GuaB Activity Is Required in Rhizobium tropici During the Early Stages of Nodulation of Determinate Nodules but Is Dispensable for the Sinorhizobium meliloti–Alfalfa Symbiotic Interaction

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate the timing of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.Fil: Collavino, Mónica Mariana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Riccillo, Pablo M.. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Grasso, Daniel Horacio. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Crespi, Martín. Centre National de la Recherche Scientifique; FranciaFil: Aguilar, Orlando Mario. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentin

Rhizobium tropici response to acidity involves activation of glutathione synthesis

Rhizobium tropici CIAT899 displays intrinsic tolerance to acidity, and efficiently nodulates Phaseolus vulgaris at low pH. By characterizing a gshB mutant strain, glutathione has been previously demonstrated to be essential for R. tropici tolerance to acid stress. The wild-type gshB gene region has been cloned and its transcription profile has been characterized by using quantitative real-time PCR and transcriptional gene fusions. Activation of the gshB gene under acid-stress conditions was demonstrated. gshB is also induced by UV irradiation. Upstream from gshB a putative σ 70 promoter element and an inverted repeat sequence were identified, which are proposed to be involved in expression under neutral and acidic conditions, respectively. Gel retardation assays indicate that transcription in acid conditions may involve protein binding to an upstream regulatory region.Facultad de Ciencias Exacta

Rhizobium tropici response to acidity involves activation of glutathione synthesis

Rhizobium tropici CIAT899 displays intrinsic tolerance to acidity, and efficiently nodulates Phaseolus vulgaris at low pH. By characterizing a gshB mutant strain, glutathione has been previously demonstrated to be essential for R. tropici tolerance to acid stress. The wild-type gshB gene region has been cloned and its transcription profile has been characterized by using quantitative real-time PCR and transcriptional gene fusions. Activation of the gshB gene under acid-stress conditions was demonstrated. gshB is also induced by UV irradiation. Upstream from gshB a putative σ 70 promoter element and an inverted repeat sequence were identified, which are proposed to be involved in expression under neutral and acidic conditions, respectively. Gel retardation assays indicate that transcription in acid conditions may involve protein binding to an upstream regulatory region.Facultad de Ciencias Exacta

The product of the <i>Rhizobium meliloti ilvC</i> gene is required for isoleucine and valine synthesis and nodulation of alfalfa

Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment.- Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv⁻ Nod⁻ mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli DJvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv⁻ and Nod⁻) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa.Facultad de Ciencias Exacta

The product of the <i>Rhizobium meliloti ilvC</i> gene is required for isoleucine and valine synthesis and nodulation of alfalfa

Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment.- Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv⁻ Nod⁻ mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli DJvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv⁻ and Nod⁻) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa.Facultad de Ciencias Exacta

Área de Producciones Intensivas. Acciones y resultados

El siguiente informe presenta los resultados de la Jornada Invernal de Lechuga desarrollada en agosto de 2022 en el Área de Producciones Intensivas del CERET en General Pico. La jornada fue organizada por el Ministerio de la Producción del Gobierno de la provincia de La Pampa, el CERET y la Agencia de Extensión INTA General Pico.AER General PicoFil: Muguiro, Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Anguil. Agencia de Extensión Rural General Pico; ArgentinaFil: Pechin, Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Anguil. Agencia de Extensión Rural General Pico; ArgentinaFil: Grasso, Rodolfo. Universidad Nacional de Rosario; Argentin

GuaB activity is required in Rhizobium tropici during the early stages of nodulation of determinate nodules but is dispensable for the sinorhizobium meliloti-alfalfa symbiotic interaction

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate the timing of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.Instituto de Biotecnologia y Biologia Molecula

Early symbiotic responses induced by Sinorhizobium meliloti ilvC mutants in alfalfa

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype, ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC‒ background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC‒ strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

Rhizobium tropici response to acidity involves activation of glutathione synthesis

Rhizobium tropici CIAT899 displays intrinsic tolerance to acidity, and efficiently nodulates Phaseolus vulgaris at low pH. By characterizing a gshB mutant strain, glutathione has been previously demonstrated to be essential for R. tropici tolerance to acid stress. The wild-type gshB gene region has been cloned and its transcription profile has been characterized by using quantitative real-time PCR and transcriptional gene fusions. Activation of the gshB gene under acid-stress conditions was demonstrated. gshB is also induced by UV irradiation. Upstream from gshB a putative σ 70 promoter element and an inverted repeat sequence were identified, which are proposed to be involved in expression under neutral and acidic conditions, respectively. Gel retardation assays indicate that transcription in acid conditions may involve protein binding to an upstream regulatory region.Facultad de Ciencias Exacta

Early symbiotic responses induced by Sinorhizobium meliloti ilvC mutants in alfalfa

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype, ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC‒ background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC‒ strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta