Caracterización funcional de factores de transcripción de tipo NF-Y y GRAS en la asociación simbiótica <i>Phaseolus vulgaris - Rhizobium etli</i>

La fijación biológica de nitrógeno que surge a partir de la relación simbiótica entre rizobios y leguminosas permite la incorporación de nitrógeno al suelo para su explotación en sistemas agropecuarios a muy bajo costo. En esta relación compleja y específica existen determinantes moleculares de las plantas que gobiernan la colonización preferencial por ciertas cepas de rizobios. Las plantas integran señales derivadas del microsimbionte y del ambiente para iniciar los programas de organogénesis de estructuras diferenciadas llamadas nódulos y el proceso de infección. Varios factores de transcripción desempeñan funciones fundamentales en estos procesos, incluyendo las proteínas de las familias GRAS y NF-Y. En el presente trabajo de tesis se generó un conjunto de datos de secuencias, estructuras génicas, relaciones filogenéticas y patrones de expresión de genes que codifican las subunidades NF-Y en Phaseolus vulgaris, la leguminosa de grano más importante utilizado para el consumo humano directo. A partir de este análisis se seleccionaron genes candidatos para estudiar su rol en la nodulación mediante genética reversa. La reducción de los niveles de mRNA de PvNF-YA1 y PvNF-YA9 condujo a la ausencia de nodulación y provocó defectos en el proceso de infección. La inducción de genes del ciclo celular en respuesta al rizobio se vio afectada en las raíces silenciadas, sugiriendo que estos factores de transcripción podrían promover el desarrollo de nódulos mediante la activación de divisiones celulares corticales que participan en la formación del primordio del nódulo. A su vez, ensayos de sobreexpresión permitieron demostrar que PvNF-YA1 contribuye a la selección de cepas de rizobio que son más eficientes en la formación de nódulos. Por otro lado, se llevó a cabo la caracterización de una proteína de la familia GRAS, denominada SIN1 (Scarecrow like 13 Involved in Nodulation), que interactúa físicamente con la subunidad PvNF-YC1, previamente vinculada al proceso de nodulación. Ensayos de silenciamiento mostraron que SIN1 desempeña una función importante tanto en la elongación de raíces laterales como en el desarrollo de nódulos fijadores de nitrógeno y en la progresión de la infección bacteriana durante la interacción simbiótica. El presente trabajo de tesis contribuye a sustentar la relevancia de los complejos de transcripción multiméricos en la capacidad de las plantas para integrar y responder a múltiples factores ambientales.Facultad de Ciencias Exacta

Caracterización funcional de factores de transcripción de tipo NF-Y y GRAS en la asociación simbiótica <i>Phaseolus vulgaris - Rhizobium etli</i>

La fijación biológica de nitrógeno que surge a partir de la relación simbiótica entre rizobios y leguminosas permite la incorporación de nitrógeno al suelo para su explotación en sistemas agropecuarios a muy bajo costo. En esta relación compleja y específica existen determinantes moleculares de las plantas que gobiernan la colonización preferencial por ciertas cepas de rizobios. Las plantas integran señales derivadas del microsimbionte y del ambiente para iniciar los programas de organogénesis de estructuras diferenciadas llamadas nódulos y el proceso de infección. Varios factores de transcripción desempeñan funciones fundamentales en estos procesos, incluyendo las proteínas de las familias GRAS y NF-Y. En el presente trabajo de tesis se generó un conjunto de datos de secuencias, estructuras génicas, relaciones filogenéticas y patrones de expresión de genes que codifican las subunidades NF-Y en Phaseolus vulgaris, la leguminosa de grano más importante utilizado para el consumo humano directo. A partir de este análisis se seleccionaron genes candidatos para estudiar su rol en la nodulación mediante genética reversa. La reducción de los niveles de mRNA de PvNF-YA1 y PvNF-YA9 condujo a la ausencia de nodulación y provocó defectos en el proceso de infección. La inducción de genes del ciclo celular en respuesta al rizobio se vio afectada en las raíces silenciadas, sugiriendo que estos factores de transcripción podrían promover el desarrollo de nódulos mediante la activación de divisiones celulares corticales que participan en la formación del primordio del nódulo. A su vez, ensayos de sobreexpresión permitieron demostrar que PvNF-YA1 contribuye a la selección de cepas de rizobio que son más eficientes en la formación de nódulos. Por otro lado, se llevó a cabo la caracterización de una proteína de la familia GRAS, denominada SIN1 (Scarecrow like 13 Involved in Nodulation), que interactúa físicamente con la subunidad PvNF-YC1, previamente vinculada al proceso de nodulación. Ensayos de silenciamiento mostraron que SIN1 desempeña una función importante tanto en la elongación de raíces laterales como en el desarrollo de nódulos fijadores de nitrógeno y en la progresión de la infección bacteriana durante la interacción simbiótica. El presente trabajo de tesis contribuye a sustentar la relevancia de los complejos de transcripción multiméricos en la capacidad de las plantas para integrar y responder a múltiples factores ambientales.Facultad de Ciencias Exacta

Annotation, phylogeny and expression analysis of the nuclear factor Y gene families in common bean (Phaseolus vulgaris)

In the past decade, plant nuclear factor Y (NF-Y) genes have gained major interest due to their roles in many biological processes in plant development or adaptation to environmental conditions, particularly in the root nodule symbiosis established between legume plants and nitrogen fixing bacteria. NF-Ys are heterotrimeric transcriptional complexes composed of three subunits, NF-YA, NF-YB, and NF-YC, which bind with high affinity and specificity to the CCAAT box, a cis element present in many eukaryotic promoters. In plants, NF-Y subunits consist of gene families with about 10 members each. In this study, we have identified and characterized the NF-Y gene families of common bean (Phaseolus vulgaris), a grain legume of worldwide economical importance and the main source of dietary protein of developing countries. Expression analysis showed that some members of each family are up-regulated at early or late stages of the nitrogen fixing symbiotic interaction with its partner Rhizobium etli. We also showed that some genes are differentially accumulated in response to inoculation with high or less efficient R. etli strains, constituting excellent candidates to participate in the strain-specific response during symbiosis. Genes of the NF-YA family exhibit a highly structured intron-exon organization. Moreover, this family is characterized by the presence of upstream ORFs when introns in the 5′ UTR are retained and miRNA target sites in their 3′ UTR, suggesting that these genes might be subjected to a complex post-transcriptional regulation. Multiple protein alignments indicated the presence of highly conserved domains in each of the NF-Y families, presumably involved in subunit interactions and DNA binding. The analysis presented here constitutes a starting point to understand the regulation and biological function of individual members of the NF-Y families in different developmental processes in this grain legume.Fil: Rípodas, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Castaingts, Mélisse. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Clua, Joaquin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Blanco, Flavio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Zanetti, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Annotation, phylogeny and expression analysis of the Nuclear factor Y gene families in common bean (<i>Phaseolus Vulgaris</i>)

In the past decade, plant nuclear factor Y (NF-Y) genes have gained major interest due to their roles in many biological processes in plant development or adaptation to environmental conditions, particularly in the root nodule symbiosis established between legume plants and nitrogen fixing bacteria. NF-Ys are heterotrimeric transcriptional complexes composed of three subunits, NF-YA, NF-YB, and NF-YC, which bind with high affinity and specificity to the CCAAT box, a cis element present in many eukaryotic promoters. In plants, NF-Y subunits consist of gene families with about 10 members each. In this study, we have identified and characterized the NF-Y gene families of common bean (Phaseolus vulgaris), a grain legume of worldwide economical importance and the main source of dietary protein of developing countries. Expression analysis showed that some members of each family are up-regulated at early or late stages of the nitrogen fixing symbiotic interaction with its partner Rhizobium etli. We also showed that some genes are differentially accumulated in response to inoculation with high or less efficient R. etli strains, constituting excellent candidates to participate in the strain-specific response during symbiosis. Genes of the NF-YA family exhibit a highly structured intron-exon organization. Moreover, this family is characterized by the presence of upstream ORFs when introns in the 5' UTR are retained and miRNA target sites in their 3' UTR, suggesting that these genes might be subjected to a complex post-transcriptional regulation. Multiple protein alignments indicated the presence of highly conserved domains in each of the NF-Y families, presumably involved in subunit interactions and DNA binding. The analysis presented here constitutes a starting point to understand the regulation and biological function of individual members of the NF-Y families in different developmental processes in this grain legume.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Transcriptional regulators of legume-rhizobia symbiosis: Nuclear Factors Ys and GRAS are two for tango

Transcription factors are DNA binding proteins that regulate gene expression. The nitrogen fixing symbiosis established between legume plants and soil bacteria is a complex interaction, in which plants need to integrate signals derived from the symbiont and the surrounding environment to initiate the developmental program of nodule organogenesis and the infection process. Several transcription factors that play critical roles in these processes have been reported in the past decade, including proteins of the GRAS and NF-Y families. Recently, we reported the characterization of a new GRAS domain containing-protein that interacts with a member of the C subunit of the NF-Y family, which plays an important role in nodule development and the progression of bacterial infection during the symbiotic interaction. The connection between transcription factors of these families highlights the significance of multimeric complexes in the fabulous capacity of plants to integrate and respond to multiple environmental stimuli.Instituto de Biotecnologia y Biologia Molecula

NIPK, a protein pseudokinase that interacts with the C subunit of the transcription factor NF-Y, is involved in rhizobial infection and nodule organogenesis

Heterotrimeric Nuclear Factor Y (NF-Y) transcription factors are key regulators of the symbiotic program that controls rhizobial infection and nodule organogenesis. Using a yeast two-hybrid screening, we identified a putative protein kinase of Phaseolus vulgaris that interacts with the C subunit of the NF-Y complex. Physical interaction between NF-YC1 Interacting Protein Kinase (NIPK) and NF-YC1 occurs in the cytoplasm and the plasma membrane. Only one of the three canonical amino acids predicted to be required for catalytic activity is conserved in NIPK and its putative homologs from lycophytes to angiosperms, indicating that NIPK is an evolutionary conserved pseudokinase. Post-transcriptional silencing on NIPK affected infection and nodule organogenesis, suggesting NIPK is a positive regulator of the NF-Y transcriptional complex. In addition, NIPK is required for activation of cell cycle genes and early symbiotic genes in response to rhizobia, including NF-YA1 and NF-YC1. However, strain preference in co-inoculation experiments was not affected by NIPK silencing, suggesting that some functions of the NF-Y complex are independent of NIPK. Our work adds a new component associated with the NF-Y transcriptional regulators in the context of nitrogen-fixing symbiosis.Fil: Clua, Joaquin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Rípodas, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Roda, Carla Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Battaglia, Marina Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Zanetti, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Blanco, Flavio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Annotation, phylogeny and expression analysis of the Nuclear factor Y gene families in common bean (<i>Phaseolus Vulgaris</i>)

In the past decade, plant nuclear factor Y (NF-Y) genes have gained major interest due to their roles in many biological processes in plant development or adaptation to environmental conditions, particularly in the root nodule symbiosis established between legume plants and nitrogen fixing bacteria. NF-Ys are heterotrimeric transcriptional complexes composed of three subunits, NF-YA, NF-YB, and NF-YC, which bind with high affinity and specificity to the CCAAT box, a cis element present in many eukaryotic promoters. In plants, NF-Y subunits consist of gene families with about 10 members each. In this study, we have identified and characterized the NF-Y gene families of common bean (Phaseolus vulgaris), a grain legume of worldwide economical importance and the main source of dietary protein of developing countries. Expression analysis showed that some members of each family are up-regulated at early or late stages of the nitrogen fixing symbiotic interaction with its partner Rhizobium etli. We also showed that some genes are differentially accumulated in response to inoculation with high or less efficient R. etli strains, constituting excellent candidates to participate in the strain-specific response during symbiosis. Genes of the NF-YA family exhibit a highly structured intron-exon organization. Moreover, this family is characterized by the presence of upstream ORFs when introns in the 5' UTR are retained and miRNA target sites in their 3' UTR, suggesting that these genes might be subjected to a complex post-transcriptional regulation. Multiple protein alignments indicated the presence of highly conserved domains in each of the NF-Y families, presumably involved in subunit interactions and DNA binding. The analysis presented here constitutes a starting point to understand the regulation and biological function of individual members of the NF-Y families in different developmental processes in this grain legume.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

A nuclear factor Y interacting protein of the GRAS family is required for nodule organogenesis, infection thread progression, and lateral root growth

A C subunit of the heterotrimeric nuclear factor Y (NF-YC1) was shown to play a key role in nodule organogenesis and bacterial infection during the nitrogen fixing symbiosis established between common bean (Phaseolus vulgaris) and Rhizobium etli. To identify other proteins involved in this process, we used the yeast (Saccharomyces cerevisiae) two-hybrid system to screen for NFYC1- interacting proteins. One of the positive clones encodes a member of the Phytochrome A Signal Transduction1 subfamily of GRAS (for Gibberellic Acid-Insensitive (GAI), Repressor of GAI, and Scarecrow) transcription factors. The protein, named Scarecrow-like13 Involved in Nodulation (SIN1), localizes both to the nucleus and the cytoplasm, but in transgenic Nicotiana benthamiana cells, bimolecular fluorescence complementation suggested that the interaction with NF-YC1 takes place predominantly in the nucleus. SIN1 is expressed in aerial and root tissues, with higher levels in roots and nodules. Posttranscriptional gene silencing of SIN1 using RNA interference (RNAi) showed that the product of this gene is involved in lateral root elongation. However, root cell organization, density of lateral roots, and the length of root hairs were not affected by SIN1 RNAi. In addition, the expression of the RNAi of SIN1 led to a marked reduction in the number and size of nodules formed upon inoculation with R. etli and affected the progression of infection threads toward the nodule primordia. Expression of NF-YA1 and the G2/M transition cell cycle genes CYCLIN B and Cell Division Cycle2 was reduced in SIN1 RNAi roots. These data suggest that SIN1 plays a role in lateral root elongation and the establishment of root symbiosis in common bean.Facultad de Ciencias Exacta

The PvNF-YA1 and PvNF-YB7 Subunits of the Heterotrimeric NF-Y Transcription Factor Influence Strain Preference in the Phaseolus vulgaris–Rhizobium etli Symbiosis

Transcription factors of the Nuclear Factor Y (NF-Y) family play essential functions in plant development and plasticity, including the formation of lateral root organs such as lateral root and symbiotic nodules. NF-Ys mediate transcriptional responses by acting as heterotrimers composed of three subunits, NF-YA, NF-YB, and NF-YC, which in plants are encoded by relatively large gene families. We have previously shown that, in the Phaseolus vulgaris × Rhizobium etli interaction, the PvNF-YC1 subunit is involved not only in the formation of symbiotic nodules, but also in the preference exhibited by the plant for rhizobial strains that are more efficient and competitive in nodule formation. PvNF-YC1 forms a heterotrimer with the PvNF-YA1 and PvNF-YB7 subunits. Here, we used promoter:reporter fusions to show that both PvNF-YA1 and PvNF-YB7 are expressed in symbiotic nodules. In addition, we report that knock-down of PvNF-YA1 and its close paralog PvNF-YA9 abolished nodule formation by either high or low efficient strains and arrested rhizobial infection. On the other hand, knock-down of PvNF-YB7 only affected the symbiotic outcome of the high efficient interaction, suggesting that other symbiotic NF-YB subunits might be involved in the more general mechanisms of nodule formation. More important, we present functional evidence supporting that both PvNF-YA1 and PvNF-YB7 are part of the mechanisms that allow P. vulgaris plants to discriminate and select those bacterial strains that perform better in nodule formation, most likely by acting in the same heterotrimeric complex that PvNF-YC1

A phylogenetically conserved group of nuclear factor-Y transcription factors interact to control nodulation in legumes

The endosymbiotic association between legumes and soil bacteria called rhizobia leads to the formation of a new root-derived organ called the nodule in which differentiated bacteria convert atmospheric nitrogen into a form that can be assimilated by the host plant. Successful root infection by rhizobia and nodule organogenesis require the activation of symbiotic genes that are controlled by a set of transcription factors (TFs). We recently identified Medicago truncatula nuclear factor-YA1 (MtNF-YA1) and MtNF-YA2 as two M. truncatula TFs playing a central role during key steps of the Sinorhizobium meliloti-M. truncatula symbiotic interaction. NF-YA TFs interact with NF-YB and NF-YC subunits to regulate target genes containing the CCAAT box consensus sequence. In this study, using a yeast two-hybrid screen approach, we identified the NF-YB and NF-YC subunits able to interact with MtNF-YA1 and MtNF-YA2. In yeast (Saccharomyces cerevisiae) and in planta, we further demonstrated by both coimmunoprecipitation and bimolecular fluorescence complementation that these NF-YA, -B, and -C subunits interact and form a stable NF-Y heterotrimeric complex. Reverse genetic and chromatin immunoprecipitation-PCR approaches revealed the importance of these newly identified NF-YB and NF-YC subunits for rhizobial symbiosis and binding to the promoter of MtERN1 (for Ethylene Responsive factor required for Nodulation), a direct target gene of MtNF-YA1 and MtNF-YA2. Finally, we verified that a similar trimer is formed in planta by the common bean (Phaseolus vulgaris) NF-Y subunits, revealing the existence of evolutionary conserved NF-Y protein complexes to control nodulation in leguminous plants. This sheds light on the process whereby an ancient heterotrimeric TF mainly controlling cell division in animals has acquired specialized functions in plants.Instituto de Biotecnologia y Biologia Molecula