followed by 40 cycles: 45 seconds at 95 °C (denaturation), 45 seconds at 58 °C (annealing) and 1´50” at 72 °C (elongation); followed by 7 min at 72 °C (extension) Supplemental Material S11. Map of vectors used in GATEWAY cloning (Invitrogen). Left vector was used as entry vector. Right vector was used as destination vector in plant construction of PvATG9 promoter. Supplementary Material 140 Supplemental Material S12.Supplemental Figure S5. Map of vectors used in GATEWAY cloning (Invitrogen). Left vector was used to RNAi construction. Right vector was used to obtain the overexpression construction (Earley et al., 2006; Valdés-López et al., 2008). Supplemental Material S13.Cloning reactions 1ul Salt solution 4.5 ul PCR product (after GenEluteTPlasmit Miniprep kit de Sigma) 0.5ul pENTR/D TOPO vector Incubate at room over night (22°C) Supplemental Material S14. Bacteria and plant transformations. A. Transformation with E.coli Top10 1. Defrost 100ul of Top10 competent cells and put on ice for 1 min and centrifuge. 2. Add 4ul of the cloning reaction to a tube and incubate for 30 min on ice 3. Pulse at 42°C for 50 sec and incubate on ice for 4 min 4. Incubate in SOC medium for 1 hour at 37°C and centrifuge and incubate with antibiotic plates overnight at 37°C B. Transformation with Agrobacterium K599 1. Defrost Agrobacteriarium Rhizogenesis K599 and add 3ul of the final plasmid 2. Put on electroporation cuvette and incubate for 30 min on ice 3. Put the electroporation cuvette in electroporator (1.8 kV, 25 μF, and 200 Ω) 4. Incubate in 500ml of SOC medium and shake for 2hrs at 28°C 5. Plate on LB-Spe100 and grow at 28°C for 2 days- Supplementary Material 141 Supplemental Material S15. (Lirua-Bertani) liquid and solid medium. Reactivo Volumen total (500ml) Tryptone 1g Yeast extract 0.5g Sodium Chloride 1g dH2O Adjust the final volumen Agar (Only solid medium) 1.5g Supplemental Material S16. PY liquid Reactivo Volumen total (100ml) Peptone 0.5g Yeast extract 0.3g dH2O Adjust the final volumen • PY medium to Wild type strain: 100ul-NAI (20ng/ml),100ul- Rifampicin (10mg/ml), 700ul-CaCl2, 500ul-Rhizobium • PY medium to GFP strain: 100ul-NAI (20ng/ml),100ul- Rifampicin (10mg/ml), 200ul-Tetracyclin(10mg/ml), 700ul-CaCl2, 500ul-Rhizobium • PY medium to GUS strain: 100ul-NAI (20ng/ml),100ul- Kanamycin (100mg/ml), 700ul-CaCl2, 500ul-Rhizobium GUS Supplemental Material S17. B&D nutrient solution composition (Broughton & Dilworth, 1971) STOCK Final molarity (uM) FORM A Ca 1,000 CaCl2 · 2H2O B P 500 KH2P04 C Fe 10 Fe‐citrate D Mg 250 MgSO4 · 7H2O K 1,500 K2SO4 S 500 Mn 1 MnSO4 · H2O B 2 H3BO4 Zn 0.5 ZnSO4 · 7H2O Cu 0.2 CuSO4 · 5H2O Co 0.1 CoSO4 · 7H2O Mo 0.1 Na2MoO4 · 2H2O Supplemental Material 18S. Cloning of PvATG9b: (A) cDNA, Aquaporine oligonucleotides (B) Promotor Amplificated. (C)Plasmid pENTR with M13 oligonucleotides. (D) Plasmid pBGWF7.0 with the promotor of PvATG9b A B C D Supplementary Material 142 Supplemental Material S19. Cloning of PvATG9b Silencing. (A) Fragment Amplificated (B) Fragment entry vector pENTR/D-TOPO (C) Plasmid PtdT with the promotor of PvATG9b A B C Supplemental Material S20. Over expression and localization isolated fragment to entry vector. (A) Fragment Amplificated of PvATG9b (B) Isolated fragment to entry vector pENTR/D-TOPO (C)Colonies in final vector. (D)Localization A B C D Supplemental Material S21. GUS essay (Jefferson, 1987) Supplemental Material S22. T- test of PvATG9b silencing roots Reactivo Volumen total(10ml) Molaridad final KH2PO4 0.615 ml 100mM KHPO4 0.385 ml 100mM EDTA 200 ul 10mM Triton X 10ul 0.1% K3Fe(CN)6 50ul 0.5mM KFe4(CN)6 50ul 0.5mM X-glux 100ul 1mM dH2O 8.59ul - tstudent PvATG9b- RNAi Vs EV Length of root Root weight Total weight Lenght of Internode Primary root Secondary root Tertiary root White node Pink node Green node t 1.692 0.82277 1.2668 1.6911 0.071 2.3697 0.41874 -2.408 -1.4217 -1.6785 df 14.205 14.148 13.295 17.808 9.6814 14.941 16.651 19 19 18 p-value 0.1125 0.4243 0.2797 0.1082 0.9448 0.0317 0.6808 0.02637 0.1713 0.1105 95 percent confidence interval: -1.509581 -0.4703962 -0.8507861 -0.2989 -2.204126 -3.17378 -39.34097 -4.1123 -1.2360907 -0.71105051 95 percent confidence interval: 12.86514 1.0568406 2.7147861 2.75579 2.3486 60.159551 58.78542 - 0.2877309 0.2360907 0.07947156 mean EV 22.27778 1.212222 3.090 3.444444 3.222222 66.6667 86.22222 0.0 0.0 mean iatg9b 16.60000 0.919000 2.158 2.216000 3.15 35.00000 76.50000 2.2 0.5 0.3157895 Supplementary Material 143 Supplemental Material S23. T- test of PvATG9b-RNAi leaves Supplemental Material S24. T-test of Overexpression of PvATG9b roots Supplemental Material S25 T-test of PvATG9b Overexpression roots tstudent PvATG9b-RNAi Vs ctrl LEAVES length Width t 2.781 3.0443 df 14.022 19.1999 p-value 0.0147 0.006617 95 percent confidence interval: 0.169329 0.1735141 95 percent confidence interval: 1.310353 0.9353748 mean EV 4.850952 2.883333 men iatg9b 4.111111 2.328889 tstudent PvATG9b- OE Vs Control-OE LEAVES length Weight t -6.2658 -4.0219 df 35.534 36.983 p-value .0003242 0.0002735 95 percent confidence interval: -1.853519 -1.7277443 95 percent confidence interval: -0.946732 -0.5701004 mean OeATG9b 6.309649 4.320351 mean OEctrl 4.909524 3.171429 tstudent OE PvATG9b Vs OE control Length of root Root weight Total weight Lenght of Internode Primary root Secondary root Tertiary root White node Pink node Green node t -0.77804 -1.451 -1.7237 -0.25109, -3.3609 -2.8603 -1.7866 -1.0702 -1.571 0.14572 df 18.656 13.949, 12.493 14.403, 12.628 11.36 18.213 18.999 18.227 17.928 p-value 0.4463 0.1689 0.1094 0.8053 0.005305 0.01506 0.09066 0.2979 0.1334 0.8858 95 percent confidenc e interval: - 9.695419 - 2.1887866 - 5.0816313 -1.850988 -8.817586 -162.76576 -196.65335 - 23.809746 63.52996 8 - 5.965301 95 percent confidenc e interval: 4.44541 9 0.422675 5 0.581631 3 1.462099 -1.904636 -21.51201 15.82001 7.698635 9.141079 6.85419 0 mean OECTRL 21.3333 3 1.464444 2.656667 3.555556 3.888889 92.77778 163.0000 10.77778 16.88889 5.111111 men OEATG9 23.95833 2.347500 4.906667 3.750000 9.250000 184.91667 253.416 18.83333 44.08333 4.666667 Supplementary Material 144 Supplemental Material 26 ANOVA of PvATg9b overexpression leaves Supplemental Material S27 PvATg9 phenotype: Silencing and Overexpression plants of 35 days. (A)Pots, (B)Roots, (C)Leaves, (D) Length and Width of leaves. Scale Bar: A & B: 7cm; C:3cm A B C D x1 Length of root Root weight Total weight Lenght of Internode Primary root Secondary root Tertiary root White node Pink node Green node MEAN CTRLOE 20.90909 1.397273 2.903636 3.54546 4.2727 90.54545 159.63636 13.81818 0.00000 0 MEAN EV 22.27778 1.212222 3.09 3.44444 3.2222 66.66667 86.22222 0 0.5 NA MEAN IATG9B 16.6 0.919 2.158 2.216 3.15 32.426 76.5 2.2 10.77778 5.11111 MEAN OEATG9B 24.95000 2.598 5.085000 3.800000 9.900000 205.82500 275.20000 52.90000 18.83333 4.66667 Sum of Squares phenotypefnl$x1 529.1304 19.28849 57.5438 23.63598 339.93 204574.9 293013.4 19668.76 29931.74 260.298 Deg. of Freedom phenotypefnl$x1 3 3 3 3 3 3 3 3 3 3 Sum of Squares Residuals 2867.49 62.24151 319.243 152.872 411.19 139805 357122.7 29931.7 3088.998 937.661 Deg. of FreedomResiduals 46 46 46 46 46 46 46 46 46 45 Residual standard error: 7.895361 1.163218 2.6344 1.82299 2.989792 55.1294 88.11094 25.5086 6084.222 4.56475 hipotesis "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" [1] "Si FValue < tablas se rechaza HO en alfa" fvalue 2.82942 [1] 4.751763 [1] 2.763846 [1] 2.370734 [1] 12.67622 [1] 22.43702 12.58075 10.0759 7.784829 4.16406 tablas 2.806845 [1] 2.806845 [1] 2.806845 [1] 2.806845 [1] 2.806845 [1] 2.806845 2.806845 2.80685 2.806845 2.80685 Supplementary Material 145 Supplemental Material S28 PvATG9b-interacting partners PvATG9b Phvul.007g194300 XP_007144916.1 PHAVU_007G194300g 1 Phvul.001g009100 XP_007160694.1 PHAVU_001G009100g 2 Phvul.001g103600 XP_007161854.1 PHAVU_001G103600g 3 Phvul.001g108101 XP_007161914.1 PHAVU_001G108101g 4 Phvul.002g249800 - PHAVU_002G249800g 5 Phvul.002g282500 XP_007159971.1 PHAVU_002G282500g 6 Phvul.002g324300 XP_007160464.1 PHAVU_002G324300g 7 Phvul.003g054600 XP_007153665.1 PHAVU_003G054600g 8 Phvul.004g026900 XP_007151210.1 PHAVU_004G026900g 9 Phvul.004g102800 XP_007152105.1 PHAVU_004G102800g 10 Phvul.005g096700 XP_007149760.1 PHAVU_005G096700g 11 Phvul.005g172400 XP_007150677.1 PHAVU_005G172400g 12 Phvul.006g125700 XP_007147450.1 PHAVU_006G125700g 13 Phvul.006g203200 - PHAVU_006G203200g 14 Phvul.007g053500 XP_007143213.1 PHAVU_007G053500g 15 Phvul.007g150800 XP_007144373.1 PHAVU_007G150800g 16 Phvul.007g162300 XP_007144514.1 PHAVU_007G162300g 17 Phvul.008g290800 XP_007142557.1 PHAVU_008G290800g 18 Phvul.009g042900 XP_007136412.1 PHAVU_009G042900g 19 Phvul.009g210564 XP_007136240.1 PHAVU_009G210564g 20 Phvul.009g236600 XP_007138778.1 PHAVU_009G236600g 21 Phvul.010g095300 XP_007135022.1 PHAVU_010G095300g 22 Phvul.011g033650 XP_007131689.1 PHAVU_011G033650g 23 Phvul.011g048200 XP_007131870.1 PHAVU_011G048200g 24 Phvul.011g065900 XP_007132090.1 PHAVU_011G065900g Supplemental Material S29 Protein features of PvATG9b-interacting partners Number of amino acids Molecular weight Theoretical pI Total number of negatively charged residues (Asp + Glu): Total number of positively charged residues (Arg + Lys) Total number of atoms Instability index Aliphatic index Grand average of hydropathicity (GRAVY) Phvul.001G009100.1.p 136 15406.07 11.15 11 31 2228 38.38 83.38 -0.602 Phvul.001G103600.1.p 493 54450.42 7.46 33 34 7790 32.9 119.47 0.726 Phvul.001G108101.1.p 493 54450.42 7.47 33 34 7789 32.65 119.86 0.727 Phvul.002G249800.1.p 358 39144.06 5.8 45 36 5588 32.86 98.52 -0.017 Phvul.002G282500.1.p 923 102634.89 6.52 139 135 14579 46.56 93.07 -0.361 Phvul.002G324300.1.p 358 39144.08 5.18 45 36 5588 27.56 98.52 -0.017 Phvul.003G054600.1.p 290 32141.22 8.32 44 46 4495 38.5 67.97 -0.694 Phvul.004G026900.1.p 281 31871.54 6.2 38 35 4442 63.39 70.39 -0.447 Phvul.004G102800.1.p 628 70469.23 8.86 48 55 9960 48.85 94.27 0.055 Phvul.005G096700.2.p 644 72365.62 9.13 83 96 10127 49.41 70.84 -0.74 Phvul.005G172400.1.p 360 39694.18 5.44 49 40 5516 52.74 77.17 -0.196 Phvul.006G125700.1.p 542 60286.89 8.36 53 56 8521 30.78 92.18 -0.092 Phvul.006G203200.1.p 37 3959.65 4.22 4 1 568 0.08 134.59 1.078 Phvul.007G053500.1.p 199 21823.07 4.82 16 13 3065 31.94 96.98 0.101 Phvul.007G150800.1.p 412 46428.98 6.12 55 51 6582 40.22 99.78 -0.092 Phvul.007G162300.1.p 382 42272.56 9.49 35 50 5955 32.32 83.77 -0.379 Phvul.008G290800.1.p 473 51716.32 8.82 40 47 7289 54.35 89.24 -0.079 Phvul.009G042900.1.p 368 41062.9 9.21 52 66 5791 28.84 71.01 -0.721 Phvul.009G210564.1.p 339 37983.41 9.25 31 43 5321 52.79 74.84 -0.242 Phvul.009G236600.1.p 893 99108.96 4.94 127 86 13717 57.49 75.91 -0.585 Phvul.010G095300.1.p 293 32296.33 6.55 35 34 4601 22.14 100.1 -0.07 Phvul.011G033650.1.p 317 39195.27 7.62 41 42 5082 45.24 79.84 -0.491 Phvul.011G048200.1.p 578 66355.97 5.65 93 76 9130 52.98 69.83 -0.561 Phvul.011G065900.1.p 358 41447.9 7.11 36 36 5863 53.47 99.44 0.013 Supplementary Material 146 Supplemental Material S30 Transmembrane domains of PvATG9b-interacting partners Phvul.001g18101.1 (No. 3), Phvul.004G102800.1 (No. 9), Phvul.006G125700.1 (No. 12), Phvul.006g203200.1 (No. 13), Phvul.007g0053500.1 (No. 14), Phvul.008G290800.1 (No. 17). Phvul.001g18101.1 (No. 3) Phvul.004G102800.1 (No. 9) Phvul.006G125700.1 (No. 12) Phvul.006g203200.1 (No. 13) Phvul.007g0053500.1 (No. 14) Phvul.008G290800.1 (No. 17) Supplemental Material S31 PvATG9(XP_007144916.1) network and summary statistics Summary statistics Number of nodes 11 Number of edges 55 Avg. Node degree 10 Avg. Local clustering coefficient 1 Expected number of edges 12 PPI enrichment p value 1,677 Supplementary Material 147 Supplemental Material S32 PvATG9b-interacting partners in String data node1 node2 node1 annotation node2 annotation coexpr. score XP_007131401.1 XP_007135446.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Ubiquitin-like protein atg12; Ubiquitin-like protein involved in cytoplasm to vacuole transport (Cvt) and autophagic vesicle formation 0.051 XP_007131401.1 XP_007141345.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_008G1878001m 0.093 XP_007131401.1 XP_007144916.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Autophagy-related protein 9; Involved in autophagy and cytoplasm to vacuole transport (Cvt) vesicle formation. Plays a key role in the organization of the preautophagosomal structure/phagophore assembly site (PAS), the nucleating site for formation of the sequestering vesicle 0.127 XP_007131401.1 XP_007148972.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Hypothetical protein; Uncharacterized protein; Encoded by transcript PHAVU_005G0299001m 0.053 XP_007131401.1 XP_007152965.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Phosphoinositide-3-kinase, regulatory subunit 4; Uncharacterized protein; Encoded by transcript PHAVU_004G1751001m 0.150 XP_007131401.1 XP_007154849.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Autophagy-related protein 18; Uncharacterized protein; Encoded by transcript PHAVU_003G1528001m 0.601 XP_007131401.1 XP_007156552.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Autophagy-related protein 2; Uncharacterized protein; Encoded by transcript PHAVU_003G2958001m 0.072 XP_007131401.1 XP_007157437.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Phosphatidylinositol 3-kinase; Belongs to the PI3/PI4-kinase family 0.157 XP_007131401.1 XP_007159813.1 Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_002G2696001m 0.093 XP_007135446.1 XP_007131401.1 Ubiquitin-like protein atg12; Ubiquitin-like protein involved in cytoplasm to vacuole transport (Cvt) and autophagic vesicle formation Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy 0.051 XP_007135446.1 XP_007148972.1 Ubiquitin-like protein atg12; Ubiquitin-like protein involved in cytoplasm to vacuole transport (Cvt) and autophagic vesicle formation Hypothetical protein; Uncharacterized protein; Encoded by transcript PHAVU_005G0299001m 0.045 XP_007135446.1 XP_007155481.1 Ubiquitin-like protein atg12; Ubiquitin-like protein involved in cytoplasm to vacuole transport (Cvt) and autophagic vesicle formation Next to brca1 gene 1 protein; Uncharacterized protein; Encoded by transcript PHAVU_003G2050001m 0.044 XP_007135446.1 XP_007157437.1 Ubiquitin-like protein atg12; Ubiquitin-like protein involved in cytoplasm to vacuole transport (Cvt) and autophagic vesicle formation Phosphatidylinositol 3-kinase; Belongs to the PI3/PI4-kinase family 0.047 XP_007141345.1 XP_007131401.1 Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_008G1878001m Ubiquitin-like modifier-activating enzyme atg7; E1-like activating enzyme involved in the 2 ubiquitin-like systems required for autophagy 0.093 XP_007141345.1 XP_007144916.1 Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_008G1878001m Autophagy-related protein 9; Involved in autophagy and cytoplasm to vacuole transport (Cvt) vesicle formation. Plays a key role in the organization of the preautophagosomal structure/phagophore assembly site (PAS), the nucleating site for formation of the sequestering vesicle 0.743 XP_007141345.1 XP_007148972.1 Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_008G1878001m Hypothetical protein; Uncharacterized protein; Encoded by transcript PHAVU_005G0299001m 0.044 XP_007141345.1 XP_007152965.1 Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_008G1878001m Phosphoinositide-3-kinase, regulatory subunit 4; Uncharacterized protein; Encoded by transcript PHAVU_004G1751001m 0.058 XP_007141345.1 XP_007154849.1 Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_008G1878001m Autophagy-related protein 18; Uncharacterized protein; Encoded by transcript PHAVU_003G1528001m 0.085 XP_007141345.1 XP_007155481.1 Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_008G1878001m Next to brca1 gene 1 protein; Uncharacterized protein; Encoded by transcript PHAVU_003G2050001m 0.042 XP_007141345.1 XP_007156552.1 Autophagy-related protein 13; Uncharacterized protein; Encoded by transcript PHAVU_008G1878001m Autophagy-related protein 2; Uncharacterized protein; Encoded by transcript PHAVU_003G2958001m 0.050 Supplemental Material S33 PvATG9b network including the 24 interacting partners Summary statistics Number of nodes 241 Number of edges 734 Avg. Number of neighbors 6.286 Network diameter 8 Network radius 4 Chracteristic path lenght 3.756 Clustering coefficient 0.756 Network density 0.27 Network heterogenity 0.584 Network centralization 0.104 Connected components 5 Supplementary Material 148 Supplemental Material S34 . Homologs in A. thaliana of PvATG9b-interacting partners Species Type Orthologue Target %id Query %id GOC Score WGA Coverage High Confidence Phvul.001G009100.1 n n n n n n n n Phvul.001G103600.1 Arabidopsis thaliana Many-to-many DTX23 (AT1G33080) 46.56 % 58.67 % n/a 0 No Phvul.001G103600.1 Arabidopsis thaliana Many-to-many DTX22 (AT1G33090) 47.57 % 59.95 % n/a 95.16 Yes Phvul.001G103600.1 Arabidopsis thaliana Many-to-many DTX20 (AT1G33100) 48.47 % 60.71 % n/a 6.02 No Phvul.001G103600.1 Arabidopsis thaliana Many-to-many DTX21 (AT1G33110) 49.19 % 61.99 % n/a 0 No Phvul.002G282500.1 Arabidopsis thaliana Many-to-many CLPC2 (AT3G48870) 81.51 % 84.07 % n/a 94.75 Yes Phvul.002G282500.1 Arabidopsis thaliana Many-to-many CLPC1 (AT5G50920) 86.98 % 87.54 % n/a 10.09 No Phvul.002G324300.1 Arabidopsis thaliana Many-to-many RPS16A (AT2G09990) 75.34 % 74.83 % n/a 100 Yes Phvul.002G324300.1 Arabidopsis thaliana Many-to-many RPS16B (AT3G04230) 71.92 % 71.43 % n/a 96 Yes Phvul.002G324300.1 Arabidopsis thaliana Many-to-many RPS16C (AT5G18380) 75.34 % 74.83 % n/a 100 Yes Phvul.003G054600.1 Arabidopsis thaliana Many-to-many EIF3G1 (AT3G11400) 64.49 % 71.38 % n/a 99.37 Yes Phvul.003G054600.1 Arabidopsis thaliana Many-to-many EIF3G2 (AT5G06000) 59.42 % 63.10 % n/a 98.3 Yes Phvul.004G026900.1 Arabidopsis thaliana Many-to-many PCO1 (AT5G15120) 54.61 % 56.94 % n/a 0 No Phvul.004G026900.1 Arabidopsis thaliana Many-to-many PCO2 (AT5G39890) 59.42 % 58.36 % n/a 97.82 Yes Phvul.004G102800.1 Arabidopsis thaliana 1-to-many SLAH3 (AT5G24030) 52.91 % 53.50 % n/a 74.68 Yes Phvul.005G096700.2 Arabidopsis thaliana 1-to-many GTE4 (AT1G06230) 41.78 % 49.38 % n/a 70.82 Yes Phvul.005G172400.1 Arabidopsis thaliana Many-to-many AT1G15670 47.35 % 47.22 % n/a 0 No Phvul.005G172400.1 Arabidopsis thaliana Many-to-many AT1G80440 48.59 % 47.78 % n/a 100 Yes Phvul.006G125700.1 Arabidopsis thaliana Many-to-many SEC1A (AT1G01980) 60.26 % 60.15 % n/a 97.7 Yes Phvul.006G125700.1 Arabidopsis thaliana Many-to-many AT1G11770 61.38 % 60.70 % n/a 97.72 Yes Phvul.006G125700.1 Arabidopsis thaliana Many-to-many AT1G30740 55.60 % 54.98 % n/a 0 No Phvul.006G125700.1 Arabidopsis thaliana Many-to-many AT4G20830 57.89 % 60.89 % n/a 95.84 Yes Phvul.006G125700.1 Arabidopsis thaliana Many-to-many AT4G20840 63.64 % 63.28 % n/a 0 No Phvul.006G203200.1 Arabidopsis thaliana 1-to-1 OST4A (AT3G12587) 81.08 % 81.08 % n/a 0 No Phvul.007G053500.1 Arabidopsis thaliana Many-to-many AT3G06035 54.50 % 54.77 % n/a 71.42 Yes Phvul.007G053500.1 Arabidopsis thaliana Many-to-many AT5G19230 43.39 % 41.21 % n/a 0 No Phvul.007G053500.1 Arabidopsis thaliana Many-to-many AT5G19240 40.20 % 40.20 % n/a 0 No Phvul.007G053500.1 Arabidopsis thaliana Many-to-many AT5G19250 54.08 % 53.27 % n/a 0 No Phvul.007G150800.1 Arabidopsis thaliana 1-to-many ACR10 (AT2G36840) 67.32 % 66.99 % n/a 100 Yes Phvul.007G162300.1 Arabidopsis thaliana 1-to-many PBL7 (AT5G02800) 69.84 % 69.11 % n/a 100 Yes Phvul.008G290800.1 n n n n n n n n Phvul.009G042900.1 Arabidopsis thaliana 1-to-1 TFIIS (AT2G38560) 49.21 % 50.54 % n/a 99.82 Yes Phvul.009G210564.1 n n n n n n n n Phvul.009G236600.1 Arabidopsis thaliana Many-to-many AT2G19240 44.02 % 41.66 % n/a 67.7 Yes Phvul.009G236600.1 Arabidopsis thaliana Many-to-many AT4G29950 50.00 % 46.36 % n/a 84.68 Yes Phvul.009G236600.1 Arabidopsis thaliana Many-to-many AT5G57210 49.25 % 40.65 % n/a 0 No Phvul.010G095300.1 n n n n n n n n Phvul.011G033650.1 n n n n n n n n Phvul.011G048200.1 Arabidopsis thaliana 1-to-many ARI2 (AT2G16090) 65.43 % 67.13 % n/a 93.54 Yes Phvul.011G048200.1 Arabidopsis thaliana 1-to-many ARI3 (AT3G27710) 56.98 % 52.94 % n/a 84.25 Yes Phvul.011G048200.1 Arabidopsis thaliana 1-to-many ARI4 (AT3G27720) 53.14 % 45.33 % n/a 72.59 Yes Phvul.011G048200.1 Arabidopsis thaliana 1-to-many ARI1 (AT4G34370) 62.48 % 64.53 % n/a 85.4 Yes Phvul.011G065900.1 Arabidopsis thaliana 1-to-1 AT5G59960 74.65 % 74.86 % n/a 97.33 Yes PUBLICATIONS AND MATERIALS OBTEINED • Scientific publication o Elsa H Quezada-Rodríguez, Homero Gómez-Velasco, Manoj-Kumar Arthikala, Miguel Lara, Antonio Hernández-López, Kalpana Nanjareddy Exploration of autophagy families in legumes and dissection of the ATG18 family with a special focus on P. vulgaris o Quezada, E. H., Arthikala, M. K., & Nanjareddy, K. (2022). Cytoskeleton in abiotic stress signaling. In Mitigation of Plant Abiotic Stress by Microorganisms (pp. 347-371). Academic Press. • Awards o Travel Grant Award to Attend Plant Biology-ASPB 2020 o Best Oral Presentation Award in Recent Advances in Chemical and Biological Sciences (VIRACBS), 2020. • Congress oral presentations o Overexpression of PvATG9 increases nodule number but affects root nodule maturation during P. vulgaris-Rhizobium tropici interaction. 2nd Latino American conference on natural and applied sciences held on April 5-7,2022. Colombia Bogotá o ATG2-ATG18 Complex During Rhizobia and Mycorrhizal Infection Process in Legumes. The Virtual Conference on Recent Advances in Chemical and Biological Sciences. DAYANANDA SAGAR COLLEGE. India, 2020. • Congress poster o Analysis of the three subfamilies of ATG18-autophagy protein and particular focus on PvATG18b in P. vulgaris (Common bean) APFED. Germany, 2022 o Understanding autophagy genes role during Rhizobia infection process in legumes. Plant Biology 2021 – ASPB. Virtual, host in US,2021 o Expression pattern of ATG9 during the symbiotic interaction between Rhizobium and beans. XVIII National Congress of Biotechnology and Bioengineering. Mexico, 2019 o Understanding the ATG18 autophagy family proteins in plants of agronomic interest. VI Latin American Protein Society Meeting. Mexico, 2019 o Identification of mycorrhizal symbiosis specific autophagy genes under TOR signal disruption in common bean. XXXII Biochemistry National Congress. Mexico, 2018. o “Analisis de expression de la familia de genes ATG en frijol durante la simbiosis con rizobia bajo la regulación de TOR” First meeting InterENES. Mexico, 2018 • Attending Congress and workshops o Target of rapamycin (TOR) signaling in photosynthetic organism. Host: Germany,2021 o Bioconductor Virtual Conference 2021. Host: Japan, 2021. o UseR!2021- virtual. The R foundation of statistical computing. Host: Austria, 2021. o Bioinformatic skills. Software Carpentry. Mexico,2021 o International Plant System Biology. EMBO virtual Workshop. Host: Germany, 2021. o 4to simposio Internacional de Bioinformática. Instituto Nacional de Salud Pública. Morelos, 2019. 150 PUBLICATION EXPLORATION OF AUTOPHAGY FAMILIES IN LEGUMES AND DISSECTION OF THE ATG18 FAMILY WITH A SPECIAL FOCUS ON Phaseolus vulgaris plants Article Exploration of Autophagy Families in Legumes and Dissection of the ATG18 Family with a Special Focus on Phaseolus vulgaris Elsa-Herminia Quezada-Rodríguez 1 , Homero Gómez-Velasco 2, Manoj-Kumar Arthikala 1, Miguel Lara 3, Antonio Hernández-López 1 and Kalpana Nanjareddy 1,*   Citation: Quezada-Rodríguez, E.-H.; Gómez-Velasco, H.; Arthikala, M.-K.; Lara, M.; Hernández-López, A.; Nanjareddy, K. Exploration of Autophagy Families in Legumes and Dissection of the ATG18 Family with a Special Focus on Phaseolus vulgaris. Plants 2021, 10, 2619. https:// doi.org/10.3390/plants10122619 Academic Editors: Olga V. Voitsekhovskaja and Cecilia Gotor Received: 7 September 2021 Accepted: 3 November 2021 Published: 29 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores Unidad León, Universidad Nacional Autónoma de México (UNAM), León C.P. 37684, Mexico; qrelsa@gmail.com (E.-H.Q.-R.); manoj@enes.unam.mx (M.-K.A.); ahernandez@enes.unam.mx (A.H.-L.) 2 Instituto de Química, Universidad Nacional Autónoma de México (UNAM), Cuidad Universitaria, Cuidad de Mexico C.P. 04510, Mexico; antropofagomer@hotmail.com 3 Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca C.P. 62271, Mexico; mflara@ibt.unam.mx * Correspondence: kalpana@enes.unam.mx; Tel.: +52-477-1940800 (ext. 43462) Abstract: Macroautophagy/autophagy is a fundamental catabolic pathway that maintains cellular homeostasis in eukaryotic cells by forming double-membrane-bound vesicles named autophago- somes. The autophagy family genes remain largely unexplored except in some model organisms. Legumes are a large family of economically important crops, and knowledge of their important cellu- lar processes is essential. Here, to first address the knowledge gaps, we identified 17 ATG families in Phaseolus vulgaris, Medicago truncatula and Glycine max based on Arabidopsis sequences and elucidated their phylogenetic relationships. Second, we dissected ATG18 in subfamilies from early plant lineages, chlorophytes to higher plants, legumes, which included a total of 27 photosynthetic organisms. Third, we focused on the ATG18 family in P. vulgaris to understand the protein structure and developed a 3D model for PvATG18b. Our results identified ATG homologs in the chosen legumes and differential expression data revealed the nitrate-responsive nature of ATG genes. A multidimensional scaling analysis of 280 protein sequences from 27 photosynthetic organisms classified ATG18 homologs into three subfamilies that were not based on the BCAS3 domain alone. The domain structure, protein motifs (FRRG) and the stable folding conformation structure of PvATG18b revealing the possible lipid-binding sites and transmembrane helices led us to propose PvATG18b as the functional homolog of AtATG18b. The findings of this study contribute to an in-depth understanding of the autophagy process in legumes and improve our knowledge of ATG18 subfamilies. Keywords: homologs; phylogeny; ATG18; FRRG motif; principal component; 3D model; expres- sion profile 1. Introduction Autophagy is a degradation process essential in the maintenance of homeostasis in eukaryotic cells and is related to a wide variety of physiological and pathophysio- logical roles, such as host defense, development, infection, and tumorigenesis [1,2]. Au- tophagy/macroautophagy is a process in which cytosolic components are sequestered within double-membrane vesicles called autophagosomes, which fuse with lysosomes or vacuoles for degradation/recycling [3]. This process is mediated by evolutionarily conserved genes known as autophagy genes (ATGs) [4], which were originally discovered in and isolated from Saccharomyces cerevisiae [5–8]. Three major intracellular autophagy pathways, namely, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), have been elucidated, and these differ in the mode of cargo delivery to the lyso- some or vacuole [9,10]. Macroautophagy can be nonselective or selective: Nonselective autophagy is a cellular response to nutrient deprivation that involves the random uptake of Plants 2021, 10, 2619. https://doi.org/10.3390/plants10122619 https://www.mdpi.com/journal/plants Plants 2021, 10, 2619 2 of 34 cytoplasm into phagophores (precursors to autophagosomes) [11], and selective autophagy is responsible for the specific removal of certain components, such as protein aggregates and damaged or superfluous organelles [12,13]. Selective autophagic degradation has been observed with several organelles, such as mitochondria [14], peroxisomes [15], lyso- somes [16], endoplasmic reticulum and nucleus [17]. In contrast, microautophagy is the least characterized type of autophagy; during this nonselective process, smaller molecules acting as substrates and the cargo for degradation are transferred into vacuole via invagi- nation of the tonoplast membrane. CMA involves molecular chaperones in the cytosol that unfold proteins and translocate them through the lysosomal membrane [18]. Research on plant autophagy has improved enormously since the first genetic analysis of plant autophagy was performed [19–24]. During the process of autophagy, ATG genes play a key role and are classified into several functional groups: The ATG1 kinase complex, the ATG9 recycling complex, the phosphatidylinositol 3-kinase (PI3K) complex and the ATG8 and ATG12 conjugation systems [12]. Autophagy/macroautophagy can be activated under nutrient-depletion conditions via the inhibition of mammalian target of rapamycin (mTOR) or the activation of AMPK. Under TOR-inhibiting conditions, ATG13 is rapidly dephosphorylated, which results in its association with ATG1 and the additional proteins ATG11 and ATG101 and thus stimulation of the autophagy process [25,26]. Phagophore expansion is driven by the transmembrane protein ATG9 along with its cycling factors ATG2 and ATG18 [27,28]. Furthermore, assem- bly of the phagophore is completed with phosphatidylinositol-3-phosphate (PI3P) by a complex containing class III phosphatidylinositol-3-kinase (PI3K), vacuolar protein sorting 34 (VPS34), ATG/VPS30/beclin-1, VPS38, ATG14 and VPS15 [28]. Phagophore expansion and maturation are completed by ATG8, which is cleaved by cysteine proteinase ATG4 to expose the C-terminal glycine residue [29]. Subsequently, the exposed glycine of ATG8 is conjugated to the membrane lipid phosphatidylethanolamine (PE) via a ubiquitin-like conjugation reaction catalyzed by ATG7 (E1-like enzyme), ATG3 (E2-like enzyme) and the ATG12-ATG5 complex (E3-like enzyme) [30–32]. The ATG8-PE adduct can be deconjugated from the membrane by ATG4 proteinase; hence, ATG8 is recycled to participate in new conjugation events [29,33]. ATG18 is an autophagy-related molecule that regulates the vacuolar shape and is conserved from yeast to higher organisms, including the human proteins WIPI1–WIPI4 [34]. While yeast has only one ATG18 gene and two other genes with WD40 repeats, the plant ATG18 family diversifies from two genes in algae to multiple genes in higher plants. The Atg18 protein is characterized by the presence of several WD-40 domains and has been predicted to form a β-propeller structure that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) [35–37]. The bind- ing of PtdIns(3)P and Atg18 is needed for the efficient recruitment of Atg8 and Atg16 during phagophore formation at the phagophore assembly site (PAS) [38]. A previous study showed that phagophore formation could also be affected in the absence of the Atg2- Atg18 complex, although other Atg proteins accumulate at the PAS [39]. The Atg2-Atg18 complex has also been shown to localize to a few specific spots on the opening edge of the isolation membrane that lie close to sites for COPII vesicle formation in the endoplasmic reticulum (ER) or ER exit sites [40,41]. Among plants, Arabidopsis contains eight ATG18 homologs, which are classified as AtATG18a–h, and multiple splice variants [42,43], and rice has six ATG18 homologs. AtATG18a is involved in oxidative, drought and salt stress [42–45]. Recent studies have also suggested the regulation of autophagy by the reversible persulfidation of AtATG18a under ER stress [46]. Similarly, ATG18 is reportedly involved in autophagy regulation under abiotic stress conditions in sweet orange (Citrus sinensis) [47], tomato (Solanum lycop- ersicum) [48] and apple (Malus domestica) [49,50]. To date, AtATG18a is the only member of the ATG18 family that has been established as an essential component of autophagy in A. thaliana. Plants 2021, 10, 2619 3 of 34 Recent studies on ATG genes conducted by Norizuki and colleagues (2019) [51] have shown the diversification of ATGs from early plant lineages to higher plants. However, legumes are a large and economically important family of flowering plants, and few studies have investigated autophagy-related aspects. The aim of the present study was to expand the previous studies to higher clades, specifically to fabaceous plants, and thus understand the current diversity and complexity of ATGs. Furthermore, we focused on the ATG18 family to understand its evolutionary relationships, diversification, expression patterns and cis-regulatory elements in many plants ranging from early plant lineages to fabaceous members. We also performed a comprehensive study of various functional and structural aspects of ATG18b in P. vulgaris. 2. Results 2.1. Identification of ATG families in P. vulgaris, M. truncatula and G. max In A. thaliana, a total of 39 ATG sequences divided into 17 families have been reported. In the present study, we identified a total of 32 genes in P. vulgaris (2n), 39 genes in M. trun- catula (2n) and 61 genes in G. max (4x) (Table 1). A BLAST analysis of Arabidopsis sequences returned 19 (59.37%) homologs in P. vulgaris, 28 (77.77%) homologs in M. truncatula and 30 (48.38%) homologs in G. max with a query coverage of 93–94% and 66–77% identity (Supplementary Information SI1). For this reason, other ortholog analysis databases were used to identify any missing ATG members. The KEGG orthology table for the autophagy pathway was the second main tool because it contains a wide variety of species, and we used this table to obtain more than 70% of genes in P. vulgaris and M. truncatula and 58% in G. max. An analysis of legumes using Ensembl Plants provided more than 70% of ATGs in the legumes under study. Other studies were performed through a HMMER analysis using Ensembl databases and the InParanoid tools in Phytozome. The obtained sequences were verified using Pfam to acquire the positions of the families, domains and repeats, and the protein motifs were determined with MEME. Additional studies were performed using EggNOG, which provided a list of orthologs, particularly in P. vulgaris (Supplementary Figure S1). We also identified 21, 17 and 15 orthologs and 10, 17 and 21 paralogs in P. vulgaris, M. truncatula and G. max, respectively. The genes identified in P. vulgaris, M. truncatula and G. max are listed in Table S1. Plants 2021, 10, 2619 4 of 34 Table 1. List of 17 autophagy gene families in A. thaliana, P. vulgaris, M. truncatula and G. max. Arabidopsis thaliana Phaseolus vulgaris Medicago truncatula Glycine max Complex Family Name ID Name ID Name ID Name ID In it ia ti o n o f au to p h ag y ATG1 complex ATG1 AtATG1a At3g61960 MtATG1a Medtr8g024100 GmATG1a.I Glyma.07g048400 GmATG1a.II Glyma.16g017300 AtATG1b At3g53930 PvATG1b Phvul.010g015100 MtATG1b Medtr4g019410 GmATG1b.I Glyma.03g069800 AtATG1c At2g37840 GmATG1b.II Glyma.01g099600 AtATG1t At1g49180 PvATG1t Phvul.010g120500 MtATG1t Medtr3g095620 GmATG1t.I Glyma.06g150700 GmATG1t.II Glyma.04g215500 ATG11 AtATG11 At4g30790 PvATG11 Phvul.003g153800 MtATG11 Medtr4g130370 GmATG11 Glyma.17g071400 ATG13 AtATG13 At3g49590 PvATG13a Phvul.008g187800 MtATG13a Medtr5g068710 GmATG13a.I Glyma.02g220700 GmATG13a.II Glyma.14g187000 AtATG13b At3g18770 PvATG13b Phvul.002g269600 MtATG13b Medtr3g095570 GmATG13b.I Glyma.05g189000 MtATG13c Medtr8g093050 GmATG13b.II Glyma.08g146700 ATG101 AtATG101 At5g66930 PvATG101 Phvul.003g248000 MtATG101 Medtr8g079240 GmATG101 Glyma.17g180900 M em b ra n e re cr u it m en t to au to p h ag o so m ez Complex ATG2-ATG18 ATG9 AtATG9 At2g31260 PvATG9a Phvul.001g159900 MtATG9a Medtr7g096680 GmATG9a.I Glyma.03g162100 GmATG9a.II Glyma.19g163500 PvATG9b Phvul.007g194300 MtATG9b Medtr1g070160 GmATG9b.III Glyma.10g035800 GmATG9b.vI Glyma.13g122200 ATG2 AtATG2 At3g19190 PvATG2 Phvul.003g295800 MtATG2 Medtr4g086370 GmATG2.I Glyma.02g133400 GmATG2.II Glyma.07g211600 ATG18 AtATG18a At3g62770 PvATG18a Phvul.001g205000 MtATG18a Medtr1g083230 GmATG18a.I Glyma.10g152500 GmATG18a.II Glyma.20g235800 GmATG18a.III Glyma.03g212100 GmATG18a.Iv Glyma.19g209200 AtATG18b At4g30510 PvATG18b Phvul.003g152800 MtATG18b Medtr4g130190 GmATG18b.I Glyma.17g070200 GmATG18b.II Glyma.02g207500 GmATG18b.III Glyma.10g126200 AtATG18c At2g40810 PvATG18c.I Phvul.009g041700 MtATG18c Medtr7g108520 GmATG18c.I Glyma.04g224300 PvATG18c.II Phvul.007g196400 GmATG18c.II Glyma.06g140400 AtATG18d At3g56440 MtATG18d Medtr1g088855 AtATG18e At5g05150 MtATG18e Medtr3g093590 GmATG18e Glyma.16g109400 AtATG18f At5g54730 PvATG18f.I Phvul.011g140900 MtATG18f Medtr2g082770 GmATG18f.I Glyma.12g214600 PvATG18f.II Phvul.005g091300 GmATG18f.II Glyma.12g136000 GmATG18f.III Glyma.13g287000 GmATG18f.IV Glyma.06g267000 AtATG18g At1g03380 PvATG18g.I Phvul.001g146700 MtATG18g Medtr1g089110 GmATG18g.I Glyma.03g148700 PvATG18g.II Phvul.007g183100 GmATG18g.II Glyma.19g152000 GmATG18g.III Glyma.20g230900 AtATG18h At1g54710 MtATG18h Medtr1g082300 GmATG18h Glyma.10g157700 Plants 2021, 10, 2619 5 of 34 Table 1. Cont. Arabidopsis thaliana Phaseolus vulgaris Medicago truncatula Glycine max Complex Family Name ID Name ID Name ID Name ID A u to p h ag o so m e fo rm at io n ATG6 AtATG6 At3g61710 PvATG6 Phvul.005g029900 MtATG6 Medtr3g018770 GmATG6.I Glyma.11g153900 GmATG6.II Glyma.04g141000 PI3K complex ATG14 AtATG14a At1g77890 PvATG14 Phvul.008g169200 MtATG14 Medtr5g061040 GmATG14.I Glyma.13g085400 GmATG14.II Glyma.14g167200 AtATG14b At4g08540 U b iq u it in -l ik e p ro te in co n ju g at io n sy st em s Ubiquitin-like conjugation (ATG8) ATG3 AtATG3 At5g61500 PvATG3 Phvul.011g006500 MtATG3 Medtr4g036265 GmATG3.I Glyma.12g005700 GmATG3.II Glyma.09g231000 AtATG4a At2g44140 PvATG4a Phvul.008g048900 MtATG4a Medtr7g081230 GmATG4a.I Glyma.18g248400 ATG4 GmATG4a.II Glyma.09g244800 AtATG4b At3g59950 ATG7 AtATG7 At5g45900 PvATG7 Phvul.011g010700 MtATG7 Medtr0003s0540 GmATG7 Glyma.12g010000 ATG8 AtATG8a At4g21980 MtATG8a Medtr2g023430 AtATG8b At4g04620 MtATG8b Medtr4g037225 GmATG8b Glyma.15g188600 AtATG8c At1g62040 PvATG8c.I Phvul.003g079300 MtATG8c Medtr4g048510 GmATG8c.I Glyma.12g098400 PvATG8c.II Phvul.006g149640 GmATG8c.II Glyma.06g306300 GmATG8c.III Glyma.09g003900 GmATG8c.IV Glyma.17g013000 GmATG8c.V Glyma.07g261000 GmATG8c.VI Glyma.15g108200 AtATG8d At2g05630 PvATG8d Phvul.011g103300 MtATG8d Medtr2g088230 AtATG8e At2g45170 MtATG8e Medtr4g101090 AtATG8f At4g16520 PvATG8f.I Phvul.003g219600 MtATG8f Medtr1g086310 GmATG8f Glyma.17g140700 PvATG8f.II Phvul.002g062200 AtATG8g At3g60640 MtATG8g Medtr4g123760 AtATG8h At3g06420 MtATG8h Medtr7g096540 AtATG8i At3g15580 PvATG8i Phvul.007g210800 GmATG8i Glyma.02g008800 ATG5 AtATG5 At5g17290 PvATG5 Phvul.008g241000 MtATG5 Medtr5g076920 GmATG5.I Glyma.14g210200 GmATG5.II Glyma.02g240700 Ubiquitin-like conjugation (ATG12) ATG10 AtATG10 At3g07525 PvATG10 Phvul.010g036300 MtATG10 Medtr8g010140 GmATG10 Glyma.03g097000 ATG12 AtATG12a At1g54210 AtATG12b At3g13970 PvATG12b Phvul.010g130300 MtATG12b Medtr8g020500 GmATG12b.I Glyma.07g038100 GmATG12b.II Glyma.16g007300 ATG16 AtATG16 At5g50230 PvATG16 Phvul.003g207100 MtATG16a Medtr3g075400 GmATG16.I Glyma.05g043700 MtATG16b Medtr4g104380 GmATG16.II Glyma.17g126200 MtATG16c Medtr4g007500 Plants 2021, 10, 2619 6 of 34 2.2. Phylogenetic Relationships, Chromosome Localization, Synteny and Ka/Ks Ratio of ATG Families in Legumes To understand the evolutionary relationships among ATGs, we generated 17 phyloge- netic trees, one for each ATG family in A. thaliana, P. vulgaris, M. truncatula and G. max as per the classification in A. thaliana. The primary protein sequences of A. thaliana, P. vulgaris, M. truncatula and G. max were aligned using Clustal Omega with the default parameters, and phylogenetic trees were obtained with the neighbor-joining method. Each of the ATG sequences was also subjected to a motif analysis, which revealed that the sequences and motifs in all the studied legumes showed high identity to their homologs in Arabidopsis. The phylogenetic tree also revealed that the majority of the ATG family distributions was predominantly composed of Medicago sequences that were more closely related to those in Arabidopsis. Among all the phylogenetic trees of ATGs developed, 11 contained only one clade (ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG10, ATG11, ATG12, ATG14 and ATG101), even if there was more than one isoform, and most of the motif P-values were greater than 1e-100. ATG8 and ATG18 were the families with the highest number of members: ATG18, eight each in Arabidopsis, Medicago and Phaseolus and 19 in G. max; ATG8, nine in Arabidopsis, eight in Medicago, six in P. vulgaris and 10 in G. max. The phylo- genetic analysis of ATG8 and ATG18 was divided into three clades with motif P-values between 1 × 10−13 and 1 × 10−90 (Figure 1). The close association of the homologs in all the species studied depicts the conservation of sequences and hence implies biological function conservation. The chromosome localization of ATGs in the A. thaliana and legume genomes was mapped using Circos (Figure 2). The distribution of ATG homologs among the chromo- somes was uneven in all the species compared. Among all 17 families, the maximal number of homologs was located on chromosome 3 in A. thaliana (8) and P. vulgaris (6), chromosome 4 in M. truncatula (6) and chromosomes 4 and 17 in G. max (6). The Ka/Ks ratio among most of the ATG sequences was lower than 1 (average 0.17), which indicates purifying selection; in contrast, the sequences of ATG8 (1.24) and two sequences (GmATG18e and GmATG18b. I) of ATG18 (1.09 and 1.04) in G. max had values higher than 1, which indicated accelerated evolution and positive selection (Figure 3). The Ka/Ks ratios suggest the conservation of ATG homologs in terms of both sequence and biological function. 2.3. Promoter Analysis and Expression Profiling of ATG Families Promoter analysis is an important method for understanding the regulatory mecha- nisms governing ATGs in response to growth and developmental issues and to environmen- tal cues. The analysis of cis-acting elements in the promoters of all 17 ATG families resulted in 44 different transcription factors. The most abundant transcription factors identified were B-Proto-Oncogene-MYB involved in the ABA response and C-Proto-Oncogene-MYC related to jasmonate signaling, and the transcription factors with the motifs ethylene re- sponse elements (ERE), TATA box, CAATT-box and G-box were found for all ATGs in A. thaliana, P. vulgaris, M. truncatula and G. max (Supplementary Figure S5). Our results also showed that the ATG8 and ATG18 families contained the highest numbers of MYB, MYC, ERE and Box 4 (ATTAAT) transcription factor-binding sites. Most of the promoters contained MeJA-, SA-, GA- and ABA-responsive elements. Furthermore, light-responsive transcription factors such as BOX-4, G-box, GT1 motif, MRE and ACE were also detected abundantly in most of the families (Figure 4). Plants 2021, 10, 2619 7 of 342021, , x FOR PEER REVIEW 7 of 42                                                             ‐               ‐                                                                                                                   ‐ Figure 1. Phylogenetic analysis and protein motifs of 17 ATG families in A. thaliana, P. vulgaris, M. truncatula and G. max. The phylogenetic tree was constructed with the neighbor-joining method with 1000 repeated bootstrap tests, p-distance and pairwise deletion in MEGA X software and visualized using EvolView. MEME was used to identify motifs of the ATG homologs in A. thaliana, P. vulgaris, M. truncatula and G. max. Plants 2021, 10, 2619 8 of 34 2021, , x FOR PEER REVIEW 8 of 42                               ‐                                 ‐                                                                                                                                                                                                                                                                                                                                                                     Figure 2. The chromosomal localization, synteny relationship and gene expression of autophagy genes were integrated into the Circos plot designed using OmicCircos. The outermost circle shows the A. thaliana (blue), P. vulgaris (green), M. truncatula (pink) and G. max (brown) chromosomes. The inner circle is a heatmap that shows the log2 RPKM values of gene expression in leaves and roots under ammonia, nitrate and urea treatments. The innermost line is the synteny of autophagy genes, but the yellow, purple and red lines represent ATG18b subfamilies I, II and III, respectively. Interestingly, we elucidated the influence of nitrogen sources on ATG expression in the legume members P. vulgaris, M. truncatula and G. max due to their ability to establish symbiotic associations with nitrogen-fixing Rhizobia. Gene expression data from the Phytozome database were retrieved for leaf and root tissues under urea as the organic source and nitrate and ammonia as inorganic sources, as depicted in Figure 2. The highest expression of ATGs was recorded in roots treated with ammonia and leaves treated with urea. ATG8i and ATG3 showed the highest abundance in all the treatments, and the lowest expression levels were recorded for ATG18b, e, c and h, ATG2 and ATG2.II in G. max and ATG3 and ATG8c in M. truncatula. The ATG18 family homologs ATG18a.II, ATG18g and ATG18h showed induced expression in all tissues under all treatments (Figures 2 and 5a). Plants 2021, 10, 2619 9 of 342021, , x FOR PEER REVIEW 9 of 42                                                                                                                                                 ‐                         ‐           ‐                                               ‐ ‐ ‐               ‐ ‐ ‐ ‐                                     ‐     ‐                                                                                     ‐           ‐     ‐   ‐   ‐    ‐       ‐ ‐           ‐   ‐                                   Ka/Ks > 1 Figure 3. Ka/Ks ratios of 17 families of ATGs in A. thaliana, P. vulagris, G. max and M. truncatula. The distribution of Ka and Ks values are obtained using TBtools. The dark blue line divides the Ka/Ks ratios lower and higher than 1 (dots in the highlighted area Ka/Ks > 1). 2021, , x FOR PEER REVIEW 10 of 42           ‐                                                                                         ‐               ‐                                                           ‐                                                                                                                                                                                                           ‐                     ‐                       ‐                                                                                                             ‐                           ‐       ‐               ‐                                                                                                                     ‐           ‐               A bu nd an ce o f t ra ns cr ip tio n fa ct or s Figure 4. Transcription factor-binding sites in the promoter regions of ATGs (2000 bp) identified using PlantCARE. Plants 2021, 10, 2619 10 of 34 2021, , x FOR PEER REVIEW 11 of 42                                                                                                               ‐                               ‐                                                                                                                                                                             ‐                                                           ‐                                                                                                                         ‐ a b Figure 5. Expression profiles of ATGs in P. vulgaris tissues. (a) The transcription abundances of P. vulgaris ATGs in different tissues and organs during different stages of development and during rhizobial infections obtained from the PvGEA database. (b) Expression data from nodulated roots (R. tropici) and mycorrhized roots (R. irregularis) obtained from RNA-seq analysis. A violin plot shows total number of up/dowregulated ATGs under nodulated/mycorrhized conditions. The highlighted box represents higher number of downregulated genes in mycorrhized condition. Plants 2021, 10, 2619 11 of 34 Furthermore, the differential expression analysis of ATGs in P. vulgaris tissues showed very low expression in young pods collected 1 to 4 days post floral senescence, whereas the fix-(inefficient) nodules collected at 21 days showed the most abundant expression of all ATGs. Interestingly, inefficient fixation increased the expression levels compared with those found with efficient fixation. Among all PvATGs, the ATG1, ATG10, ATG13b, ATG18c and ATG18g.I genes showed the lowest expression in all the analyzed tissues, and a total of 16 ATGs were found to be expressed in most of the tissues (Figure 5a; Supplementary Information SI2). Following the interesting observation of ATG expression in nodules, we analyzed the expression of ATGs using our previously generated RNA-seq data of Rhizobium/mycorrhiza-inoculated P. vulgaris roots. The results were interesting: Six ATGs were upregulated and 16 ATGs were downregulated in mycorrhized roots, and nine ATGs were upregulated and 12 ATGs were downregulated in nodulated roots (Figure 5b; Supplementary Information SI2). The expression of ATG10 was found to be specifically induced in mycorrhized roots, ATG12 was highly induced and ATG18g.l was highly suppressed under both symbiotic conditions. The RNA-seq data was validated using RT-qPCR for PvATG2, PvATG8i, PvATG9 and PvATG10. 2.4. Identification of ATG18 Families in Plants Through an extensive study aiming to identify and analyze the ATG18 family, we selected 27 plant species starting from the early plant lineage Chlorophyta, Charophyta, liverworts, mosses and higher plants such as monocots and dicots. As with other ATGs, the ATG18 family is also well conserved in all the studied plant species; herein, a total of 280 genes and amino acid sequences were identified and retrieved from various databases. Initially, we identified the ATG18 homologs through a BLAST search of NCBI, and we then used the Pfam database to ensure the presence of WD40 repeats in the characteristic ATG18 members. The identified members were named using the aliases registered in the legume information system, NCBI, Phytozome, InParanoid, EGGNOG and Ensembl (Supplementary Information SI3). The genes with the same names were distinguished by adding a Roman numeral: The number I indicated the closest sequence to that in NCBI. For the primitive plants Physcomitrella patens, Chara braunii, Chlamydomonas reinhardtii, Dunaliella salina, Volvox carteri, Klebsormidium nitens, Micromonas pusilla, Ostreococcus lucimarinus, Ostreococcus tauri and Coccomyxa subellipsoidea, we retained the same names that were reported by Norizuki and colleagues [51]. Starting from the most primitive photosynthetic organisms of Chlorophyta, all the members studied had two ATG18 homologs except C. subellipsoidea, which had three ATG18 genes. Charophyta (C. braunii), liverworts (Marchantia polymorpha) and mosses (P. patens) had two, four and eight genes, respectively. Among monocots, we found that Oryza sativa had the lower number of genes (8), and Z. mays had the highest number of genes (31). Arabidopsis had a total of eight ATG18 members, and the 12 legumes considered here together had a total of 180 genes belonging to the ATG18 family. P. sativum had a minimum of six, and a maximum of 27 genes were found in L. angustifolius. The details of the ATG18 homologs in every species are listed in Tables 2 and 3. Plants 2021, 10, 2619 12 of 34 Table 2. List of ATG18 homologs in early plant lineages. Chlorophyta Charophyta Liverworts Bryophyta Monocots Arabidopsis Dunaliella salina Volvox carteri Ostreococcus tauri OOstreococcus lucimarinus Micromonas pusilla Coccomyxa subellipsoidea Chlamydomonas reinhardtii Chara braunii Klebsormidium nitens Marchantia polymorpha Physcomitrella patens Oryza sativa Zea mays Triticum aestivum Arabidopsis thaliana S u b fa m il y I A DsATG18 (Dusal.0227s00002.1) VcATG18 (Vo- car.0005s0363) OtATG18 (Ot06g00830) OlATG18 (OlATG18.3284. fragment) MpuATG18 (Mpu- ATG1849616) CsubATG18 (CsATG18.65175) CrATG18 (Cre10.g425750.t1) CbATG18 (CHBRA95g00960) KnATG18 (kfl00229.0060) MpoATG18a.I (MARPO.0005s0065) PpATG18 (Ph- pat.005G022700) OsATG18a (XP.015621196) ZmATG18a (Zm00001d011920) TaATG18a.I (CDM86058) AtATG18a (AT3G62770) MpoATG18a.II (MARPO.0001s0033) PpATG18 (Ph- pat.006G095100) TaATG18a.II (AGW81806) PpATG18 (Ph- pat.017G015900) TaATG18a.III (Traes.3B.19AF6BFF0) TaATG18a.IV (TRAES.3B.113DC4275) ZmATG18b.IV (Zm00001d042215.T002) ZmATG18b.V (GRMZM2G143211) C ZmATG18c.I (AQK90439) TaATG18c.I (Traes.3DS.985ED34D7) AtATG18c (AT2G40810) ZmATG18c.II (Zm00001d008691) TaATG18c.II (Traes.3AS.71D103050) ZmATG18c.III (GRMZM2G069177) TaATG18c.III (TraesCS3B02G110900) ZmATG18c.IV (AQK90440) TaATG18c.IV (CDM81498) D OsATG18d.I (XP.015620970) TaATG18d (AGW81809) AtATG18d (AT3G56440) E OsATG18eII (XP.015639564) AtATG18e (AT5G05150) S u b fa m il y II B DsATG18 (Dusal.0460s00003) VcATG18 (Vo- car.0020s0155) OtATG18 (Ot06g00720) OlATG18 (OlATG18.41442. fragment) MpuATg18 (Mpu- ATG18.156491. fragment) CsubATG18 (CsATG18.3880. fragment) CrATG18 (Cre10.g457550) KnATG18 (kfl00404.0130) MpoATG18b (MARPO.0027s0044) PpATG18 (Ph- pat.007G038400) OsATG18b (XP.015627655) ZmATG18b.I (NP_00114563.1) TaATG18b (Traes.6AL.DDF2EBF31) AtATG18b (AT4G30510) ZmATG18b.II (XP.020408852) TaATG18e.I (Traes_6BL_B2A8BBB52) ZmATG18b.III (Zm00001d018355) TaATG18e.II (Traes.6DL.9F29527A0) S u b fa m il y II I F CsubATG18 (CsATG18.63899) CbATG18 (CHBRA141g00400) KnATG18 (kfl00046.0070) MpoATG18f (MARPO.0006s0048) PpATG18 (Ph- pat.008G022700) OsATG18f.I (XP.015621123) ZmATG18f.I (ONM37261) TaATG18f.I (Traes.3B.F4F2FC6FA) AtATG18f (AT5G54730) PpATG18 (Ph- pat.020G070000) OsATG18f.II (XP.025877429) ZmATG18f.II (ONM37262) TaATG18f.II (Traes.3DL.E400E521A) PpATG18 (Ph- pat.023G024100) OsATG18f.III (LOC.Os05g33610) ZmATG18f.III (Zm00001d043239) TaATG18f.III (TraesCS3D02G318200) PpATG18 (Ph- pat.024G018700) ZmATG18f.IV (ONM37265) TaATG18f.IV (CDM84501) ZmATG18f.V (PWZ31673) TaATG18f.V (Traes.3B.7A23DFB41) TaATG18f.VI (Traes.3AL.B27F0D4FF) G ZmATG18g.I (AQK85845) AtATG18g (AT1G03380) ZmATG18g.II (AQK85860) ZmATG18g.III (AQK93836) ZmATG18g.IV (AQK93828) ZmATG18g.V (AQK93834) ZmATG18g.VI (AQK85849) Plants 2021, 10, 2619 13 of 34 Table 2. Cont. Chlorophyta Charophyta Liverworts Bryophyta Monocots Arabidopsis Dunaliella salina Volvox carteri Ostreococcus tauri OOstreococcus lucimarinus Micromonas pusilla Coccomyxa subellipsoidea Chlamydomonas reinhardtii Chara braunii Klebsormidium nitens Marchantia polymorpha Physcomitrella patens Oryza sativa Zea mays Triticum aestivum Arabidopsis thaliana S u b fa m il y II I G ZmATG18g.VII (GRMZM2G078468) ZmATG18g.VIII (PWZ17532) ZmATG18g.IX (AQK93830) ZmATG18g.X (AQK93829) ZmATG18g.XI (AQK93835) ZmATG18g.XII (AQK85856) ZmATG18g.XIII (AQK93827) H ZmATG18h.I (XP.008649626) TaATG18h.I (Traes.1BL.45E2558BB.1) AtATG18h (AT1G54710) OsATG18h (XP.015639663) ZmATG18h.II (PWZ11786) TaATG18h.II (TraesCS1A02G254200.1) ZmATG18h.III (XP.008656294) TaATG18h.III (Traes.1DL.DB75BFD8A.1) TaATG18h.IV (Traes.1AL.C4A651390.1) Table 3. List of ATG18 homologs in legumes. Genestoids Dalbergioids Milletioids Robinioids IRLC Lupinus angustifolius Arachis duranensis Arachis ipaensis Glycine max Vigna angularis Vigna radiata Phaseolus vulgaris Lotus Japonica Cicer arietinum Cajanus cajan Medicago truncatula Pisum sativum Trifolium pratense S u b fa m il y I A LaATG18a.I (XP.019421581.1) AdATG18a.I (XP.015939789.1) AiATG18a (XP.016174738.1) GmATG18a.I (Glyma.10G152500.1) VaATG18a.I (VIGAN03G286700) VrATG18a.I (VRADI08G12430) PvATG18a (Phvul.001G205000.1) CaATG18a.I (XP.004495714.1) CcATG18a.I (XP.020209984.1) MtATG18a (Medtr1G083230.1) PsATG18a (PSAT0S3233G0120.1) TpATG18a.I (TRIPR.GENE96259) LaATG18a.II (XP.019452261.1) AdATG18a.II (XP.015967701.1) GmATG18a.II (Glyma.20G235800.1) VaATG18a.II (XP.017412432.1) VrATG18a.II (VRADI03G05850) CaATG18a.II (XP.004494924.1) CcATG18a.II (C.CAJAN.10296.1) TpATG18a.II (TRIPR.GENE33973) LaATG18a.III (XP.019419463.1) GmATG18a.III (Glyma.03G212100.1) VaATG18a.III (VANG04G16030.1) CaATG18a.III (C.CA.05407.1) CcATG18a.III (XP.020212010.1) TpATG18a.III (PNX79795.1) LaATG18a.IV (XP.019441771.1) GmATG18a.IV (Glyma.19G209200.1) VaATG18a.IV (VANG06G12920.1) LaATG18a.V (XP.019441170.1) LaATG18b.III (TanjilG.02747) LjATG18b.II (Lj5g3v1496760.1) CaATG18b.V (Ca.04089) LjATG18b.III (Lj0g3v0083309.1) CaATG18b.VI (CC4958C.Ca14068.1) LjATG18b.IV (Lj1g3v4912170.1) C LaATG18c.I (XP.019430950.1) AdATG18c (XP.015945005.1) AiATG18c (XP.016181861.1) GmATG18c.I (Glyma.04G224300.1) PvATG18c.I (Phvul.009G041700.1) CaATG18c (C.CA.03673) PsATG18c (PSAT5G069920.1) TpATG18c.I (TRIPR.GENE13965) LaATG18c.II (XP.019417508.1) GmATG18c.II (Glyma.06G140400.1) PvATG18c.II (Phvul.007G196400.1) MtATG18c (Medtr7G108520.1) TpATG18c.II (PNX92525.1) LaATG18c.III (LUP000470) LjATG18c (Lj1G3V1112870.1) D LaATG18d (XP.019430946.1) VaATG18d.I (VIGAN04G120000) VrATG18d.I (VRADI0239S00050) CaATG18d (XP.004502800.1) CcATG18d.I (XP.029129536.1) MtATG18d (Medtr1G088855.1) VaATG18d.II (VANG0200S00330.1) VrATG18d.II (XP.022632145.1) CcATG18d.II (XP.020229011.1) E VrATG18d.VI (XP.022632144.1) MtATG18e (Medtr3G093590.1) Plants 2021, 10, 2619 14 of 34 Table 3. Cont. Genestoids Dalbergioids Milletioids Robinioids IRLC Lupinus angustifolius Arachis duranensis Arachis ipaensis Glycine max Vigna angularis Vigna radiata Phaseolus vulgaris Lotus Japonica Cicer arietinum Cajanus cajan Medicago truncatula Pisum sativum Trifolium pratense S u b fa m il y II LaATG18b.I (XP.019441874.1) AdATG18b (XP.015933286.1) AiATG18b (XP.016200540.1) GmATG18b.I (Glyma.17G070200.1) VaATG18b.I (VIGAN01G240600) VrATG18b.I (VRADI07G21660) PvATG18b (Phvul.003G152800.1) LjATG18b.I (Lj4G3V2018270.1) CaATG18b.I (XP.027192941.1) MtATG18b (Medtr4G130190.1) PsATG18b (PSAT0S2826G0080.1) TpATG18b.I (PNX94509) B* LaATG18b.II (XP.019441865.1 GmATG18b.II (Glyma.02G207500.2) VaATG18b.II (XP.017411081.1) VrATG18b.II (XP.014510099.1) CaATG18b.II (XP.004507771.1) TpATG18b.II (PNY02700.1) GmATG18b.III (Glyma.10G126200.1) VaATG18b.III (XP.017411091.1) CaATG18b.III (XP.027192940.1) VaATG18b.IV (VANG11G12160.2) CaATG18b.IV (ICC4958.CA.21790.1) VaATG18b.V (XP.017411074.1) GmATG18e (Glyma.16G109400.1) * VrATG18d.III (XP.014522590.1) LaATG18f.I (XP.019437124.1) AdATG18f.I (ARADU.XJ3JE.1) AiATG18f.I (XP.016170472.1) GmATG18f.I (Glyma.12G214600.1) VaATG18f.I (VIGAN05G145500) VrATG18f.I (XP.014522059.1) PvATG18f.I (Phvul.011G140900.1) LjATG18f (Lj3G3V1544540.1) CaATG18f.I (XP.004487613.1) CcATG18f.I (XP.020229318.1) MtATG18f (Medtr2G082770.1) PsATG18f (PSAT5G249880.1) TpATG18f (TRIPR.GENE36798) S u b fa m il y II I F LaATG18f.II (XP.019453655.1) AdATG18f.II (XP.015936500.1) AiATG18f.II (ARAIP.FRI7H.1) GmATG18f.II (Glyma.12G136000.1) VaATG18f.II (XP.017425518.1) VrATG18f.II (XP.014494161.1) PvATG18f.II (Phvul.005G091300.1) CaATG18f.II (XP.027187641.1) CcATG18f.II (XP.020229320.1) LaATG18f.III (OIW15456.1) GmATG18f.III (Glyma.13G287000.1) VaATG18f.III (VIGAN08G077000) VrATG18f.III (XP.022634400.1) CaATG18f.III (XP.004487612.1) CcATG18f.III (C.CAJAN32508.1) LaATG18f.IV (XP.019453653.1) GmATG18f.IV. (Glyma.06G267000.1) VaATG18f.IV (VANG1095S00020.1) VrATG18f.IV (VRADI02G09460.1) CaATG18f.IV (CA.00864.1) CcATG18f.IV (XP.020235274.1) CcATG18f.V (XP.020229319.1) CcATG18f.VI (XP.020229316.1) LaATG18g.I (XP.019441802.1) AdATG18g (XP.015951046.1) AiATG18g (XP.016184366.1) GmATG18g.I (Glyma.03G148700.1) VaATG18g.I (XP.017419622.1) VrATG18g (VRADI03G00450) PvATG18g.I (Phvul.001G146700.1) LjATG18g (Lj1G3V4404380.1) CaATG18g.I (CA.09934.1) CcATG18g.I (XP.020211839.1) MtATG18g.I (Medtr1G089110.1) PsATG18g (PSAT6G169560.1) TpATG18g.I (TRIPR.GENE16922) G LaATG18g.II (XP.019441803.1) GmATG18g.II (Glyma.19G152000.1) VaATG18g.II (KOM38883.1) PvATG18g.II (Phvul.007G183100.1) CaATG18g.III (CA.08309) CcATG18g.II (C.CAJAN09614.1) TpATG18g.II (TRIPR.GENE2713) GmATG18g.III (Glyma.20G230900.1) VaATG18g.III (VI- GAN.VANG07G05180.1) CcATG18g.III (KYP70659.1) LaATG18h.I (XP.019421306.1) AdATG18h.I (XP.015939933.1) AiATG18h.I (XP.016205481.1) GmATG18h (Glyma.10G157700.1) VaATG18h.I (KOM55039.1) VrATG18h (VRADI08G12840.1) LjATG18h (Lj5G3V1451080.1) CaATG18h.I (XP.027189075.1) CcATG18h.I (XP.020233978.1) MtATG18h (Medtr1G082300.1) PsATG18h (PSAT6G148560.1) TpATG18h.I (PNY09258.1) H LaATG18h.II (XP.019421307.1) AdATG18h.II (XP.015939934.1) AiATG18h.II (XP.016176031.1) VaATG18h.II (VANG06G10190.1) CaATG18h.II (XP.027189076.1) CcATG18h.II (C.CAJAN06885.1) TpATG18h.II (PNY17060.1) LaATG18h.III (XP.019421305.1) AdATG18h.III (XP.015968551.1) AiATG18h.III (XP.016176030.1) CaATG18h.III (CA.09238.1) CcATG18h.III (XP.020233954.1) TpATG18h.III (PNY12850.1) LaATG18h.IV (XP.019452235.1) AiATG18h.IV (XP.016176032.1) CcATG18h.IV (XP.029125824.1) LaATG18h.V (TANJILG.10103) LaATG18h.VI (OIW07130.1) LaATG18h.VII (XP.019452236.1) LaATG18h.VIII (XP.019452234.1) LaATG18h.IX (OIW12695.1) * Sequence ID with assigned the letter but belongs to other ATG18 Subfamily. Plants 2021, 10, 2619 15 of 34 2.5. Principal Component Analysis of the ATG18 Family Multidimensional scaling analysis using Bios2mds demonstrates the similarity be- tween 280 ATG18 protein sequences from 27 different species. The plot clearly shows that orthologs (genes with closely related sequences and having the same function in different species) are more similar than paralogs (genes that have similar sequences but have differ- ent functions in the same species). The plots show that all ATG18 sequences were grouped into three clusters (Figure 6 and Supplementary Figure S3A). The principal components (PCs) allowed us to construct graphs with PC1, PC2 and PC3, and we then applied the K-means method. Cluster I formed a subfamily with ATG18a, c, d and e members from all the higher plant species studied. Cluster II contained only ATG18b homologs, and cluster III contained ATG18f, g and h members. Cluster III consisted of 3 groups: Lower plants formed a distant group, the second group contained the monocot-derived proteins, and the third group harbored all dicots except Arabidopsis, which was more similar to monocots than dicots. Lower plant species were found to be distributed mostly in clusters I and II with the exception of K. nitens, C. subellipsoidea, M. polymorpha and P. patens, which were also grouped in cluster III but exhibited more similarities among themselves than with higher plants. These clusters were named subfamilies I, II and III for convenience. 2021, , x FOR PEER REVIEW 22 of 42                                   ‐                                                                                 ‐                                                     ‐                               ‐     ‐                                                                                                           ‐                                                                                                                                                           ‐                     ‐                     ‐     ‐                                           ‐     ‐       ‐       Figure 6. Three-dimensional representation of 280 ATG18 proteins from different plant species analyzed by multidimensional scaling using Bios2mds. The ATG18 subfamilies are color-coded as follows: Subfamily I (yellow), subfamily II (purple) and subfamily III (red). PC, principal component. The axes are the principal components (PC): x-axis (PC1), y-axis (PC2) and z-axis (PC3). Plants 2021, 10, 2619 16 of 34 2.6. Phylogenetic Relationships of the ATG18 Family in Plants To understand the evolutionary relationship among primitive and advanced dicot plant species, a multiple sequence alignment of 280 ATG18 amino acid sequences was performed. The aligned sequences were used to generate phylogenetic trees based on the maximum likelihood and Bayes methods using MEGA and Phangorn software (Figure 7 and Supplementary Figure S3B). The largest clade was subfamily III followed by subfamily I, which was mainly composed of ATG18 a, c, d and e. Subfamily II harbored ATG18b. Subfamilies II and III consisted of the Bryopsida, Charophyceae, Klebsormidiophyceae, Mamiellophyceae and Trebouxiophyceae plants, which is important for understanding the divergence of ATG18 homologs. 2021, , x FOR PEER REVIEW 23 of 42                                                                                                                                           ‐                                                                                                                                               ‐                                                                                                                                                                                                                                     Figure 7. Phylogenetic tree of ATG18 proteins in plants. The protein sequences were aligned using Clustal Omega, and the phylogenetic tree was constructed using the ML method in MEGA X software with 1000 bootstrap replications. A total of 280 sequences of ATG18 are differentiated into subfamilies: Subfamily I (yellow), subfamily II (purple) and subfamily III (red). The plant species are differentiated by letters: A. thaliana (At), M. polymorpha (Mpo), O. sativa (Os), Triticum aestivum (Ta), Zea mays (Zm), Arachis duranensis (Ad), Arachis ipaensis (Ai), Cajanus cajan (Cc), Lotus japonicus (Lj), Cicer arietinum (Ca), Lupinus angustifolius (La), Pisum sativum (Ps), Vigna angularis (Va), Vigna radiata (Vr) and Trifolium pratense (Tp), P. patens, C. braunii (Cb), C. reinhardtii (Cr), D. salina (Ds), V. carteri (Vc), K. nitens (Kn), M. pusilla (Mpu), O. lucimarinus (Ol), O. tauri (Ot) and C. subellipsoidea (Cs). The branch lengths are labeled. Plants 2021, 10, 2619 17 of 34 2.7. Analysis of the Primary Structure and the Secondary Structure Predictions of the ATG18 Family in Plants For the detection of motifs in 280 aa sequences, we identified four main motifs using MEME software. Motif 1 (SGVHLYKLRRGATNAVIQDIAFSHDSQWJAISSSKGTVHIF) contained 41 aa, and the motif sequence matched that of the WD40 family (PF00400) and β propeller clan 186 (CL0186) in the Pfam database. The InterProScan results also showed that motif 1 belongs to the superfamily WD40 (IPR036322), WD40 repeat-like (SSF50978) and breast carcinoma amplified sequence 3 (PTHR13268). Motif 2 (VIAQFRAHTSPISALCFDPS- GTLLVTASVHGHNINVFRIMP) contained 41 aa and was similar to motif 1 but contained an additional domain (WD40/YVTN repeat-like domain, IPR015943). Moreover, motifs 3 (VRCSRDRVAVVLATQIYCYBA) and 4 (GYGPMAVGPRWLAYASNPPLLSNTGRLSPQN) did not belong to any protein family (Figure 8). 2021, , x FOR PEER REVIEW 24 of 42                                                                                                           β                                                ‐                       ‐             ‐                     ‐                   ‐                         Su bf am ily I Su bf am ily II Figure 8. Cont. Plants 2021, 10, 2619 18 of 342021, , x FOR PEER REVIEW 25 of 42                                                                                                                                                                                                 ‐                         ‐                                                     ‐                                       ‐                                               Su bf am ily II I Figure 8. Protein motifs of the ATG18b family from different plant species. The conserved motifs were identified with MEME. The amino acid sequence of the ATG18 family is represented by lines, and the motifs identified using TBtools are shown with boxes: Motif 1 (green), motif 2 (yellow), motif 3 (dark green) and motif 4 (pink). The motif sequences were further analyzed with PfamScan to identify the repeats, domains and families. Subfamily I was characterized by motifs 1 and 4, which consisted of WD40 and ANAPC4_WD40 repeats. These motifs also had two domains and eight families, although these Pfam family results are not representative of the subfamily. Subfamily II had motifs 1, 2 and 4, and we detected WD40 and ANAPC4_WD40 repeats in all the members. Only the green alga O. tauri contained leucine-rich repeats (LRR9 and LRR4). A total of four domains were identified: Gel_WD40, which was the largest, a defensin domain and PQQ and SecA preprotein crosslinking domains. Subfamily II also consisted of three families in six plants (Figure 9; Supplementary Information SI4). Plants 2021, 10, 2619 19 of 342021, , x FOR PEER REVIEW 26 of 42                     ‐                                                                                                                           ‐                                                   ‐                                                           ‐                                                             ‐       ‐                  β‐               ‐       ‐                                                                                                 ‐                                                                                                                                         ‐                       Figure 9. Repeats, domains and families of ATG18b sub-families. (a) The ATG18 protein functions were determined using Pfam, and the proteins were divided into subfamilies: Subfamily I (yellow), subfamily II (purple) and subfamily III (red). (b) Pfam identifiers and their annotations. Subfamily III had all four motifs, and we found PD40 repeats along with WD40 and ANAPC4_WD40 repeats. Among the 27 plant species analyzed, nine of them had 12 domains and ATP synthase was specific Z. mays. Breast carcinoma amplified sequence 3 (BCAS3) is a characteristic domain found in most members (Figure 9; Supplementary Information SI4). The secondary structure of ATG18 was determined by protein alignment using JPred software. Here, we found that the sequence of ATG18h in A. thaliana was the largest sequence in the alignment with 927 aa. The protein contains seven blades with four beta blades commonly found in the WD40 family (Supplementary Figure S4). This sequence composition was 1% alpha-helix (H), 29% beta-sheet and 68% coil. ATG18 sequences have four antiparallel β-strands, which are named blades [52]. The beta-sheets in ATG18 proteins contain flexible loops that facilitate molecule binding. AtATG18h has an LHRG sequence in the same place where the alignments have the FRRG sequence, and we found the BCAS3 domain with Phe17 (Figure 10). The sequence alignment performed to identify the FRRG motif revealed that FRRGs appeared in subfamily II, which consists of ATG18b. In addition, subfamily I contained the LRRG or VRRG sequences, whereas subfamily III contained LQRG, LHRG or LYRG sequences. The sequences that appear in ATG18 contain the same pattern of two polar and neutral amino acids in the center of the sequence between two neutral and nonpolar amino acids. ATG18b in subfamily II has the conserved sequence for PtdInsP binding, and other subfamilies likely also show PtdInsP binding (Figure 10, Supplementary Figure S4). Plants 2021, 10, 2619 20 of 34 2021, , x FOR PEER REVIEW 27 of 42           ‐                       ‐                                    β‐               ‐             ‐                 ‐                                                                                               ‐                                                             ‐                                                                                                                             ‐                               ‐               a  b  c  d  Figure 10. Three-dimensional structural model of PvATG18b determined by molecular dynamics simulation and alignment of ATG18 protein sequences of P. vulgaris. (a) The PvATG18b protein structure preserves seven blades of four β-strands. (a–d) In the colored rainbow, the N-terminus is shown in blue, the C-terminal is shown in red, the FRRG repeat (F218-G221) is colored pink, the conserved T131 residue is shown in orange and S246 Ser is presented in blue. The region consisting of site I PI(3)P and site II PI(3,5)P2 are shown in the gray circle. (b) PvATG18b protein structure rotated 180◦ and showing the CD loop (S269-T288) in yellow. (c) PvATG18b protein structure surfaces (positive and negative charges are shown in blue and red, respectively) showing a nonspecific electrostatic charge. (d) The FRRG repeat position is highlighted with the following colors: Subfamily I (yellow), subfamily II (purple) and subfamily III (red). 2.8. Microsynteny of ATG18 in P. vulgaris To explore the origins and evolutionary processes of the P. vulgaris ATG18 family genes, a comparative synteny map between the eight PvATG18 homologs and 15 other genomes was constructed. The species compared in this study were based on their availability in the GCV database. The classification of the ATG18 family was based on the subfamilies obtained by multidimensional scaling (Figure 6). Plants 2021, 10, 2619 21 of 34 2.8.1. Subfamily I ATG18a was highly conserved in all species with the exception of A. ipaensis. SPATA 20 (legfed_v1_0.L_1H5ZXB) is tandemly duplicated in P. vulgaris. In contrast, the lyase dihydroneopterin aldolase (legfed_v1_0.L_2MWVJ4) was only found in P. vulgaris in the syntenic block. Other genes conserved in the syntenic block were related to cell cycle regulation, transcriptional regulation, transcription factors, zinc finger proteins and other structural motifs involved in peroxisomal and mitochondrial import (Supplementary Figure S5A). ATG18c was not located in the syntenic block in L. albus, M. truncatula, P. sativum or V. angularis. Genes related to ABC transport, vacuolar iron transport, proteins with WD40 repeats involved in protein–protein interactions, cytochrome P450, oxidoreductases and zinc-binding dehydrogenase were highly conserved in the syntenic block. T. pratense and P. lunatus show duplication of oxidoreductases and zinc-binding dehydrogenase family proteins (Supplementary Figure S5B). ATG18c II was not located in the syntenic block in L. japonicus. Transcriptional regula- tor SUPERMAN-like (legfed_v1_0.L_Tx802x and legfed_v1_0.L_NLQvfk) were specific to P. vulgaris. Furthermore, an uncharacterized protein (legfed_v1_0.L_2ffJFT) was found to have undergone duplications in G. max, indicating a putative functional role. Pre-mRNA- splicing factor (legfed_v1_0.L_1Bt8v9) was specifically found in milletioid members of legumes, such as P. vulgaris, G. max, G. soja and V. unguiculata (Supplementary Figure S5C). 2.8.2. Subfamily II ATG18b was not located in L. japonicus or V. angularis. L. japonicus exhibited inversions in the syntenic block involving the synthesis of pectic cell wall components, ATPases and DUF788 proteins, which have been proven to be involved in autophagy regulation. ATG11 was also found in the same syntenic block (Supplementary Figure S5D; Supplementary Information SI5). 2.8.3. Subfamily III ATG18f.I was identified in most of the species compared, and most of the flanking genes were conserved. An important observation from this syntenic block is the tandem duplication of Histone H2A (legfed_v1_0.L_0mwghf) in all species except Arachis and Lotus. Fe(II)-dependent dioxygenase-like (legfed_v1_0.L_81S90D) was missing in L. albus and L. angustifolia (Supplementary Figure S6A; Supplementary Information SI5). ATG18g.I was only found in P. vulgaris, C. cajan, G. max, L. japonicus and V. angularis, and in the other species, the circadian clock-regulated growth regulator Zinc knuckle family protein (legfed_v1_0.L_001qtq) was found in the same syntenic block. The most significant feature of this block was the repeated duplication of disease resistance-responsive dirigent- like protein family protein (legfed_v1_0.L_08frmp) in all the species except V. angularis. In Arachis species, the clustering of vacuolar protein-sorting protein (legfed_v1_0.L_0c0sd2) and breast carcinoma amplified sequence 3 protein (legfed_v1_0.L_cdgcy6) with other genes was an important observation (Supplementary Figure S6B; Supplementary Informa- tion SI5). ATG18g.II was missing in L. albus and was well conserved in other species. In Arachis, gene clusters involving FANTASTIC FOUR 3-like (legfed_v1_0.L_xmq5fm) protein were found associated with shoot meristem growth (Supplementary Figure S6C; Supplementary Information SI5). 2.9. ATG18 Protein Characterization As mentioned previously, ATG18 homologs in P. vulgaris were also divided into three subfamilies with the characteristic motifs FRRG in PvATG18b, VRRG in PvATG18a and PvATG18c, LQRG in PvATG18f and LHRG in PvATG18g. Characterization of the PvATG18 homologs revealed that PvATG18b had the lowest molecular weight, was stable with an Plants 2021, 10, 2619 22 of 34 isoelectric point of 8.86 and had a high aliphatic index. High-molecular-weight proteins were specifically found in subfamily III (Supplementary Table S2). Prediction of the subcellular localization of ATG18 homologs showed that ATG18a, c.I, c.II, g.I and g.II were localized in the cytoplasm, and ATG18f.I and f.II were located in the ER membrane and plasma membrane. Only ATG18c homologs were localized to the lumen of lysosomes. ATG18b was unique because it was found in the mitochondrial inner membrane, inner membrane space and ER membrane (Supplementary Table S2). Furthermore, only three of the PvATG18 proteins had a transmembrane helix spanning the aa 44–67 in PvATG18b and located between the aa 12 and 34 in PvATG18f.I and the aa 7 and 26 in PvATG18f.II (Supplementary Figure S7). Furthermore, we predicted the putative phosphorylation sites in PvATG18 homologs and found that these were located on the amino acids threonine and serine in all sequence alignments (Supplementary Figure S8). 2.10. Protein Structure Prediction and Molecular Dynamics Simulation of ATG18b in P. vulgaris The above-described analysis implies that PvATG18b is the functional ortholog of AtATG18b; hence, we attempted to understand the structure of this protein using the Robetta Server. This model was submitted to 2.1-µs-long unbiased MD to evaluate the predicted protein model (Figure 11a). In the simulation, we monitored the root mean square deviation (RMSD) of the model protein. The graph clearly indicates a change in the RMSD during the first 1.8 µs of simulation, but the RMSD then reached a plateau. This finding indicates that after 1.8 µs of simulation, the 3D structural model of PvATG18b represents a stable folding conformation (Figure 11b). The model shows the seven-bladed β-propeller architecture conserved among the ATG18 family of proteins [52]. The PvATG18 protein structure consists of seven blades formed by antiparallel β-stands connected by short loop regions. The blades are listed with the numbers 1 to 7 beginning at the C-terminus, whereas the β-stands are named with letters from an inner to outer location as A to D. These structures were similar to those observed with the biophysical characterization of PROPPIN ATG18 in Pichia angusta [52]. We also found a CD loop (S269 to T288) located between the two phosphoinositide-binding sites and the FRRG motif at positions F218, R219, R220 and G221 between blades 5 and 6 (Figure 10d). PROPPINs are WD-40 family propeller proteins that act as scaffolds for protein–protein interactions. The binding of PvATG18b to PtdIns(3,5)P2 and PtdIns3P might be mediated by additional protein–protein interactions, as observed in Kluyveromyces lactis [37]. Earlier models of PROPPINS pre- dicted the insertion of two loops into the membrane in a perpendicular orientation in the phagophore membrane through nonspecific electrostatic interactions [53,54]. Our results for PvATG18 reveal the previously reported nonspecific electrostatic interaction in the protein structure and the presence of one transmembrane motif (Figure 10b,c.) 2021, , x FOR PEER REVIEW 29 of 42                                                   ‐ ‐                                                                                                                                                         ‐                                                                                       ‐                                                                               ‐                                                             ‐μ ‐                                                                              μ                                μ                                         ‐   β‐                                          β‐   ‐                                     ‐      β‐                     ‐                               ‐                                           ‐               ‐                               ‐                                                 ‐                       ‐                           ‐                                         ‐                                                       a  b  Figure 11. ATG18 structure. (a) Three-dimensional structural model of Atg18b before (gray) and after (purple) running the molecular dynamics simulation. (b) RMSD of the modeled ATG18b protein over a time period of 2.1 µs. Plants 2021, 10, 2619 23 of 34 3. Discussion Autophagy is recognized as a highly selective cellular clearance pathway that helps maintain homeostasis in eukaryotic cells. The genes involved in autophagy are highly conserved from yeast to humans, and the process is the result of the interaction of these ATGs and other associated genes. The number of identified ATGs shows a marked variation among different species. In yeast, a total of 41 genes have been identified to date, and several studies on plant ATGs have also identified a varied number of genes. In the present investigation, we attempted to perform a comprehensive study for identifying ATG families in three important legume species, namely, P. vulgaris, M. truncatula and G. max. Furthermore, we focused on the ATG18 gene family, the largest of all the families, to identify and phylogenetically compare 27 plant species starting from early plant lineages, chlorophytes to higher plants including legumes. 3.1. Autophagy Genes in Legumes Are Highly Conserved Using Arabidopsis ATGs as a reference, we retrieved ATG homologs in all the species listed in various databases, including Phytozome, and the sequences were confirmed to be affiliated with ATG-like homologs by analyzing their Pfam matches in the Pfam database. We identified a total of 32, 28 and 61 ATG homologs in P. vulgaris, M. truncatula and G. max, respectively. The identified homologs could be classified into 17 families based on their phylogenetic relationships and motifs. The phylogenetic analysis revealed that homologs in Medicago were located closer to Arabidopsis than those in other species. Unlike in yeast, which contains a single copy of each family, many of the gene families have multiple copies. ATG1 has 4, 3, 2 and 6 homologs in Arabidopsis, Medicago, Phaseolus and Glycine, respectively, ATG13 has 2 homologs in Arabidopsis, Medicago and Phaseolus (2 in each) and 4 homologs in G. max, ATG9 has 2 or 4 homologs in Medicago, Phaseolus and G. max and ATG14 and ATG4 have 2 homologs in Arabidopsis and 2 homologs in G. max. The analysis of larger families revealed that ATG8 has 9, 6, 7 and 10 homologs in Arabidopsis, Medicago, Phaseolus and G. max, respectively, and that ATG18 has 8 homologs in Arabidopsis, Medicago and Phaseolus (8 in each) and a maximum of 19 homologs in G. max. Similar results were also obtained with O. sativa [55], Nicotiana tabacum [56], Vitis vinifera [57], Musa acuminate [58] and Setaria italic [59]. However, in most of the families, the homologs were placed in one clade, which clearly showed sequence similarity and the derivation of statistically reliable pairs of possible orthologous proteins sharing similar functions from a common ancestor, consistent with the results from a previous study conducted by Kellogg (2001) [60]. Furthermore, the ATG families identified constituted a relatively complete autophagic machinery in forming the complexes, namely, the ATG1 kinase complex, class III PI3K complex, ATG9 recycling complex, Atg8-lipidation system and Atg12-conjugation system. ATG17 is an important accessory protein along with ATG31-ATG29, which acts as a scaffold/modulator in linking the ATG1-ATG13 complex to the phagophore assembly site in yeast. Homologs of the ATG17-ATG31-ATG29 subcomplex were not detected in Arabidopsis. However, single orthologs of ATG11 and ATG101 were identified, and ATG11 reportedly contains a short cryptic ATG17-like domain with weak identity to yeast ATG17 [61]. The identification of ATG homologs in the present study revealed one homolog of ATG11 and one homolog of ATG101 in all the legumes analyzed. For further exploration of the origin and evolutionary process of ATGs, a comparative synteny map that depicted the presence of 160 genes in Arabidopsis and three legumes compared was constructed. The results suggested that the majority of ATGs had a common ancestor. The Ka/Ks ratio is an important genetic parameter for determining whether positive Darwinian selection is related to gene differentiation [62]. Positive Darwinian se- lection will retain the advantages of nonsynonymous mutations, and purification selection will gradually remove deleterious nonsynonymous mutations. Herein, the Ka/Ks ratio among most of the ATG sequences was lower than 1 (average of 0.17), indicating purifying selection; in contrast, the sequences of ATG8 (1.24) and two ATG18s (1.09 and 1.04) in G. max had higher values, indicating accelerated evolution and positive selection. Plants 2021, 10, 2619 24 of 34 Plant macroautophagy is a process in which macromolecules and cellular components are recycled in lytic vacuoles to be reused. Recycling is crucial for the maintenance of cellu- lar homeostasis by acting as a quality control mechanism under nonstressful conditions and is stimulated under stress conditions [63]. Stress-induced autophagy is well documented in some plant species. Our study of the transcription factors binding to the ATGs revealed that several light-responsive transcription factors, such as BOX-4, G-box, GT1-motif, MRE and ACE, were abundant in most of the ATGs. Furthermore, cis-acting elements related to circadian control were also identified. Phytohormones play key roles in different plant processes, including stress responses. The ATGs analyzed exhibited TF-binding sites for EREs, ABA-responsive ABREs, MeJA-responsive CGTCA motifs, auxin-responsive TGA elements and gibberellin-responsive GARE motifs. Ethylene is considered a key regulator of autophagy in petal senescence in petunia, and ERF5 is also shown to induce autophagy by binding to ATG8 and ATG18h under drought stress in tomato. Upregulation of au- tophagy by low concentrations of salicylic acid is found to delay methyl jasmonate-induced leaf senescence in Arabidopsis [64–66]. In addition, several wound-responsive, pathogen- responsive, flavonoid biosynthetic gene regulation-related and meristem-specific elements were also detected. Based on all the results, the involvement of autophagy in the regulation of plant responses to biotic and abiotic stresses is undeniable. 3.2. Autophagy Genes Are Responsive to Nitrate To assess the differential expression pattern and responsive nature of ATGs to the presence of different nitrate sources, we developed heatmaps using the data retrieved from databases and from a previous RNA-seq analysis performed by our research group. The differential expression pattern in Phaseolus tissues showed that most of the ATGs were expressed in all tested tissues. Nitrogen is an essential component of life that is needed for building proteins and DNA, and despite its abundance in the atmosphere, only limited reserves of soil inorganic nitrogen are accessible to plants, and this nitrogen is primarily in the forms of nitrate and ammonium. Legumes have a unique ability to establish a symbiotic association with nitrogen-fixing rhizobia. Due to our understanding of the evolution of ATGs in legumes, we opted to understand the response of both arial and root tissues of these legumes to different nitrate sources. The expression patterns showed that the highest expression was found in roots treated with ammonia and leaves treated with urea. ATG18 homologs a, g and h were specifically induced in all tissues and by all treatments, indicating the nitrate-responsive nature of these genes. Furthermore, an analysis of the differential expression patterns of ATGs in Phaseolus tissues revealed that the highest expression level was noted in 21-day fix (-) nodules, which could be due to the involvement of the autophagic process in providing the necessary amino acids for the synthesis of nitrogen in the absence of the symbiont. In yeast and other eukaryotes, it has been proven that nitrogen deficiency induces autophagy. A recent study using yeast cells also suggested that autophagy sustains glutamate and aspartate synthesis during nitrogen starvation [67]. RNA-seq data from early symbiosis with rhizobia and mycorrhizae showed differential ATG expression, and more ATGs were upregulated in rhizobia-inoculated roots than in mycorrhizae-inoculated roots. This analysis provided candidate genes that could play pivotal roles in symbiosis. The involvement of ATG6/beclin has previously been reported in P. vulgaris during rhizobial infection progression and arbuscule maturation [68]. 3.3. The ATG18 Family Is Highly Conserved and Has a Broader Sequence-Based Classification Atg18 is one of the autophagy-related molecules responsible for autophagic processes and is conserved from yeast to higher organisms [34]. ATG18 proteins belong to the PROP- PINs (β-propellers that bind polyphosphoinositides) family and work as PI3P effectors. Earlier studies that focused on the identification of ATG genes in primitive and higher plants showed that each family is represented by only one gene for each component of the Plants 2021, 10, 2619 25 of 34 core autophagy machinery. ATG8 and ATG18 are exceptions and have multiple homologs with lower redundancy in Arabidopsis and P. patens [51]. ATG18 was the family with the highest number of homologs; hence, we chose this family for a comprehensive analysis of the family from the early plant lineage to legumes. The multiple sequence alignment and phylogeny of ATG18 homologs resulted in separation of the homologs into three clades. Each of the clades had subfamily members, as determined by the multidimensional scaling projection of 280 ATG18 homologs in 27 photosynthetic organisms. Unlike previous studies by Norizuki and colleagues [51], the classification of the ATG18 family was not based on the BCAS3 domain alone. Knockout of the BCAS3 gene in Dictyostelium resulted in a reduction in early autophagosomes compared with that found in wild-type cells [69]. In the present study, due to the multidimensional scaling projection of the retrieved sequences, we classified the ATG18 sequences into three subfamilies. Subfamily I contained ATG18a, ATG18c, ATG18d and ATG18e homologs, subfamily II had only ATG18b and subfamily III had ATG18f, ATG18g and ATG18h members. All homologs with BCAS3 were found to be clustered within subfamily III. Subfamily II, which contained only ATG18b homologs, had few members but was detected in all the plant species investigated in this study, which suggested the sequence and functional conservation of these proteins. Among the early photosynthetic organisms, we identified at least one homolog in subfamilies I and II, but significant divergence was detected, particularly within subfamily III. Among monocots, O. sativa had 8 homologs, whereas 32 and 21 homologs were found in Z. mays and T. aestivum, respectively. The analysis of dicots revealed 8 homologs in each of Arabidopsis, L. japonicus, M. truncatula and P. vulgaris, whereas Arachis sp. had 9 and 10. The maximum number of homologs was recorded in C. cajan (18), G. max (18), C. arietinum (20), Vigna sp. and L. angustifolius (27). The legume family includes one of the most agroeconomically important plant crops after Poaceae [70]. Of the three subfamilies within Fabaceae, Papilionoideae is the largest, the most recently evolved and monophyletic. Because Papilionoideae includes the most important cultivated legumes, we sought to determine the members of this subfamily in different clades. In the present study, the maximum number of homologs (27) was identified in L. angustifolius, which belongs to the genistoid clade and exhibited an early divergence at approximately 56.4 ± 2 mya. Furthermore, in Arachis species, we found less than half of the ATG18 homologs, indicating possible deletions. Among the members of the next recent (45 mya) clade, which consisted of milletoids, an increase in the number of homologs (18) was detected, which might be due to whole-genome duplication in G. max. However, P. vulgaris had only eight members of ATG18, indicating possible divergence prior to whole- genome duplications, whereas Vigna sp. was found to have high numbers of homologs. Furthermore, more recent robinioid (48.3 ± 1.0 mya) and IRLC (39.0 ± 2.4 mya) clade members had fewer members with the exception of the tribe Vicieae, whose gene numbers were due to genome expansion and related genomic events. In contrast, syntenic relations were not disrupted due to differences in genome sizes [71,72]. A phylogenetic analysis revealed that the ATG18 homologs of Chlorophyta, Charophyta, Marchantiophyta and Bryophyta were always grouped together, and similar results were obtained for monocots and dicots. However, in a comparison of a broad class of species, it is often not simple to precisely define orthologous genes or genomic loci in a straightforward manner, and this analysis is complicated due to gene duplication, recurring polyploidy and extensive genome rearrangement [73]. 3.4. The ATG18 Protein Structure Predicts Possible Functional Diversification In addition, the prediction of the primary and secondary structures of the proteins strengthens the classification of ATG18 proteins into subfamilies. The protein size, motif structure and changes in FRRG motifs among the ATG18 homologs were identified as the fundamental features that contribute to the classification. The changes in the FRRG motifs found in members of subfamily II comprising ATG18b to LRRG, VRRG in subfamily I, LQRG, LHRG or LYRG in subfamily III indicate functional diversification. The WD40 Plants 2021, 10, 2619 26 of 34 domain is among the top ten most abundant domains in eukaryotic genomes and is also ranked as the top interacting domain in S. cerevisae [74] (Stirnimann et al., 2010). Based on the SMART database, the human genome contains approximately 349 WD40 domain- containing proteins [75]. The presence of the WD40 domain in ATG18 homologs could indicate their involvement in cellular functions. Proteins containing WD40 domains are known to be involved in signal transduction, vesicular trafficking, cytoskeletal assembly, cell cycle control, apoptosis, chromatin dynamics and transcription regulation due to their ability to bind and thus function as interchangeable substrate receptors to target different substrates and recruit different substrates in distinct modes [76]. In C. elegans, ATG18 and WIPI 1/2 (WD-repeat protein interacting with phosphoinositides) in mammals have FRRGs and EPG-6 and WIPI 3/4 have LRRGs. The substitution of the FRRG motif by FTTG and FKKG does not allow PtdInsP binding; however, the changes in LKKG and LTTG still allow PtdInsP binding [77], implying a possible functional diversification of ATG18 homologs. The studies conducted thus far also demonstrate the involvement of ATG18 homologs in abiotic stress responses in plants [42–50]. 3.5. ATG18 Family in P. vulgaris In P. vulgaris, a total of eight ATG18 homologs were identified in the current study and were also classified into three subfamilies. While the functional roles of these subfamilies were not determined in this study, the involvement of these proteins in diversified cellular functions cannot be ruled out. All the subfamilies showed conserved phosphorylation sites but different subcellular localizations. The conserved nature of serine/threonine sites could indicate the functional roles corresponding to several cellular responses in P. vulgaris. In yeast, Pichia pastoris, Atg18 phosphorylation in the loops in the propeller structure of blades 6 and 7 decreases its binding affinity to phosphatidylinositol 3,5-bisphosphate. The association of ATG18 with the vacuolar membrane is inhibited until dephosphorylation [78]. A recent study in Arabidopsis showed that the phosphorylation of ATG18a by brassinosteroid insensitive 1- associated receptor kinase 1 (BAK1) suppresses autophagy and attenuates plant resistance against necrotrophic pathogens [79]. The microsynteny of P. vulgaris ATG18 homologs showed that subfamily I members were highly conserved across the compared species and were flanked by genes involved in cell cycle regulation, transcriptional regulation, cellular transport and metal ion binding. Furthermore, subfamily II was flanked by the ATPase and DUF788 proteins, which have been proven to be involved in autophagy regulation. ATG11, which is a part of the ATG13- ATG1 complex in autophagy initiation, was also found in the same syntenic block. The subfamily III syntenic block contained conserved genes related to histones, circadian clock, growth and vacuolar transport. 3.6. PvATG18b Could Be the Homolog of AtATG18b In accordance with a well-established fact, the most important feature of ATG18 proteins is the presence of the FRRG motif and its ability to bind to phosphoinositide. Among P. vulgaris ATG18 homologs, the FRRG motif was found only in ATG18b belonging to subfamily II. Hence, we propose PvATG18b as the functional homolog of A. thaliana ATG18b. We also hypothesize that other ATG18 homologs might be involved in other molecular recognition events through binding to surface molecules that play a distinctive role in autophagy, and similar findings have been observed with human ATG18 homologs, e.g., WIPI 1/WIPI 2 with FRRG repeats and WIPI 3/WIPI 4 with LRRG repeats bind to various PtdIns and thus play distinct roles in autophagy [76,80]. We then performed a molecular dynamic simulation of PvATG18b that is unique to ATG models in legumes. Our model shows the stable folding conformation of the seven-bladed β-propeller architecture. PvATG18b is composed of 359 amino acids, and we found the CD loop (S269 to T288) in blade 6. While this loop sequence differs among species, it forms an amphipathic alpha-helix and might insert into a membrane to allow Plants 2021, 10, 2619 27 of 34 two lipid-binding sites (PtdIns3P and PtdIns(3,5)P2) [81]. Additionally, PvATG18b contains the FRRG repeat and helps form the site for binding to lipids. The FRRG repeat is in F218 to G221 and is conserved in ATG18b to form the PROPPIN family. The FRRG motif (Phe-Arg- Arg-Gly) in ATG18 proteins has been studied in mammals, yeast and C. elegance [79,82]. In Kluyveromyces lactis, the mutation of the blade 6 β3-β4 loop affects the loss of liposome binding, and the flexible loop coordinates two distinct lipid-binding sites [83]. Previous studies with S. cerevisiae have demonstrated that loops A and B of blade 7 are the locations where ATG2 interacts with ATG18. Further research should be performed to understand the interaction of ATG18 with ATG2 and thus ensure the binding site and vacuole scission function of PvATG18b. 4. Materials and Methods 4.1. Identification of ATG Families in Legumes Arabidopsis (taxid: 3702) ATG family gene sequences were retrieved from the Araport (https://www.araport.org; accessed on 13 May 2020) and TAIR (https://www.arabidopsis. org; accessed on 15 May 2020) databases through Phytozome v.13. Using these sequences, a BLAST [84] (http://www.ncbi.nlm.nih.gov; Stephen et al., 1997; accessed on 19 May 2020) search was conducted to identify the homologs of ATG genes in Phaseolus vulgaris v 2.1 (taxid: 3885), Medicago truncatula Mt4.0v1 (taxid: 3880) and Glycine max Wm82.a2.v1 (taxid: 3847). The stringency of the search was maintained by keeping the mean BLAST results within a query coverage of 93.85% and 67.78% identity. The detection of homologs was further optimized using other programs, such as KEGG (www.genome.jp/kegg/; accessed on 2 June 2020) [85], Ensembl Plants (https:// plants.ensembl.org; accessed on 4 June 2020) [86], HMMER suite server (http://hmmer.org; accessed on 4 June 2020) [87] and InParanoid 4.1 [88]. Additionally, we examined the ontology IDs for all ATG families using KOG (EuKaryotic Orthologous subfamilies) in the EggNOG v5.0 database [89] (http://eggnog.embl.de; accessed on 7 June 2020) and Protein ANalysis THrough Evolutionary Relationships (PANTHER v14.0, http://www. pantherdb.org; accessed on 10 June 2020) and Pfam IDs were identified in Portal v33.1 (http://pfam.xfam.org/about accessed on 30 October 2020). The ATG18 protein family was studied in 27 photosynthetic organisms, 13 dicots (legumes), 3 monocots and 10 plants through the evolution of land plants from an algal ancestor. We obtained the ATG18 protein sequences of monocotyledonous crops such as Zea mays (taxid: 4577), Triticum aestivum (taxid: 4565) and Oryza sativa (rice, taxid: 4530) and legumes such as Arachis duranensis (peanut, taxid: 130453), Arachis ipaensis (taxid: 130454), Cajanus cajan (taxid: 3821), Lotus japonicus (taxid: 34305), Cicer arietinum (taxid: 3827), Lupinus angustifolius (taxid: 3871), Pisum sativum (pea, taxid: 3888), Vigna angularis (taxid: 3914), Vigna radiata (taxid: 157791) and Trifolium pratense (red clover, taxid: 57577) through a BLAST analysis of the NCBI, Phytozome, LegumeInfo (https://legumeinfo.org; accessed on 18 June 2020), KEGG, InParanoid, Ensembl, EggNOG and Pfam databases. Additionally, we used the Norizuki report of early-divergent plant lineages to extract the ATG18 protein sequences of Bryopsida (Physcomitrella patens, taxid: 3218), Charophyceae (Chara braunii, taxid: 69332), Chlorophyceae (Chlamydomonas reinhardtii, taxid: 3055, Dunaliella salina, taxid: 3046), (Volvox carteri, taxid: 3067), Klebsormidiophyceae (Klebsormidium nitens, taxid: 105231), Mamiellophyceae (Micromonas pusilla, taxid: 38833; Ostreococcus lucimarinus, taxid: 242159; Ostreococcus tauri, taxid: 70448) and Trebouxiophyceae (Coccomyxa subellipsoidea, taxid: 248742) [51]. 4.2. Alignment and Phylogenetic Tree Analyses The protein sequences of ATG families were aligned using Clustal Omega (1.2.4) [90] (www.clustal.org and www.ebi.ac.uk; accessed on 5 July 2020) with the default parameters. The phylogenetic tree was a neighbor-joining tree without distance corrections, and we extracted the outputs from the tree and generated circular phylogram and cladogram tree images using EvolView. The different phylogenetic trees were combined with the MEME Plants 2021, 10, 2619 28 of 34 results for all sequences, and the final details were obtained using Inkscape software [91] (https://www.evolgenius.info/evolview/; accessed on 6 July 2020). Multiple sequence alignment of 280 intraspecies protein sequences of ATG18 family members was performed using Clustal Omega. The phylogenetic analysis was performed using MEGA X with the maximum likelihood method and Bayes analyses with 1000 bootstrap replicates and the default parameters [92]. Phangorn and APE packages in R were used to build the phylogenetic trees [93,94]. In Phangorn, we used the Akaike information criterion and the Whelan and Goldman matrix (WAG) as the substitution model. 4.3. Chromosome Localization, Synteny and Ka/Ks Calculation The chromosomal localization of ATG family homologs in A. thaliana, P. vulgaris, M. truncatula and G. max was verified using NCBI. Furthermore, Ensembl Plants was used to compare and explore the gene alignments and generate a segment to link the genomes. The synteny relation of ATG genes was drawn using OmicCircos in R36 [95]. The macro- and microsynteny of the ATG18 family was developed using the Genome Context Viewer (GCV) in the Legume information system [96] (https://legumeinfo.org/ lis_context_viewer/instructions; accessed on 16 July 2020). The CDSs and protein sequences were obtained from Phytozome and used to calculate the synonymous substitutions (Ks) and nonsynonymous substitutions (Ka) with TBtools software (https://github.com/CJ-Chen/TBtools; accessed on 26 July 2020). Using the data table, we developed a graph of the Ka and Ks values for all ATG families in P. vulgaris, M. truncatula and G. max using the ggplot2 R packages (https://ggplot2.tidyverse.org/; accessed on 28 July 2020). 4.4. Promoter Analysis, Expression Profiling and Transcriptome of ATG Families The 2000-bp upstream sequences of ATG genes were retrieved from Phytozome, and these sequences were used as query sequences in PlantCARE software (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 2 August 2020). The results were analyzed, and the most abundant transcription factors were identified using ggplot2 in R. ATG gene expression data for A. thaliana, M. truncatula and G. max were extracted from Phytozome to determine the differential expression of the genes under different nitrogen treatments [97]. Data on the differential expression of genes in P. vulgaris under nitrogen treatments and after fixation and inoculation with Rhizobium tropici (CIAT899) were obtained from the PvGEA website (https://plantgrn.noble.org/PvGEA/; accessed on 2 July 2020). We calculated the log2 values of the RPKM values for the comparison. To show the data for A. thaliana, M. truncatula and G. max, we used the OmicCircos package and constructed subfamilies using the synteny graph. However, for P. vulgaris, we constructed an independent heatmap of ggplot2 because the amounts of treatments and tissues were higher. The expression data for ATGs under rhizobial and mycorrhizal symbiotic conditions were obtained from our previous global transcriptomic analysis [98]. A heatmap of the fold change values was constructed using the ggplot2 package. 4.5. Quantitative Real-Time PCR Analysis Four genes were selected for RT-qPCR analysis, which was performed to validate the RNA-seq data. High-quality total RNA was isolated from frozen root tissues using TRIzol reagent (Sigma) according to the manufacturer’s instructions. RNA integrity was verified by gel electrophoresis and RNA concentration was assessed using a NanoDrop spectrophotometer (Thermo Scientific). RNA was treated with DNase to eliminate DNA contamination (1 U/µL; Roche, USA) according to the manufacturer’s instructions. Reverse- transcription quantitative PCR (RT-qPCR) analysis was performed using a DNA-free RNA and iScriptTM One-Step RT-PCR Kit with SYBR® Green (Bio-Rad) according to the manufacturer’s instructions. To confirm the absence of DNA contamination, a sample Plants 2021, 10, 2619 29 of 34 lacking reverse transcriptase was included. Relative expression values were calculated using the 2-∆Ct method, where the quantification cycle (Cq) value equals the Cq value of the gene of interest minus the Cq value of the reference gene [99]. Gene-specific primers were used for RT-qPCR analysis (Table S3). PvEF1α and PvIDE were used as reference as described previously by Arthikala et al. [100]. The relative expression values were normalized with respect to two reference genes EF1α and IDE as described previously by Vandesompele et al. [101]. The values presented are averages of three biological replicates, and each data set was recorded using triplicate samples. 4.6. Principal Components Analysis of the ATG18 Family Based on multiple alignments of ATG18 protein sequences, we converted the infor- mation into a distance matrix calculated using the bios2mds packages (https://CRAN.R- project.org/package=bios2mds; accessed on 3 July 2020) in R. The matrix used was BLO- SUM62 (BLOcks of Amino Acid SUbstitution Matrix), and sequences with 62% identity were obtained. Using the same packages, we obtain the K-means and principal components to generate the multidimensional scaling projection and thus define the subfamilies within the protein family. 4.7. Detection of Motifs, Domains, Repeats, Families and Secondary Protein Structure of the ATG18 Family ATG sequences were analyzed for a repeated sequence motif pattern using Multiple Expectation Maximization for Motif Elicitation [102] (http://meme-suite.org/tools/meme; accessed on 18 July 2020) in the classical motif discovery mode and using a limit of three motifs. The secondary structures of the proteins were developed after alignment with Clustal Omega using the online tool JPred in FASTA format. To obtain the repeats, domains and families, a Pfam scan of EMBL-EBI was performed (https://www.ebi.ac.uk/Tools/ pfa/pfamscan/; accessed on 26 August 2020). 4.8. Microsynteny and Protein Sequence Parameters of ATG18 in P. vulgaris The computed parameters for PvATG18, including the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, grand average of hydropathicity (GRAVY), phosphoryla- tion sites, predicted transmembrane helixes and subcellular localization, were obtained us- ing ProtParam, PSORT, THMHMM and NetPhos 51 (https://web.expasy.org; accessed on 5 July 2020). The ATG positions were extracted from Phytozome, and microsynteny calcula- tions were generated using GCV v1.2.0 [103] (https://legumeinfo.org/lis_context_viewer/; accessed on 6 August 2020). 4.9. ATG18b Protein in P. vulgaris The 3D structure of the PvATG18b protein was determined using the Robetta server [102]. Comparative models were built from structures detected and aligned using HHSEARCH, SPARKS and Raptor [104–107]. The loop regions were assembled from fragments and optimized to fit the aligned template structures. The final structure prediction was selected using the lowest-energy model as determined by a low-resolution Rosetta energy function. The final 3D image was colored with Quimera [108]. 5. Conclusions The present study was carried out to understand the diversification of ATG genes during plant evolution with special emphasis on legumes and P. vulgaris. In the present study, we identified 32, 39 and 61 core ATG genes in P. vulgaris, M. truncatula and G. max, respectively. The ATG genes were conserved across the species, but the higher plants revealed great redundancy. Most of the ATGs in Phaseolus were found to be nitrate responsive and were differentially expressed under rhizobial and mycorrhizal symbiosis, implying their possible role during symbiosis. Further, analysis ATG18 of the family in Plants 2021, 10, 2619 30 of 34 27 photosynthetic organisms showed their classification into three subfamilies based on the sequence. In Phaseolus, ATG18 members belonging to all the three subfamilies were conserved. Comparison of Phaseolus ATG18b structure to the crystal structure in Arabidopsis showed conserved FRRG sequence. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10122619/s1, Figure S1: Percentage of legume ATG homologs in different software pro- grams. Figure S2: Validation of expression patterns of ATGs of symbiont-colonized P. vulgaris roots by RT-qPCR analysis. Figure S3A: Representation of 280 ATG18 proteins from different plant species analyzed by multidimensional scaling using Bios2mds. Figure S3B: Phylogenetic tree of ATG18 in plants. Figure S4: Secondary structure prediction using JPred. Figure S5: Microsynteny analysis of ATG18 (Subfamily I & II) in P. vulgaris. Figure S6: Microsynteny analysis of ATG18 (Subfamily III) in P. vulgaris. Figure S7: Phosphorylation sites of ATG18 in P. vulgaris identified using NetPhos. Figure S8: Prediction of transmembrane helices in PvATG18 proteins using TMHMM. Table S1: List of identifiers of the genes, transcripts, and proteins of each ATG in P. vulgaris, Table S2: ATG18 protein characterization in P. vulgaris. Table S3: List of Oligos for RT-Qpcr. Supplementary information: Supplementary Information SI1: Analysis of ATG genes homologs in P. vulgaris, M. truncatula, G. max in different databases; Supplementary Information SI2: Expression profiles of ATGs in P. vulgaris; Supplementary Information SI3: Analysis of ATG18 homologs; Supplementary Information SI4: Family, repeats, motifs and domain positions in legumes; Supplementary Information SI5: Alignment and synteny of ATG genes between A. thaliana and legumes using the comparative genomics in Ensembl. Author Contributions: Conceptualization, K.N. and E.-H.Q.-R.; methodology, E.-H.Q.-R., H.G.-V. and K.N.; software, E.-H.Q.-R.; validation, K.N., M.-K.A. and A.H.-L.; formal analysis, E.-H.Q.-R. and H.G.-V.; investigation, K.N. and E.-H.Q.-R.; resources, E.-H.Q.-R. and H.G.-V.; writing—original draft preparation, E.-H.Q.-R.; writing—review and editing, K.N., E.-H.Q.-R., M.-K.A. and M.L.; visualization, K.N., E.-H.Q.-R. and M.-K.A.; supervision, K.N.; project administration, K.N.; funding acquisition, K.N., M.-K.A. and M.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Dirección General de Asuntos del Personal Académico, DGAPA/PAPIIT-UNAM grant no. IN211218 to K.N. Partially supported by CONACyT project CF-MI-20191017134234199/316538 to M.-K.A., DGAPA/PAPIIT-UNAM grant no. IN216321 to K.N. and DGAPA/PAPIIT-UNAM grant no. IN205619 to M.L. Institutional Review Board Statement: Not applicable. 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