稻黄单胞菌碳代谢和hrp基因间调控关联性及机理研究

Xanthomonas oryzae cause bacterial blight and bacterial leaf streak of rice, which are most serious diseases of rice. In order to parasitize the host rice, the bacteria injects repertoires of T3SS effectors (T3SEs) into plant cells via a type-III secretion system (T3SS) encoded by an hrp cluster, to obtain nutrients from rice. Our previous research has revealed that mutation of the fbaB gene in Embden-Meyerhof pathway (EMP) of Xanthomonas oryzae pv. oryzicola (Xoc) affected expression of the hrpG, hrpX and hrpE genes and HrpX has a feedback regulation on expression of the fbaB gene. To investigate molecular mechanisms how the carbohydrate metabolisms regulate the expression of hrp genes. We seek to: isolate positive or negative hrp regulators affected by FbaB from two Tn5-inserted mutant libraries by a gusA reporter system combining with a transcriptome sequencing using Xoc wild type strain and the fbaB mutant; analyze regulatory relations among these unknown hrp regulators,hrpG,hrpX and hrpE; reveal the regulation mechanisms how the unknown hrp regulators regulate the expression of hrpG, hrpX and hrpE in transcriptional level or post transcriptional level by EMSA and Nothern blot; verify whether HrpX protein has the ability to bind the fbaB promoter by EMSA; identify the genes of carbohydrate metabolism pathway whose expression are changed when the hrpG, hrpX and hrpE gene are deleted by transcriptome sequencing. Analysis about the correlations of carbohydrate metabolism and T3SS will has a important scientific significance to reveal the parasitic and pathogenic mechanism of Xanthomonas oryzae when they interact with host rice.

稻黄单胞菌引起重要致灾白叶枯病和条斑病。稻黄单胞菌为了寄生致病水稻，可借助hrp基因簇形成的三型分泌系统分泌（T3S）的效应蛋白（T3SEs），从水稻中获得营养。前期发现，水稻条斑病菌糖酵解途径的fbaB基因突变影响hrpG、hrpX和hrpE基因的表达，HrpX对fbaB基因具有反馈调控作用，为揭示碳代谢途径对T3S的调控作用，拟利用转录组学和gusA报道体系，筛选受FbaB影响的hrp调控子，阐明这些调控子对hrpG、hrpX和hrpE的调控关系；通过Northern杂交和EMSA途径，阐明调控子对hrpG、hrpX和hrpE转录或转录后的调控作用；利用EMSA分析HrpX对fbaB启动子的结合作用；利用转录组学数据，分析hrpG、hrpX和hrpE缺失后碳代谢有变化的基因。稻黄单胞菌-水稻互作中的碳源代谢与T3S的关联性分析，对于揭示稻黄单胞菌的寄生性和致病性机理具有重要的科学意义。

稻黄单胞菌引起重要致灾白叶枯病和条斑病。稻黄单胞菌为了寄生致病水稻，可借助hrp基因簇形成的III型分泌系统（T3SS）分泌效应蛋白，从水稻中获得营养。前期发现，水稻条斑病菌糖酵解途径的fbaB基因突变影响hrpG、hrpX和hrpE基因的表达，HrpX对fbaB基因具有反馈调控作用，但是，调控关系和分子机理未知。本研究构建了5个PTS基因的突变体，明确了2个果糖PTS基因fruA和fruK涉及正调控Xoc的毒性。利用转录组学，分析了fbaB和hrpE基因突变体后差异表达的基因。通过Tn5转座子介导的突变体库，鉴定了7个基因调控fbaB的转录表达，分析了这些基因在Xoc致病性中的作用，发现其中4个基因的突变体，Xoc在寄主水稻上的毒性显著降低。发现vemR基因涉及正调控fbaB基因的表达，研究结果表明，vemR是Xoc在寄主水稻上致病性和非寄主烟草上激发HR反应所必需的；vemR基因缺失显著影响Xoc的游动性、胞外多糖的产量和细菌的形态；VemR的互作蛋白AtoC能够磷酸化VemR，vemR基因也涉及正调控hrcC、hrcV、hrpD6、hpaB及hrpG等5个hrp基因的表达。同时，研究也发现HrpD5涉及反馈调控hrpX的表达以及碳代谢的抑制子RsmA正调控hrp基因的转录表达。这些研究结果为稻黄单胞菌-水稻互作中的碳源代谢与T3S的关联性分析提供了新的线索，对于揭示稻黄单胞菌的寄生性和致病性机理具有重要的科学意义。