Estudio de la participación de la fosfatidilinositol 3-cinasa durante la interacción simbiótica entre <i>Phaseolus vulgaris</i> y <i>Rhizobium tropici</i>

La nodulación es un modelo muy interesante de la diferenciación celular y del desarrollo en plantas así como también de la interacción de las leguminosas con microorganismos simbiontes. En nuestro grupo estudiamos los mecanismos de señalización durante la organogénesis de los nódulos en las raíces de Phaseolus vulgaris L. (poroto común, también conocido como frijol). El poroto es de gran importancia económica y una fuente de proteínas para la nutrición de más de 500 millones de personas en los países en desarrollo (Graham y Vance, 2003). Las leguminosas establecen asociaciones simbióticas con bacterias gram-negativas (rizobios) y con hongos arbusculares (HA) que facilitan la adquisición de nutrientes tales como el nitrógeno y el fósforo. La simbiosis entre las leguminosas y los rizobios da lugar a la formación de nódulos en la raíz y a la fijación simbiótica de nitrógeno (Denarie y Cullimore, 1993). La interacción del microsimbionte se inicia con un diálogo molecular que involucra flavonoides liberados por la raíz, los cuales inducen en la bacteria la síntesis de un lipo-quitooligosacárido conocido como Factor Nod (FN), el cual es a su vez es liberado por la bacteria hacia la raíz. El FN actúa como un regulador de crecimiento de la planta, disparando los procesos de diferenciación que culminan con la formación de las estructuras simbióticas, llamadas nódulos. Dentro de los nódulos las bacterias simbióticas se diferencian y se engloban en simbiosomas, los cuales son estructuras especializadas intracelulares desarrolladas de novo dentro de nódulos de las raíces (Oldroyd y Downie, 2008).Facultad de Ciencias Exacta

Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume <it>Phaseolus vulgaris </it>(common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of <it>Phaseolus vulgaris</it>.</p> <p>Results</p> <p>Small RNA libraries were generated from roots, flowers, leaves, and seedlings of <it>P. vulgaris</it>. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies.</p> <p>Conclusions</p> <p>This work represents the first massive-scale RNA sequencing study performed in <it>Phaseolus vulgaris </it>to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of <it>P. vulgaris </it>miRNAs in relation to those of other legumes.</p

Phosphatidylinositol 3-kinase function at very early symbiont perception: a local nodulation control under stress conditions?

Root hair curling is an early and essential morphological change required for the success of the symbiotic interaction between legumes and rhizobia. At this stage rhizobia grow as an infection thread within root hairs and are internalized into the plant cells by endocytosis, where the PI3K enzyme plays important roles. Previous observations show that stress conditions affect early stages of the symbiotic interaction, from 2 to 30 min post-inoculation, which we term as very early host responses, and affect symbiosis establishment. Herein, we demonstrated the relevance of the very early host responses for the symbiotic interaction. PI3K and the NADPH oxidase complex are found to have key roles in the microsymbiont recognition response, modulating the apoplastic and intracellular/endosomal ROS induction in root hairs. Interestingly, compared with soybean mutant plants that do not perceive the symbiont, we demonstrated that the very early symbiont perception under sublethal saline stress conditions induced root hair death. Together, these results highlight not only the importance of the very early host-responses on later stages of the symbiont interaction, but also suggest that they act as a mechanism for local control of nodulation capacity, prior to the abortion of the infection thread, preventing the allocation of resources/energy for nodule formation under unfavorable environmental conditions.Instituto de Fisiología y Recursos Genéticos VegetalesFil: Robert, German. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Cátedra de Fisiología Vegetal; ArgentinaFil: Muñoz, Nacira Belen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Cátedra de Fisiología Vegetal; ArgentinaFil: Alvarado-Affantranger, Xochitl. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Saavedra, Laura. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Cátedra de Fisiología Vegetal; ArgentinaFil: Davidenco, Vanina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Cátedra de Fisiología Vegetal; ArgentinaFil: Rodríguez-Kessler, Margarita. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Estrada-Navarrete, Georgina. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Sanchez, Federico. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Lascano, Hernan Ramiro. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Cátedra de Fisiología Vegetal; Argentin

Nodulin 41, a novel late nodulin of common bean with peptidase activity

<p>Abstract</p> <p>Background</p> <p>The legume-rhizobium symbiosis requires the formation of root nodules, specialized organs where the nitrogen fixation process takes place. Nodule development is accompanied by the induction of specific plant genes, referred to as nodulin genes. Important roles in processes such as morphogenesis and metabolism have been assigned to nodulins during the legume-rhizobium symbiosis.</p> <p>Results</p> <p>Here we report the purification and biochemical characterization of a novel nodulin from common bean (<it>Phaseolus vulgaris </it>L.) root nodules. This protein, called nodulin 41 (PvNod41) was purified through affinity chromatography and was partially sequenced. A genomic clone was then isolated via PCR amplification. PvNod41 is an atypical aspartyl peptidase of the A1B subfamily with an optimal hydrolytic activity at pH 4.5. We demonstrate that PvNod41 has limited peptidase activity against casein and is partially inhibited by pepstatin A. A PvNod41-specific antiserum was used to assess the expression pattern of this protein in different plant organs and throughout root nodule development, revealing that PvNod41 is found only in bean root nodules and is confined to uninfected cells.</p> <p>Conclusions</p> <p>To date, only a small number of atypical aspartyl peptidases have been characterized in plants. Their particular spatial and temporal expression patterns along with their unique enzymatic properties imply a high degree of functional specialization. Indeed, PvNod41 is closely related to CDR1, an <it>Arabidopsis thaliana </it>extracellular aspartyl protease involved in defense against bacterial pathogens. PvNod41's biochemical properties and specific cell-type localization, in uninfected cells of the common bean root nodule, strongly suggest that this aspartyl peptidase has a key role in plant defense during the symbiotic interaction.</p

An Autophagy-Related Kinase Is Essential for the Symbiotic Relationship between Phaseolus vulgaris and Both Rhizobia and Arbuscular Mycorrhizal Fungi

Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis. Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAi-mediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganismsInstituto de Fisiología y Recursos Genéticos VegetalesFil: Estrada-Navarrete, Georgina. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Cruz-Mireles, Neftaly. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Lascano, Hernán Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Estudios Agropecuarios (UDEA); ArgentinaFil: Lascano, Hernán Ramiro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales. ArgentinaFil: Alvarado-Affantranger, Xóchitl. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Laboratorio Nacional de Microscopía Avanzada; MéxicoFil: Hernández-Barrera, Alejandra. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Barraza, Aarón. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Olivares, Juan E. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Arthikala, Manoj-Kumar. Universidad Nacional Autónoma de México. Escuela Nacional de Estudios Superiores-Unidad León; MéxicoFil: Cárdenas, Luis. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Quinto, Carmen. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; MéxicoFil: Sanchez, Federico. Universidad Nacional Autónoma de México. Instituto de Biotecnología. Departamento de Biología Molecular de Plantas; Méxic

A comprehensive, improved protocol for generating common bean (Phaseolus vulgaris L.) transgenic hairy roots and their use in reverse-genetics studies.

Generating transgenic hairy roots has been the preferred strategy for molecular studies in common bean (Phaseolus vulgaris L.), since generating stable knockout lines in this species is challenging. However, the number of plants producing hairy roots following the original protocol published in 2007 is usually low, which has impeded progress. Since its initial publication, the original protocol has been extensively modified, but these modifications have not been adequately or systematically reported, making it difficult to assess the reproducibility of the method. The protocol presented here is an update and expansion of the original method. Importantly, it includes new, critical steps for generating transgenic hairy roots and using them in molecular analyses based on reverse-genetics approaches. Using this protocol, the expression of two different genes, used as an example, was significantly increased or decreased in approximately 30% of the transformed plants. In addition, the promoter activity of a given gene was observed, and the infection process of rhizobia in transgenic hairy roots was monitored successfully. Thus, this improved protocol can be used to upregulate, downregulate, and perform promoter activity analysis of various genes in common bean transgenic hairy roots as well as to track rhizobia infection

An Autophagy-Related Kinase Is Essential for the Symbiotic Relationship between Phaseolus vulgaris

Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis. Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAi-mediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganisms

The Class II trehalose 6-phosphate synthase gene PvTPS9 modulates trehalose metabolism in Phaseolus vulgaris nodules.

Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS) of common bean (Phaseolus vulgaris), was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1%) of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant

Agrobacterium rhizogenes transformation of the Phaseolus spp.: A tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: R coccineus, P lunatus, and P acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions