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When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N2 in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH4NO3, the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system. The symbiotic N2 fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N2 fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation., Instituto de Biotecnología y Biología Molecular, Facultad de Ciencias Agrarias y Forestales

To achieve fertilization, pollen tubes have to protect and properly deliver sperm cells through the pistil to the ovules. Pollen tube growth is a representative example of polarized growth where new components of the cell wall and plasma membrane are continuously deposited at the tip of the growing cell. The integrity of the cell wall is of fundamental importance to maintain apical growth. For this reason, pollen tube growth has become an excellent model to study the role of polysaccharides and structural cell wall proteins involved in polar cell expansion. However, quantification of structural polysaccharides at the pollen tube cell wall has been challenging due to technical complexity and the difficulty of finding specific dyes. Here, we propose simple methods for imaging and quantification of callose, pectin, and cellulose using specific dyes such as Aniline Blue, Propidium Iodide, and Pontamine Fast Scarlet 4B. Fil: Sede, Ana Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Wengier, Diego Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Borassi, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Estevez, Jose Manuel. Instituto del Milenio para la Biología Integrativa; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Andrés Bello; Chile Fil: Muschietti, Jorge Prometeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina

La plasticidad estructural de los carbohidratos es mayor a la de los aminoácidos, pero nuestro entendimiento de cómo esa plasticidad se relaciona con la funcionalidad biológica es aún muy limitada. Los pelos radicales y los tubos polínicos son células individuales que requieren una gran síntesis de pared celular para poder sostener la expansión apical también denominada crecimiento apical. Las paredes celulares en las células vegetales son estructuras complejas compuestas en su mayoría por polisacáridos, incluyendo una red de microfibrillas de celulosa-xiloglucanos, pectinas y O-glicoproteínas ricas en hidroxiprolina (HRGPs) tales como extensinas (EXTs) y arabinogalactanos asociados a proteínas (AGPs). En esta tesis se propone individualizar la respuesta de señalización que permite el crecimiento polarizado en tubos polínicos en Arabidopsis thaliana. Mediante la utilización de redes transcripcionales modulares ya hemos identificado componentes necesarios para el crecimiento polarizado en tubos polínicos. Estas redes establecen un punto de partida para explorar la funcionalidad biológica de los HRGPs, extensinas y receptores quinasa con dominio extracelular de tipo extensina (PERK; proline-rich extensin-like receptor kinase) asociados al crecimiento polarizado. En esta tesis estudiamos en tubos polínicos cómo las proteínas PERK afectan al crecimiento polarizado mediante la inhibición bioquímica y por métodos de genética reversa. Los genes candidatos fueron analizados en un contexto más amplio mediante métodos moleculares específicos y microscopía confocal. The structural plasticity of carbohydrates is greater than that of amino acids but our understanding for how this plasticity relates to biological function is still limited. Root hairs and pollen tubes are single cells that require intensive cell-wall amendments to accommodate apical cell expansion in a process known as tip growth. Plant cell walls are intricate structures comprised largely of polysaccharides, including a network of cellulose microfibrils-xyloglucans, pectins, and of highly glycosylated hydroxyproline-rich O-glycoproteins (HRGPs), such as extensins (EXTs) and arabinogalactan proteins (AGPs). We propose to functionally dissect the signaling feedback loop that allows for polarized growth in Arabidopsis thaliana. We have employed transcriptional modular networks to identify components necessary for polarized growth of pollen tubes. These predictions provide a framework to explore the biological function of HRGPs and proline-rich extensin-like receptor kinase (PERK) involved in tip growth. We targeted these processes by biochemical inhibition and by different modes of reverse genetics, and assessed blockage of polarized growth. Suitable candidate genes were put into a larger biological context through targeted molecular approaches and through high-definition confocal microscopy. Fil: Borassi, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina