RavA/VemR Two Component System Regulates Virulence And Motility Of Xanthomonas Campestris Pv. Campestris

Xanthomonas campestris pv.campestris(Xcc)can cause black rot in cruciferous plants.The virulence of Xcc depends on various virulence factors,including extracellular polysaccharides,extracellular enzymes,biofilms,exercise capacity,and type III secretion system effector proteins.Xcc has a unipolar flagellum as its locomotor organ.With the help of chemokines,it moves in the direction of the environment that is conducive to its own survival,so that bacteria can achieve advantages and avoid harmness.Meanwhile,the fimbriae can adhere to the semi-solid surface,which is conducive to the movement of bacteria in the plant.Flagella-mediated Swimming depends on the expression of flagellin,and the expression of flagellin fli C is regulated by two σ factors(Rpo N2 and Fli A).Two-component systems(TCSs)are key systems for bacteria to adapt to the external environment(including the host).It consists of signal-sensing components histidine kinase(HK)and response regulator(RR).Vem R is an atypical RR,which has only a phosphorylation receiving domain and no output domains.Our previous research found that Vem R is an important regulator of Xcc motility and virulence,but its cognate HK is not yet clear.There is no gene encoding HK in the context of vem R genome,so its cognate HK cannot be predicated by bioinformatics.We previously found that rav A mutation changed bacterial pathogenicity,motility and other phenotypic characteristics,which is consistent with the vem R mutant phenotypes,suggesting that Rav A may be the cognate HK of Vem R.This study aims to clarify the regulation pathway of bacterial virulence and flagellin fli C expression by the vem R,and to find the cognate HK of Vem R.The main results are as follows:⑴ Based on the bacterial two-hybrid assays,protein pull-down experiments and rpo N2 promoter activities,we found that Vem R can interact with Fle Q to regulate the expression of rpo N2-vem R-fle Q operon.⑵ The results of genetic and biochemical experiments(bacterial two-hybrid assays,protein pull-down experiments and phosphorylation assays)also showed that Rav A can physically interact with Vem R and phosphorylate Vem R.Therefore,we identified Rav A as the paired HK of Vem R.⑶ Using promoter-reporter systems to detect gene promoter activities,we found that RavA/VemR TCS regulates the expression of flh F-fle N-fli A via Rpo N2/Fle Q,and regulates fli C expression,thereby affecting flagellar movement.⑷ We also analyzed the relationship between RavA/VemR and the global regulatory factor Clp and found that Clp was located downstream of RavA/VemR to regulate the pathogenicity of Xcc,but both independently regulate the flagellar movement.⑸ Based on the phenotypes such as virulence and some virulence-related factors(including motility,biofilm formation,EPS synthesis,and extracellular enzyme activity)of vem R mutants,we suggest that Vem R may be similar to Clp,and act as a global regulator that regulates Xcc various behavious.In summary,RavA/VemR TCS regulates the activity of Fle Q,and infulences the expression of rpo N2-vem R-fle Q.The changes of Fle Q expression and activity can regulate the promoter activity of flh F-fle N-fli A operons,which can regulate fli C expression and affect Xcc motility and virulence.