Mutation Analysis Of The Predicted Two-component Regulator Encoding Genes In Xanthomonas Oryzae Pathovar Oryzicola

Xanthomonas oryzae pathovar oryzicola(hereafter Xoc)is one of the main pathogenic bacteria of rice,bacterial leaf streak(hereafter BLS)cause by Xoc can reduce 5%to 30%of the rice yield.The disease was first discovered in the Philippines in 1918,and today this disease can be found in Asia,Africa and Oceania.Xoc can infect riee in various growth stage,and occur mainly in the early growth stage of rice.Xoc use two-component systems(hereafter TCSs)to sense environmental changes and respond the changes.For systematically study the function of TCSs'regulator in Xoc,26 putative regulator encoding genes were chosen as candidate genes,and the non-polar mutant of 18 of them were successfully obtained.Phenotype and virulence assay results of the mutants indicate that the virulence of 18 mutants decreased significantly compared with wild type GX01,the extracellular polysaccharide(EPS)production and motility of four mutants(NK0553,NK1021,NK1499 and NK3260)reduced significantly and the protease activity raised significantly,while NK2154 produced opposite phenotype compares to the four mutants.EPS production and motility of two mutants(NK0270 and NK3241)reduced significantly.EPS production of two mutant(NK0607 and NK1979)was significantly reduced,while protease activity raised significantly.Protease activity of NK0863 was significantly increased.EPS production of NK3622 was enhanced significantly.In order to study the exact relationship between the regulator genes and pathogenic,XOC1021 has been chosen for further functional analysis,because EPS production,motility and virulence of NK1021 were reduced significantly,its protease activity was increased significantly.XOC1021 and XOC1020 encode paired sensor and regulator of TCS,respectively,and were annotated as phoB and phoR,separated by 58 bp,arranged in the same transcriptional orientation.RT-PCR results showed that XOC1020 and XOC1021 were in the same transcription unit.The protein encode by XOC1021 contains a REC domain and a Trans_reg C domain.Exogenous fragment which contains the whole XOC 1021 sequence have been cloned into plasmid pLAFR3,and the complement was obtained by importing the recombinant plasmids into NK1021.The empty vector pLAFR3 import into the NK1021 was named PNK1021.Stress tolerance experiment results show that NK1021 was sensitive to sodium dodecyl sulfonate,sodium chloride,Cu2+ and phenol.CNK1021 can complement the defects of NK1021 in virulence and phenotype alterations.Homology comparison result show that phoB widely exists in Xanthomonas spp.,they may have similar function.18 TCSs' regulator genes in Xoc GX01 were study by mutation,some regulator genes may regulate EPS production,protease activity,motility and virulence in Xoc GX01.This study shows that phoB is related to the pathogesis,and involved in EPS production,protease activity,motility and stress tolerance of Xoc GX01.