| Xanthomonas campestris pathovar campestris(Xcc)is one of the most important pathogens and the causal agent of black rot of crucifers that affects all cultivated brassicas.The virulence of Xcc towards plants depends on pathogenic factors that include extracellular enzyme,EPS,type three effectors and motility.The current research showed that pathogenic factors in Xcc is regulated by the single-domain response regulators(SD-RRs),such as Vem R regulates mobility and EPS.There are sixty genes annotated as single-domain response regulators in the genome of Xanthomonas campestris pv.campestris(Xcc)and it is unknown about their function and molecular mechanism on pathogenic factors except reported Vem R.Two of these mutants with Tn5 gus A5 insertions in the open reading frames XC＿1183 and XC＿1184 were implicated in motility,and they encoded two SDRRs PilG and PilH.Here,we present evidence demonstrating the role of two SDRRs PilG and PilH in the antagonistic control of swimming motility and swarming motility in Xcc.Using informative mutagenesis,we reveal PilG positively regulates swimming motility while and negatively regulating swarming motility.Conversely,PilH negatively regulates swimming behaviour while and positively regulating swarming motility.Additionally,we present evidence,using mutagenesis that PilG and PilH are involved in other cellular processes,including cell adhesion,chemotaxis and virulence.By transcriptome analyses we confirm these observations as PilG is shown to downregulate many genes involved chemotaxis and flagellar biosynthesis but these similar genes were upregulated by PilH.To further study the relationship between bacterial function and PilG or PilH,PilG confirmed directly interaction with 7 proteins and PilH was tested it showed interactions with 7 protein by the co-immunoprecipitation(Co-IP)and bacterial two-hybrid experiment.These proteins interacted with PilG or PilH were shown to have roles in signalling,regulation and importantly motility.Moreover,PilG’s target protein Che A(XC＿2284)(or PilH’s target protein XC＿1050)maybe compose a two-component system with PilG(or PilH)as a histidine kinase,so a pull-down biotinylated protein-protein assay was performed and showed that Che A(or XC＿1050)accordingly captured more PilG(or PilH)in the presence of ATP.These findings suggested that PilG and PilH may function as an intermediary in signal transduction.In order to further analyze impact of the target proteins,we selected one of proteins for target proteins of PilG and PilH to perform mutant analysis and found that PilG’s target protein Pil B(XC＿1060)negatively regulates swarming motility,while and PilH’s target protein Pil I(XC＿1185),like PilH,positively regulates swarming motility and negatively regulates swimming in Xcc,suggesting that PilG and PilH may affect motility by interaction with target proteins.To further investigate the role of conserved sites in function,we assessed the effects of Asp-21 and Asp-64 of PilG,and Asp-9 and Asp-52 of PilH on basis of the results using bioinformatics analysis.Interestingly,no change were seen in swarming motility when the Asp-21 and Asp-64 of PilG were substituted,but alterations were seen in swimming motility,indicating that they were two conserved sites of PilG;alterations were seen in swarming and swimming motility as well as the pilH mutant when the Asp-9 and Asp-52 of PilH were substituted,indicating that they were two conserved sites of PilH.In addition to PilG and PilH,we found that SD-RR XC＿1160 was related to swarming motility and EPS in previous work whereas unknown mechanism,and then explored the regulatory mechanism of XC＿1160 in molecular level.Firstly,we confirm XC＿1160 interacts with Col S and XC＿1355 in proteome of Xcc by the co-immunoprecipitation(co-IP)and bacterial two-hybrid.Col S,a classical histidine kinase,interacted with XC＿1160 to be stronger in the presence of ATP by the pull-down biotinylated protein-protein assay and ITC,indicating that XC＿1160 and Col S are shown to have roles in signal transduction.Double mutation in XC＿1160 and Col S reveal that production of EPS similar to wild type while and XC＿1160 positively regulating,furthermore,a drop in production of EPS was observed by overexpression of Col S in Xcc.These findings indicated that XC＿1160 and Col S contribute to the regulation of EPS and this relationship appears to be epistatic,namely,XC＿1160 reduced the phosphorylation of Col S to regulate production of EPS.However,Col S and XC＿1160 were no impact on the expression of gum gene and further research is how to affect production of EPS.XC＿1355,a DNA binding protein and interacted with XC＿1160,positively regulates swarming motility and has an influence over the expression of genes involved in motility,but it’s unclear whether XC＿1160 impacts the expression of motility-related genes by XC＿1355.In order to further investigate the role of conserved sites,we performed functional analysis on the possible conserved sites(D15,S58,D59,T87,K108)of XC＿1160 protein.The results showed that no change was seen in swarming motility and EPS when the Ser-58 of XC＿1160 were substituted,but others were no significance for the regulation of swarming motility but had a production of EPS similar to the mutant of XC＿1160,indicating that four conserved sites are important for function and the relationship need to be more in-depth study between protein structure and function.In conclusion,this study mainly analyzed the regulatory mechanism of three SD-RRs for pathogenic factors motility and EPS in Xcc and the effect of their conserved sites on protein function.This research enriched the regulatory mechanism of SD-RRs for motility and EPS in pathogens,and provided a theoretical basis for the prevention on black rot of crucifers in the future. |
