Metabolomics and Transcriptomics Identify Multiple Downstream Targets of Paraburkholderia phymatum σ54 During Symbiosis with Phaseolus vulgaris

Abstract

RpoN (or σ54) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include α- and β-rhizobia. Our previous studies showed that an rpoN mutant of the β-rhizobial strain Paraburkholderia phymatum STM815T formed root nodules on Phaseolus vulgaris cv. Negro jamapa, which were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of P. phymatum, transcriptomics was combined with a powerful metabolomics approach. The metabolome of P. vulgaris root nodules infected by a P. phymatum rpoN Fix− mutant revealed statistically significant metabolic changes compared to wild-type Fix+ nodules, including reduced amounts of chorismate and elevated levels of flavonoids. A transcriptome analysis on Fix− and Fix+ nodules—combined with a search for RpoN binding sequences in promoter regions of regulated genes—confirmed the expected control of σ54 on nitrogen fixation genes in nodules. The transcriptomic data also allowed us to identify additional target genes, whose differential expression was able to explain the observed metabolite changes in numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the rpoN mutant, and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an ntrB mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype, as fewer but heavier nodules were formed on P. vulgaris roots.

Lack of the Flavin-Containing Monooxygenase FmoA Partially Impairs the Symbiotic Interaction of Mesorhizobium Huakuii with Astragalus Sinicus

“…Flagellar motility is a critical environmental adaptation for the plant-associated bacteria such as Rhizobium that allows bacteria to escape adverse conditions and populate new environments [27]. The upregulation of flagellar genes in this case might be due to the hostile environment in which the bacteroids with low nitrogenase activity are embedded [28]. In the fmoA mutant-induced nodules, many symbiosomes were aberrant and the bacteroid membrane showed incrassation (Fig.…”

Functional Genomics Approaches to Studying Symbioses between Legumes and Nitrogen-Fixing Rhizobia

“…Besides genes known to be involved in nitrogen assimilation, such as the key regulatory gene in control of nitrogen metabolism ntrC, amtB (encoding an ammonium transporter), and the ure cluster (encoding a urease), genes associated with important traits for legume infection, such as exopolysaccharide (EPS) production and motility, were upregulated [ 43 ]. Moreover, a recent comparative transcriptome analysis between P. vulgaris nodules induced by P. phymatum wild-type (Fix + ) and by an rpoN mutant (Fix − ) strain has confirmed the importance of RpoN in controlling the expression of nif genes and identified potential additional target genes of this alternative sigma factor in nodules [ 70 ].…”

“…Recently, the first metabolomic study of the beta-rhizobia P. phymatum during symbiosis with P. vulgaris was published [ 70 ]; it showed that nodules formed by beta-rhizobia also accumulated compounds such as glutamine, chorismate, and arginine, when compared with the roots. In the same study, P. vulgaris nodules infected by the wild type (Fix + ) and by the Fix − rpoN mutant strain showed the accumulation of flavonoids in the nodules elicited by the Fix − strain.…”

“…Thus far, to our knowledge, only one study has integrated different -omics technologies for studying beta-rhizobial symbiosis. Therein, transcriptomics and metabolomics were applied to shed light on the role of the key regulator of symbiosis P. phymatum RpoN in P. vulgaris nodules [ 70 ]. In several cases, metabolite measurements in Fix + (wild type) and Fix − ( rpoN mutant) nodules confirmed transcript changes observed by RNA-seq analysis.…”

Plant Metabolomics in the Global Scenario of Food Security: A Systems-Biology Approach for Sustainable Crop Production

“…Similarly, symbiotic interactions represent another relevant issue. Bean ( Phaseoulus vulgaris ) root nodules inoculated with a mutant strain of the rhizobium, Paraburkholderia phymatum , revealed significantly higher levels of flavonoids, a class of phytoalexins [ 9 ]. The topic of plant resistance to pests was also studied.…”