The Mutation Study Of Chemoreceptor Genes In Xanthomonas Oryzae PV.Oryzae PXO99~A

Bacterial leaf blight?BLB?,caused by Xanthomonas oryzae pv.oryzae?Xoo?,is a kind of rice bacterial diseases.It has occurred world widely and resulted in reducing or losing production of rice.Xoo invades the rice leaf typically through natural openings such as hydathodes and wounds and then colonizes the vascular tissues and propagates in the xylem.The researches of Xoo pathogenesis will be helpful to develop new drugs and methods to prevent the BLB.It was found that chemotaxis of bacteria is an important trait for virulence and plays a more important role in the early stages of infection process.However,since the chemoreceptors,also known as methyl-accepting chemotaxis proteins?MCP?,and their genes have significant bacterial specificity and diversity,the function and mechanism of MCP remains unclear in Xoo.In order to elucidate MCP functions in Xoo,a preliminary gene function study was described in this study that a series of genes encoding MCPs of Xoo PX099A.The main results as follows:1.The mcp genes was analyzed by bioinformatics methods and show that there are 11 mcp genes in PX099A,most of which are typical mcp genes.Their products consist of two transmembane domains,a ligand biding domain?LBD?and a mythyle accepting domain?MA?.PXO₀0916,one of which,has been reannotated encoding an atypical MCP.By using the RT-PCR technology,it proved that PXO₀0916 gene in the genome annotation is not integrity and a REC domain was found in the newly annotated open reading frame.Thus,PXO₀0916 may be a novel MCP in PX099A.2.Based on double exchange method pK18mobsacB suicide plasmid,the mutants have been constructed.At first,according to the flanking sequence of mcp genes,primers were designed and then amplified for cloning the up-and down-stream sequences of the target genes to pK18mobsacB plasmid.Secondly,candidates of mutants were constructed by triparental conjugation.Last,mutants have been verified through the selection markers and PCR.Eventually,11 chemoreceptor genes mutants?two of them for our laboratory has been constructed?and an MCP gene cluster mutants which involved seven mcp genes have been acquired.3.The chemotaxis assay showed that wild type strain PX099A tends to sucrose,maltose,glucose,xylose,fructose,lactose,galactose,ribose,malic acid,indole acetic acid,abscisic acid,rice leaf extract,alanine,valine,leucine,serine and threonine and methionine and tryptophan,arginine and aspartic acid,asparagine,phenylalanine,lysine,glutamic acid.On this basis,the chemotaxis assay was conducted on 11 single mcp gene knockout mutants and 1 cluster knockout mutants,showing that one MCP was able to sense more than one compound and one compound was sensed by more than one MCP.4.A variety of phenotype assays were performed on 11 single mcp gene knockout mutants,1 cluster gene knockout mutants and some complemented strains.It was found that the deletion of PXO₀0054 gene affected the colonization ability of the strain on the seed surface.The deletion of PXO₀0041,PXo₀0045 and PXO₀6142 caused a significant decrease in the swimming mobility of bacteria.The absence of PXo₀0916,PXo₀4751 and PXO₀6142 gene resulted in a significant decrease in the bacterial swarmming activity.After the deletion of PXO 00045,PXO 00046 and PXO₀0054 gene,the SDS tolerance ability of the strain decreased slightly,while the resistance of PXO 00916 and PXO₀4751 to SDS was significantly enhanced.The PXO₀0039 and PXO₀0041 genes contribute to the formation of bacterial biofilm,and the ability of the two genes to form biofilm after deletion is reduced.Notably,there was a significant decrease in the colonization ability of the strain on the seed surface,motility and biofilm formation when mcp gene cluster was knocked out.Through the above results,the mcp gene function of PX099A strain was preliminarily explored,which provided clues for further research on MCP function,and provided a new strategy for the prevention and treatment of bacterial leaf blight.