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【目的】决定水稻条斑病菌(Xanthomonas oryzaepv.oryzicola)在非寄主植物上激发过敏反应(hypersensitive response,HR)和在寄主水稻上致病性(pathogenicity)的hrp基因簇是受hrpG和hrpX基因调控的,但还不清楚hrpG和hrpX基因是否共同决定着所有hrp基因的表达。【方法】本文通过基因敲除方式获得了水稻条斑病菌的hrpG和hrpX基因的双突变体。【结果】烟草和水稻上测定结果显示,双突变体与单突变体一样,均在烟草上失去HR激发能力和丧失在水稻上的致病性;相应地,功能互补后双突变体恢复至野生表型。细菌在水稻悬浮细胞、hrp诱导培养基XOM3和营养丰富的培养基NB中生长后的RT-PCR结果显示,NB中hrp基因低水平表达,XOM3和水稻细胞能够高水平诱导hrp基因表达。无论何种生长条件,hrpG单突变体中hrcC、hrcT、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcJ和hrpG基因不表达;hrpX单突变体中hpa2、hrcC、hpa3、hrpE和hrpG基因表达,而hpa1、hrcT、hpaB和hrcJ基因不表达;hrpG和hrpX双突变体中hrcC、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcT、hrcJ和hrpG基因不表达。【结论】这提示,水稻条斑病菌的hrcC、hrpE和hpa3基因不受hrpG和hrpX基因单独或同时调控,而hrcT基因受HrpG调控。由此推测,水稻条斑病菌Ⅲ型分泌系统关键组份的表达有可能通过另外的信号途径进行调控,这为进一步分析Ⅲ型分泌途经的形成提供了线索。
【Objective】 The objective of this study was to determine whether the hrp gene cluster of Xanthomonas oryzaepv. Oryzicola inducing hypersensitive response (HR) in non-host plants and pathogenicity in host rice was regulated by hrpG and hrpX genes It is unclear whether the hrpG and hrpX genes together determine the expression of all hrp genes. 【Method】 Double mutants of hrpG and hrpX genes were obtained from the rice leaf spot pathogen by gene knockout. 【Result】 The results of tobacco and rice assays showed that the double mutants, like the single mutants, all lost HR-induced ability to tobacco and lost their pathogenicity on rice. Correspondingly, double mutants recovered to wild after complementary functions Phenotype. The results of RT-PCR showed that the hrp gene was expressed at low level in NB, and the hrp gene was induced at high level in XOM3 and rice cells by RT-PCR after the bacteria were cultured in rice suspension cells, hrp induction medium XOM3 and nutrient-rich medium NB. Regardless of the growth conditions, hrcC, hrcT, hpa3 and hrpE genes were expressed in the hrpG single mutant while hpa1, hpa2, hpaB, hrcJ and hrpG genes were not expressed; the hpa2, hrcC, hpa3, hrpE and hrpG genes in the hrpX single mutant HpaB, hpaB and hrcJ genes were not expressed; hrcC, hpa3 and hrpE genes were expressed in hrpG and hrpX double mutants, while hpa1, hpa2, hpaB, hrcT, hrcJ and hrpG genes were not expressed. 【Conclusion】 This suggests that hrcC, hrpE and hpa3 genes of S. cerealis are not regulated by hrpG and hrpX genes individually or simultaneously, while hrcT gene is regulated by HrpG. Therefore, it is speculated that the expression of the key components of the type III secretion system of S.potatum may be regulated by other signaling pathways, which provides a clue for further analysis of the formation of type III secretion pathway.