hrpD6基因决定白叶枯病菌在烟草上的过敏反应和在水稻上的致病性

hrpD6 gene determines Xanthomonas oryzae pv.oryzae to trigger hypersensitive response in tobacco and pathogenicity in rice

【目的】白叶枯病菌hrp基因簇由包括hrpD6在内的26个hpa-hrp-hrc基因组成,与植物互作后形成Ⅲ型分泌系统（T3S）,将T3S效应分子注入寄主细胞中从而决定在非寄主上的过敏反应（HR）和在水稻上的致病性。但hrpD6基因是否参与了白叶枯病菌在非寄主上的过敏反应（HR）和在水稻上的致病性（pathogenicity）还不清楚。【方法】借助同源重组方法,本研究对白叶枯病菌hrpD6基因进行了突变。【结果】PCR和Southern杂交结果显示,hrpD6基因被成功敲除。烟草上测定结果显示,hrpD6突变体ΔPhrpD6丧失了HR激发能力。致病性测定发现,ΔPhrpD6在水稻苗期不能形成水渍症状,在成株期水稻上不具有致病性,并且细菌生长能力显著下降。功能互补结果显示,hrpD6基因可恢复ΔPhrpD6在烟草上激发HR和在水稻上的致病性以及在水稻组织中的生长能力。RT-PCR结果显示,hrpD6基因的转录表达不仅受水稻诱导,而且受hrpG和hrpX基因调控。不仅如此,hrpD6基因突变还影响T3S效应分子hpa1基因的转录表达和Hpa1蛋白的分泌,暗示hrpD6基因对hpa1基因转录表达具有调控作用。【结论】hrpD6基因的缺失导致白叶枯病菌不能激发烟草产生HR和和丧失在水稻上的致病性,主要是HrpD6对hpa1基因转录表达具有调控作用,并影响T3S效应分子Hpa1的分泌。这些结果为进一步分析hrpD6是否参与T3S分泌装置的形成和调控其它hrp基因的转录表达从而决定病菌在非寄主上的HR和在水稻上的致病性,提供了科学线索。

[Objective]Xanthomonas oryzae pv.oryzae（Xoo） possesses a type III secretion system（T3S）,encoded by a hpa-hrp-hrc cluster,including hrpD6,to inject T3S effectors into plant cells to trigger hypersensitive response（HR） in nonhost tobacco and pathogenicity in susceptible host rice.However,it is unclear what roles of Xoo hrpD6 gene plays in HR in tobacco and in pathogenicity in rice.[Methods]In this study,we constructed a deletion mutant of hrpD6 gene by using marker-exchange method.PCR and Southern blot analysis demonstrated that the hrpD6 gene was knocked out successfully.[Results]in planta assays indicated the hrpD6 mutant,ΔPhrpD6,lost the ability to induce HR in tobacco,to trigger water-soaked symptoms in seedlings rice and to cause bacterial blight in adult rice.Importantly,the bacterial growth in rice tissues was tremendously reduced.Complementation assays confirmed that hrpD6 gene could restore HR induction in tobacco,pathogenicity and bacterial growth in rice to the mutant ΔPhrpD6.Reverse transcriptional polymerase chain reaction（RT-PCR） revealed that the expression of hrpD6 was not only induced by rice cells,but also controlled by hrpG and hrpX.Intriguyingly,the expression of hpa1,encoding a harpin protein,was found to be dependent on hrpD6,implying that hrpD6 regulates the expression of hpa1.Immunobloting assay confirmed that the mutation of hrpD6 affect the secretion of Hpa1 through T3S.[Conclusion]The mutant lost the ability of triggering hypersensitive response in nonhost tobacco and pathogenicity in host rice is due to that hrpD6 regulates the expression of hpa1 gene and the mutation in hrpD6 affects the secretion of T3S effectors,like Hpa1,through T3SS.Our results provide molecular clues to understand whether hrpD6 is involved in the formation of T3S apparatus and in regulation of other hpa-hrp-hrc gene expression or not for HR induction in tobacco and pathogenicity in rice.