Investigation Of The Mechanism By Which RpfC Regulats RpfF Expression In Xanthomonas Campestris Pathovar Campestris

Xanthomonas campestris pathovar campestris(Xcc)is the causal agent of the black rot disease of cruciferous plants including many important crops such as cabbage,Chinese cabbage,radish and rape.Xcc is one of the model bacterial pathogen for studying the molecular mechanism of microbe-host plant interactions.The pathogenicity of Xcc strictly relies on the production of several virulence factors including exopolysaccharide(EPS)and extracellular enzymes.The production of EPS and extracellular enzymes in Xcc is tightly regulated by the Rpf/DSF quorum sensing system(QS).The Xcc Rpf/DSF quorum sensing system,encoded by rpf(regulator of pathogenicity)gene cluster,is a cell-cell signaling system in which the Rpf C,Rpf G and Rpf F act as key players.Rpf F,an enoyl Co A hydratase,is responsible for the synthesis of a signal molecule known as diffusible signal factor(DSF).Rpf C and Rpf G constitute a two-component system and is responsible for the reception and transmission ofthe DSF signal,and control the production of EPS,extracellular enzymes,cell movement and biofilm formation.Shortly after the discovery of the Rpf/DSF quorum sensing system,Dr.Dow and his colleagues found that inactivation of rpf C resulted in the over-production of DSF via an unknown mechanism.Several years later,Professor Zhang Lian-Hui's Lab found that Rpf C can bind to the DSF synthesis enzyme Rpf F specifically and thus inhibits the enzyme activity of Rpf F.This finding can explain why inactivation of rpf C resulted in over-production of DSF.However,in addition to protein-protein interaction,is it also possible that Rpf C controls DSF synthesis by regulating the level of Rpf F? To answer this question,in this study,we use Western blotting analysis to detect and compare the accumulation of Rpf F protein in the wild-type strains and the rpf C deletion mutant.The results showed that the accumulation of Rpf F protein in the rpf C deletion mutant was ?2.8-fold higher than that in the wild-type strain,while the level of Rpf F in the complementary strain was similar to that of the wild-type strain,demonstrating that rpf C has the ability to inhibit the expression of Rpf F.More importantly,this result reveals that increasing of Rpf F protein level upon Rpf C lacking should be another reason for “inactivation of rpf C resulted in over-production of DSF”.To investigate the molecular basis for the increased accumulation of Rpf F upon rpf C deletion,we detected and compared the rpf F m RNA level in the wild-type strain and the rpf C deletion mutant using Northern blotting analysis.To our surprise,Northern results showed that the rpf F m RNA level in the wild-type strain and the rpf C deletion mutant are similar,indicating that the increase of Rpf F protein accumulation in the rpf C deletion mutant was not caused by the increase of rpf F m RNA level.In accordance with the Northern blotting results,GUS assay of the rpf F promoter-gus A fusion reporter strain revealed that the transcription and translation of rpf F were not affected by rpf C deletion.These results indicate that Rpf C may be able to accelerate Rpf F degradation.To confirm this hypothesis,we detected and compared the half-life of Rpf F in the wild-type strain and the rpf C deletion mutant by using quantitative Western blotting analysis.The results showed that the half-life of Rpf F protein in rpf C deletion mutants is approximately 16 times longer than that in wild-type strains(2 h vs.32 h).The mechanism by which Rpf C promoting Rpf F degradation remains to be further investigation.