Investigation Of The Effect Of RpfC Deletion On HrpX And HrpG Expression In Xanthomonas Campestris Pv.campestris

Xanthomonas campestris pathovar campestris(Xcc),the causal agent of the black rot disease of cruciferous crops including kale,cabbage and radish,is a model bacterium for studying the molecular mechanism of the interactions between plant pathogenic bacteria and their host plants.It has been shown that the Rpf/DSF quorum sensing system(QS),which is encoded by the rpf(regulator of pathogenicity factor)gene cluster,and the Type ? secretion system(T3SS),which is encoded by the hrp(hypersensitive response and pathogenicity)gene cluster,are the two most important virulence systems of Xcc.Previously,by using a promoter-gus A fusion reporter system,the previous work of this project studied demonstrated in Xcc that the expression of hrp X and hrp G,two key regulatory genes of T3 SS,is positively regulated by the DSF receptor Rpf C of Rpf/DSF quorum sensing system.The purpose of this study is to determine whether the accumulation of Hrp X and Hrp G proteins was affected by rpf C deletion using Western blotting analysis.To this end,firstly this study used a quantitative blotting analysis to detect and compare the accumulation of Hrp X in wild-type strains and rpf C deletion mutants.The results showed that under the minimal medium MMX culture conditions,the accumulation of Hrp X in rpf C deletion mutant decreased about 74% compared to that in the wild-type strain,indicating that Rpf C had a positive effect on the accumulation of Hrp X.More importantly,Western blotting results showed that,in the rpf C deletion mutant,a signal band with a smaller molecular weight was detected,in addition to the band corresponding to the full-length Hrp X.We speculate that this smaller signal band may be a processing product of the full-length Hrp X.This result indicated that deletion of rpf C triggers the post-translational processing of Hrp X.Similar to the situation in the minimal medium MMX culture condition,under the rich medium NYG culture condition,the accumulation of Hrp X in the rpf C deletion mutant was also significantly lower than that in the wild-type strain,but no processed product of Hrp X was detected.It seems that the effect of rpf C deletion on Hrp X varies in different environmental conditions.Northern blotting results showed that the hrp X mRNA level in the rpf C deletion mutant was significantly reduced compare to that in the wild-type strain.Next,this study used the same method to detect and compare the accumulation of Hrp G in the wild-type strain and the rpf C deletion mutant.This study found that,regardless of MMX or NYG culture conditions,rpf C deletion resulted in a significant decrease in Hrp G accumulation,but the decreased extent of Hrp G was not as big as that of Hrp X.Surprisingly,although the accumulation of Hrp G in the rpf C deletion mutant was significantly lower than that in the wild-type strain,Northern blotting results showed that no significant difference in the levels of hrp G mRNA in the rpf C deletion mutant and the wild-type strain was observed,indicating that the decreased Hrp G accumulation in the rpf C deletion mutant should be due to the translation of hrp G mRNA or the stability of Hrp G is affected by Rpf C.On this basis,this study used Northern blotting to detect the accumulation of mRNA in the hrp gene cluster(hrp A?hrp F)positively regulated by Hrp X in wild-type strains and rpf C deletion mutants and rpf C supplementary strains.It was found that the deletion of rpf C in MMX medium would cause down-regulation of hrp B,hrp C,hrp D,and hrp F mRNA accumulation.The results of this experiment indicate that Rpf C may affect the type 3 secretion system through various regulatory pathways.Furthermore,this study investigated the effect of rpf C deletion on the expression of the global transcriptional regulatory protein Clp by using Western blotting analysis and found that rpf C deletion did not affect the accumulation of Clp.In summary,in this study,the effect of deletion of rpf C,a gene encoding the DSF receptor Rpf C of the Rpf/DSF quorum sensing system,on the expression of the two key regulatory proteins Hrp X and Hrp G of the T3 SS,was investigated by using Western blotting analysis.This leads to the finding,for the first time,that in certain conditions Hrp X undergoes a post-translational processes,in which Rpf C played an important role.This study is of great significance to further study the pathogenic mechanism of Xanthomonas campestris pathovar campestris.