Functional Characterization Of Three GGDEF-Domain Proteins In Xanthomonas Oryzae Pv.oryzae

In this study, we addressed the role of cyclic di-GMP（c-di-GMP） signaling in virulence expression of Xanthomonas oryzae pv. oryzae（Xoo）, the causal agent of bacterial blight disease of rice. As well as being a plant pathogenic bacterium of global importance, Xoo is a model pathogenic organism for molecular studies of plant–microbe interactions. We have applied Xoo-rice as a model system to study the function of GGDEF-domain proteins, a diguanylate cyclase（DGC） responsible for the synthesis of c-di-GMP, by analysis of gene-knockout mutants of Xoo wild type strain PXO99 A. As a first step towards functionally characterizing only GGDEF-domain proteins in Xoo PXO99 A, we inactivated PXO₀0466, PXO₀2019 and PXO₀3378 by following non-marker mutagenesis strategy, thus three single gene-knockout mutants ?PXO₀0466, ?PXO₀2019 and ?PXO₀3378 were created. Further, using these genes with their different combinations three double（?PXO₀0466/?PXO₀2019, ?PXO₀0466/?PXO₀3378, and ?PXO₀2019/?PXO₀3378） and one triple（?PXO₀0466/?PXO₀2019/?PXO₀3378） gene-knockout mutants were generated by homologous recombination method. We then applied these single and multiple gene-knockout mutants for identifying their roles in regulating different virulence factors responsible for Xoo pathogenesis.To gauge any changes in the mutants to virulence traits, all the single, double and triple gene knockout mutants or WT strain were clip-inoculataed in the leaves of rice（Oryza sativa var. japonica）, and lesion lengths were measured. The results showed all mutants had increased lesion lengths of bacterial blight of rice. Such virulence assays showed consistent with the hypothesis that deletion of DGCs results in lower potential levels of c-di-GMP, leading to an increase in virulence. Our current study on virulence assay showed consistent with this hypothesis. In the swimmimg motility assay, all the single mutants, one double mutant（?PXO₀0466/?PXO₀2019）, and the triple mutant（?PXO₀0466/?PXO₀2019/?PXO₀3378） showed bigger swimmimg zone, whereas smaller swimmimg zone for the double mutants（?PXO₀0466/?PXO₀3378） and（?PXO₀2019/?PXO₀3378）. Bacterial attachment by biofilm formation was also examined. Interestingly, all the tested mutants showed enhanced biofilm production, in contrast to the single mutant ?PXO₀3378. To gauge another important virulence trait exopolysaccharides（EPS） production by the inactivated mutants, EPS secretion were quantified and showed significant differences between the mutants and WT. The single mutant ?PXO₀33788 and double mutants ?PXO₀0466/?PXO₀3378 and ?PXO₀2019/?PXO₀3378 exhibited reduced EPS productions.In summary, we have observed that deletion of PXO₀0466 and PXO₀2019 often caused similar phenotypical changes, and there were some additive effects in their double mutant, suggesting that PXO₀0466 and PXO₀2019 are potential functional DGCs to negatively regulate swimming motility and EPS production. In contrast, PXO₀3378 might not be an active DGC. Instead, it appeared to mediate the signaling pathway of PXO₀0466 and PXO₀2019 as a c-di-GMP receptor. Next, we will characterize their activities through biochemical approaches. These studies will help to elucidate the function of other GGDEF-domain encoding-genes in Xoo.