稻黄单胞菌HrcT调控hrp-hrc-hpa基因表达的机理研究

Xanthomonas oryzae, the causal agent of bacterial blight and leaf streak diseases in rice, injects repertoires of T3SS effectors (T3SEs) into plant cells via a type-III secretion system (T3SS), encoded by an hrp cluster, to trigger hypersensitive response (HR) in nonhost or resistance plants and pathogenicity in susceptibile plants. Our previous research has revealed that the expression of HrcT, a key component of the T3SS apparatus, is not regulated by HrpG that positively regulates the expression of HrpX. To investigate molecular mechanisms of the expression of hrp genes by HrcT, we seek to: isolate positive or negative regulators from a Tn5-inserted mutant library of the hrpG mutant where the hrcT promoter was fused with a gusA reporter; analize regulatory relations among HrcT, HrpG and HrpX in isolated Tn5-inserted mutants and the hrcT mutant by using gusA reporter fused with hrcT, hrpG and hrpX promoters; verify whether HrcT protein has or not the ability to bind the hrpX promoter or to degrade hrpX mRNA by EMSA and Nothern blot; examine the expression levels of HrcT in candidate regulators that play roles in pathogenicity, T3SS formation and secretion of T3SEs of the pathogen; and confirm whether or not the HrcT proteins of other typical plant pathogenic bacteria possess such the abilities. This project will not only highlight our understanding on regulation of T3SS formation and secretion, but also provide fundamental basis to design new green chemicals for controlling plant bacterial diseases using HrcT as a target.

稻黄单胞菌是白叶枯病和条斑病的病原菌，借助hrp基因簇形成III型分泌系统（T3SS）,将效应蛋白（T3SE）注入植物细胞中从而导致水稻产生抗（感）病性。前期研究发现，HrcT除是T3SS的组份外，其表达不受HrpG调控，并对其它hrp基因表达有调控作用。为揭示HrcT调控hrp基因的机理，本项目拟分析hrcT基因启动子，借gusA报道系统在hrpG突变体中筛选hrcT基因表达的调控子；在候选调控子与hrcT突变体中分析hrcT、hrpG和hrpX间的调控关系；纯化HrcT蛋白，通过EMSA和Northern杂交分析HrcT蛋白对hrpX的转录或转录后调控作用；在候选调控子中分析hrcT表达变化对病菌致病性、T3SS形成和分泌T3SE的影响；研究其它植物病原细菌的 hrcT是否具有类似功能。这不仅开辟T3SS调控的新认识，而且为利用HrcT蛋白开发植物细菌病害防治新型药物具有重要的科学价值

水稻条斑病菌引起细菌性条斑病，其借助hrp基因簇形成III型分泌系统（T3SS），将效应蛋白（T3SE）注入植物细胞中从而导致水稻产生抗（感）病性。hrp基因的诱导表达受HrpG和HrpX调控。前期研究发现，T3SS的组分蛋白HrcT对一些hrp基因的表达有调控作用，但是，调控关系和分子机理未知。本研究证明HrcT除是T3SS的组分蛋白外，也涉及正调控hrpX的转录表达。hrcT基因的转录表达主要受2个启动子（T1和T3）的调控，T1为hrpB转录单元的启动子，T3位于hrpB7基因中，为其自身的启动子。HrcT蛋白的N端能够结合在hrpX的启动子区，正调控hrpX的转录表达。研究结果也显示，仅来自植物病原黄单胞菌的hrcT基因能够互补Xoc的hrcT突变体。这些研究不仅开辟了T3SS调控的新认识，而且为利用HrcT蛋白开发植物细菌病害防治新型药物具有重要的科学价值。本项目另一研究结果显示，Lon蛋白能够通过HrpX负调控hrp基因的转录表达，以及Lon蛋白在Xoc菌体内能够降解HrcT蛋白。推测Lon的存在对于HrcT蛋白起到平衡的作用。lon基因缺失降低了Xoc在寄主水稻上的致病性和在非寄主烟草上激发HR反应的能力。这些研究结果为Xoc毒性调控网络的解析提供了新的线索。