The Analysis Of The Min System Negatively Regulating Hrp Genes In Xanthomonas Oryzae Pv.Oryzae

Xanthomonas oryzea pv.oryzae(Xoo)and Xanthomonas oryzae pv.oryzicola(Xoc)are two pathovars in Xanthomonas oryzae,are capable of infecting host rice and causing bacterial leaf blight(BLB)and bacterial leaf streak(BLS),respectively.These are the most serious bacterial diseases in rice.The type III effectors(T3Es)are injected into rice cell by the type III secretion system(T3SS)encoded by the hrp gene clusters to elicit the resistance or susceptibility in rice.In order to achieve quantitative detection at proteins level in X.oryzae,this study established a set of protein level detection system that can be applied to X.oryzae using pHM1 vector as the original vector.The protein expression vectors pH1-flag,pH2-flag,pHM1-flag,pH1-3myc and pH2-3myc were constructed.The hrpG gene of Xoo and the hrpG gene,hrpX gene of Xoc were selected as the target genes and their successful expression were achieved at different levels.Bacterial cell division is regulated by a number of regulators,including the Min system consisting of MinC,MinD,and MinE.In order to explore the regulation mechanism between the Min system and the hrp genes,I used the previously constructed Min system gene mutants to prove that the MinCDE system can negatively regulates the transcriptional expression of the hrp gene cluster by affecting the upstream regulatory genes hrpG and hrpX at the post-transcriptional and translational level based on q-RT PCR and Western blotting,respectively.By introducing the Xoo virulence regulation networks,it was found that the MinCDE system negatively regulates the hrp gene expression by inducing rpfG,colS and clp.The phenotype showed that the motility level of MinCDE system-related gene mutants were significantly decreased compared with wild type,and the amount of biofilm formation were significantly increased.It was confirmed that the loss of MinCDE system can affect the motility and biofilm formation of Xoo.In this study,i designed a modular plasmid system for protein expression analysis,it can be applied to achieve the gene expression at different levels of X.oryzae and providing an effective working system for subsequent expression regulation,mutant complementation and gene function related research.Meanwhile i explore the mechanism of negative regulation of T3 SS gene expression by MinCDE system and link the Min system to cell division and pathogenicity related T3 SS,which provides a new idea for the revealing of T3 SS upstream regulatory network.