Project description:The Bradyrhizobium japonicum NtrC regulatory protein influences gene expression in response to changes in intracellular nitrogen status. Under conditions of low nitrogen, phosphorylation of NtrC results in up-regulation of a number of genes involved in nitrogen metabolism and nitrogen acquisition. To better define the exact nature of NtrC’s influence on gene expression, a ntrC mutation was created in B. japonicum and transcriptional profiling was performed by DNA microarray analysis of both the mutant and wild type strains. Bradyrhizobium japonicum USDA 110 and a ntrC mutant in the USDA 110 background were cultured in minimal medium supplemented with either 10mM glutamate (low nitrogen) or 10mM ammonium and 10mM glutamate (high nitrogen) as nitrogen sources. Four comparisons were performed: wild type high nitrogen vs. mutant high nitrogen, wild type low nitrogen vs. wild type high nitrogen, wild type low nitrogen vs. mutant low nitrogen, and mutant low nitrogen vs. mutant high nitrogen. For each of the four comparisons, three biological replicates were prepared for each strain and dye swap replications were performed for each hybridization producing a total of six arrays per comparison and 24 arrays in total.
Project description:An initializing step in the rhizobia-legume symbiosis is the secretion of flavonoids by plants that leads to the expression of nodulation genes in rhizobia. Here we report the genome-wide transcriptional response of Bradyrhizobium japonicum to genistein, an isoflavone secreted by soybean. About 100 genes were induced in the wild type. This included all nod box-associated genes, the flagellar cluster and several genes that are likely to be involved in transport processes. To elucidate the role of known regulators, we analysed mutant strains. This revealed that the two-component response regulator NodW is essential for induction of almost all genistein-inducible genes, with the exception of 8 genes. The phenotype of the nodW mutant could be partially suppressed by overexpression of NwsB, which is also a two-component response regulator. These data indicate that genistein has a much broader function than mere induction of nod genes. Keywords: transcriptional analysis, genistein induction Overall design: 3 biological replicates of wild type (uninduced) and mutant strain 613 (nodW mutant; induced with genistein) were compared.
Project description:An initializing step in the rhizobia-legume symbiosis is the secretion of flavonoids by plants that leads to the expression of nodulation genes in rhizobia. Here we report the genome-wide transcriptional response of Bradyrhizobium japonicum to genistein, an isoflavone secreted by soybean. About 100 genes were induced in the wild type. This included all nod box-associated genes, the flagellar cluster and several genes that are likely to be involved in transport processes. To elucidate the role of known regulators, we analysed mutant strains. This revealed that the two-component response regulator NodW is essential for induction of almost all genistein-inducible genes, with the exception of 8 genes. The phenotype of the nodW mutant could be partially suppressed by overexpression of NwsB, which is also a two-component response regulator. These data indicate that genistein has a much broader function than mere induction of nod genes. Keywords: transcriptional analysis, genistein induction Overall design: 3 biological replicates of wild type uninduced and wild type induced with genistein were compared.
Project description:An initializing step in the rhizobia-legume symbiosis is the secretion of flavonoids by plants that leads to the expression of nodulation genes in rhizobia. Here we report the genome-wide transcriptional response of Bradyrhizobium japonicum to genistein, an isoflavone secreted by soybean. About 100 genes were induced in the wild type. This included all nod box-associated genes, the flagellar cluster and several genes that are likely to be involved in transport processes. To elucidate the role of known regulators, we analysed mutant strains. This revealed that the two-component response regulator NodW is essential for induction of almost all genistein-inducible genes, with the exception of 8 genes. The phenotype of the nodW mutant could be partially suppressed by overexpression of NwsB, which is also a two-component response regulator. These data indicate that genistein has a much broader function than mere induction of nod genes. Keywords: transcriptional analysis, genistein induction Overall design: 3 biological replicates of wild type (uninduced) and mutant strain Δ901 (overexpressed nwsB; induced with genistein) were compared.
Project description:An initializing step in the rhizobia-legume symbiosis is the secretion of flavonoids by plants that leads to the expression of nodulation genes in rhizobia. Here we report the genome-wide transcriptional response of Bradyrhizobium japonicum to genistein, an isoflavone secreted by soybean. About 100 genes were induced in the wild type. This included all nod box-associated genes, the flagellar cluster and several genes that are likely to be involved in transport processes. To elucidate the role of known regulators, we analysed mutant strains. This revealed that the two-component response regulator NodW is essential for induction of almost all genistein-inducible genes, with the exception of 8 genes. The phenotype of the nodW mutant could be partially suppressed by overexpression of NwsB, which is also a two-component response regulator. These data indicate that genistein has a much broader function than mere induction of nod genes. Keywords: transcriptional analysis, genistein induction 3 biological replicates of wild type (uninduced) and mutant strain 613 (nodW mutant; induced with genistein) were compared.
Project description:An initializing step in the rhizobia-legume symbiosis is the secretion of flavonoids by plants that leads to the expression of nodulation genes in rhizobia. Here we report the genome-wide transcriptional response of Bradyrhizobium japonicum to genistein, an isoflavone secreted by soybean. About 100 genes were induced in the wild type. This included all nod box-associated genes, the flagellar cluster and several genes that are likely to be involved in transport processes. To elucidate the role of known regulators, we analysed mutant strains. This revealed that the two-component response regulator NodW is essential for induction of almost all genistein-inducible genes, with the exception of 8 genes. The phenotype of the nodW mutant could be partially suppressed by overexpression of NwsB, which is also a two-component response regulator. These data indicate that genistein has a much broader function than mere induction of nod genes. Keywords: transcriptional analysis, genistein induction 3 biological replicates of wild type uninduced and wild type induced with genistein were compared.
Project description:An initializing step in the rhizobia-legume symbiosis is the secretion of flavonoids by plants that leads to the expression of nodulation genes in rhizobia. Here we report the genome-wide transcriptional response of Bradyrhizobium japonicum to genistein, an isoflavone secreted by soybean. About 100 genes were induced in the wild type. This included all nod box-associated genes, the flagellar cluster and several genes that are likely to be involved in transport processes. To elucidate the role of known regulators, we analysed mutant strains. This revealed that the two-component response regulator NodW is essential for induction of almost all genistein-inducible genes, with the exception of 8 genes. The phenotype of the nodW mutant could be partially suppressed by overexpression of NwsB, which is also a two-component response regulator. These data indicate that genistein has a much broader function than mere induction of nod genes. Keywords: transcriptional analysis, genistein induction 3 biological replicates of wild type (uninduced) and mutant strain Δ901 (overexpressed nwsB; induced with genistein) were compared.
Project description:The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp. strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment. The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD1, nodD2, and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Nodulation assays of nodD1, nodD2, or nolA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers. The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF. nodD1 mutants displayed a 2-day nodulation delay, whereas nodD2 strains were indistinguishable from the wild type. Translational nodA-lacZ, nodD1-lacZ, nodD2-lacZ, and nolA-lacZ fusions were created. Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein. In a nodD1 or nodD2 background, basal expression of the nodA-lacZ fusion increased two- to threefold. The level of expression of the nodD2-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD1, nodD2, and nodD1 nodD2 backgrounds independently of the addition of the inducer genistein. nolA was required for increased expression of the nodD2-lacZ fusion. These data suggest that a common factor is involved in the regulation of nodD2 and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp. strain NC92 in which negative regulation is mediated by the products of the nodD1 and nodD2 genes.
Project description:Soybean plants establish symbiotic relationships with soil rhizobia which form nodules on the plant roots. Nodule formation starts when the plant roots exudate isoflavonoids that induce nod gene expression of a specific Bradyrhizobium. We examined the specific indigenous rhizobia that form nodules with the soybean cultivars Peking and Tamahomare in different soils. PCR-RFLP analysis targeted to the 16S-23S rRNA gene internal transcribed spacer (ITS) region of the bacterial type of each root nodule showed that Bradyrhizobium japonicum (USDA110-type) and Bradyrhizobium elkanii (USDA94-type) had high compatibility with the Tamahomare and Peking cultivars, respectively. We grew 93 recombinant inbred lines (RIL) of soybean seeds derived from the cross between Peking and Tamahomare in three different field soils and identified the indigenous rhizobia nodulating each line using the same PCR-RFLP analysis. QTL analysis identified one QTL region in chromosome-18 with a highly significant additive effect that controls compatibility with both B. japonicum USDA110 and B. elkanii USDA94. We also measured the amount of daidzein and genistein secretion from roots of the 93 RILs by HPLC analysis. QTL analysis showed one QTL region in chromosome-18 controlling genistein secretion from roots and coinciding with that regulating compatibility of specific indigenous rhizobia with soybean. The amount of genistein may be a major regulatory factor in soybean-rhizobium compatibility.
Project description:Initial interaction between rhizobia and legumes actually starts via encounters of both partners in the rhizosphere. In this study, the global expression profiles of Bradyrhizobium japonicum USDA 110 in response to soybean (Glycine max) seed extracts (SSE) and genistein, a major soybean-released isoflavone for nod genes induction of B. japonicum, were compared. SSE induced many genomic loci as compared with genistein (5.0 microM), nevertheless SSE-supplemented medium contained 4.7 microM genistein. SSE markedly induced four predominant genomic regions within a large symbiosis island (681 kb), which include tts genes (type III secretion system) and various nod genes. In addition, SSE-treated cells expressed many genomic loci containing genes for polygalacturonase (cell-wall degradation), exopolysaccharide synthesis, 1-aminocyclopropane-1-carboxylate deaminase, ribosome proteins family and energy metabolism even outside symbiosis island. On the other hand, genistein-treated cells exclusively showed one expression cluster including common nod gene operon within symbiosis island and six expression loci including multidrug resistance, which were shared with SSE-treated cells. Twelve putatively regulated genes were indeed validated by quantitative RT-PCR. Several SSE-induced genomic loci likely participate in the initial interaction with legumes. Thus, these results can provide a basic knowledge for screening novel genes relevant to the B. japonicum- soybean symbiosis.