Identification Of Genes Controlling The Expression Of HrpG And HrpX In Xanthomonas Campestris Pv. Campestris

The hrp(hypersensitive reaction and pathogenicity)genes control the ability of pathogenic bacteria to cause disease in host plants and to elicit the hypersensitive response in nonhost plant.Up to now,hrpG and hrpX are the key regulatory genes which control the expression of hrp genes in Xcc,while the regulators upstream of hrpG and hrpX are nearly unknown.Therefore, identification of the genes that regulate hrpG and hrpX is important to investigate the regulatory mechanism of hrp genes expression and dissect the pathogenic mechanism of phytopathogenic bacteria.In this study,the sucrose sensitive suicide gene sacB was used as a reporter.The reporter plasmids pL6sacBhrpX and pL6sacBhrpG were transferred through tri-parental conjugation into 253 mutants of which corresponding genes were annotated as regulatory genes in Xcc 8004.Six candidate regulatory genes(XC3799,XC2253,XC3056,XC1734,XC2650 and XC2251)were screened out.On the base of getting 6 candidate regulatory genes,the confirmation system was set up to identify these genes furthermore by using the gene gus as a reporter.First,the GUS reporter plasmids pL6gushrpX and pL6gushrpG were constructed and then were transferred together with the other two GUS reporter plasmids pLGX and pLGG through tri-parental conjugation into 6 mutants(073G12,125H08,151D11,NK1668,PK2558 and NK2168)of candidate regulatory genes.The GUS activities were determined in medium MMX.The results showed that the expression of hrpG and hrpX genes(the GUS activities)in mutants 073G12,125H08,151D11,NK1668 and NK2168 were significantly lower than that in wild type Xcc 8004.It indicated that hrpG and hrpX genes of Xcc were positively regulated by the corresponding five genes(XC3799,XC2253,XC3056,XC1734 and XC2251)of the above mutants,but not by XC2650 of PK2558.The trans-complemented stains of mutants 125H08(XC2253)and 151D11(XC3056)were constructed.The phenotypes of the mutants and complemented stains were tested.The assay results demonstrated that XC2253 and XC3056 affected the virulence of Xcc,but not nearly to hypersensitive reaction(HR)on pepper ECW-10R.The production of exopolysaccharide,the activities of extracellular protease,amylase and cellulase were not affected.The motility and biofilm formation of gene XC2253 were affected,but that of XC3056 were not.