水稻白叶枯病菌毒性基因调控hrp基因表达的分析  

Analysis of virulence genes regulating hrp genes expression in Xanthomonas oryzae pv.oryzae

作　　者：王艳艳[1] 马文秀[1] 蔡璐璐[1] 邱建敏[1] 宋丽[1] 邹丽芳[1] 陈功友[1] 

机构地区：[1]上海交通大学农业与生物学院,上海200240

出　　处：《植物病理学报》2016年第4期492-506,共15页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助项目(31371905;32470235);公益性行业(农业)科研专项(201303015-02)

摘　　要：水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)侵染寄主水稻,引起水稻白叶枯病(bacterial leaf blight,BLB)。病原菌主要依赖hrp基因簇编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)将效应蛋白(T3SS effectors,T3SEs)注入水稻细胞中,激发水稻的抗(感)病性。同源性搜索结果显示,植物病原黄单胞菌中已鉴定的一些毒性基因在Xoo的代表菌株PXO99A中保守存在。为了明确这些毒性基因对hrp基因表达调控的影响,本研究利用pK18mob-W介导的定点突变方法,成功获得了14个毒性基因的突变体;在突变体中,利用hrp∶∶gusA融合表达体系,通过GUS活性定量测定检测了hrpG、hrpX和hrpB1的启动子活性;通过荧光定量PCR技术,检测了这3个基因的mRNA水平。结果显示,双组分调控系统ColR/ColS、RpfC/RpfG和转录调控子Clp负调控hrpG和hrpB1的表达;Trh和Xrv A通过HrpG-HrpX途径正调控hrpB1的表达;HpaR1和Fur不依赖于HrpG仅通过HrpX正调控hrpB1的表达。这些调控关系的鉴定为解析水稻黄单胞菌hrp调控网络提供了新的线索。Xanthomonas oryzae pv. oryzae （Xoo） causes bacterial leaf blight （BLB） in host plant rice （Oryza sativa）. The pathogen mainly depends on a type HI secretion system （T3SS） encoded by a hrp gene cluster to inject T3SS effectors （T3SEs） into rice cells and thus triggers a resistance or susceptibility reaction in rice. Ho- mology search results have demonstrated that some virulence related genes identified in other Xanthomonas strains are conserved in PXO99A, a representative strain of Xoo. In order to determine the regulatory effects of these virulence related genes on hrp gene expression, fourteen mutants of virulence related genes were constructed by employing a site-directed insertion mutagenesis method with pK18mob-W vector. The promoter activities of hrpG, hrpX and hrpB1 were measured in these mutants through GUS quantitative assays by using a hrp： ：gusA fusion as a reporter. In accordingly, the mRNA levels of hrpG, hrpX and hrpB1 in the mutants were determined by real-time quantitative PCR. The results showed that two-component signal transduction system ColR/ColS, rpfC/rpfG, and transcriptional regulator Clp negatively regulated hrpG and hrpB1 expression; Trh and XrvA positively regulated hrpB1 expression through HrpG-HrpX pathway; HpaR1 and Fur positively regulated hrpB1 expression merely through HrpX pathway whereas independent of HrpG. In conclusion, the results provide some new clues for understanding of the hrp regulation network in X. oryzae.

关 键 词：水稻白叶枯病菌 HRP基因 gusA基因 基因调控 

分 类 号：S435.111[农业科学—农业昆虫与害虫防治]