The PhyR-σEcfG signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum

PhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens. Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF sigma factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF sigma factor sigma(EcfG) are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and sigma(EcfG) control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein-protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/sigma(EcfG) regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium-legume interaction via yet undisclosed mechanisms.