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【目的】在十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)的致病因子中,III型分泌系统(Type III Secretion System,T3SS)是至关重要的致病系统,III型效应物通过III型分泌系统直接转运到寄主植物细胞内。本研究通过效应物水平转移的特征获得候选基因,旨在鉴定一个新的依赖于III型分泌的效应物。【方法】以缺失了N-端58个氨基酸的Avr Bs1作为报告系统构建效应物鉴定报告质粒p LJB3176,导入Δavr Bs1和Δhrc V,通过检测报告菌株在辣椒ECW-10R上的过敏反应来鉴定XC3176是否为III型效应物。构建XC3176融合GUS报告质粒p LGUS3176,导入野生菌株Xcc 8004、Δhrp G和Δhrp X,通过测定菌株GUS活性检测hrp G、hrp X对XC3176的调控作用。构建XC3176缺失突变体和互补菌株,通过剪叶法接种检测XC3176对Xcc 8004致病性的影响。【结果】XC3176融合Avr Bs1报告菌株在非寄主辣椒ECW-10R上能引发过敏反应,GUS活性检测显示Δhrp G/p LGUS3176、Δhrp X/p LGUS3176比8004/p LGUS3176的GUS酶活显著降低,致病性检测显示突变体Δ3176与野生型Xcc 8004相比在寄主满身红萝卜上的病斑长度有显著减少,互补菌株C3176的病斑长度能补回到野生型水平。【结论】XC3176是依赖于hrc V分泌的III型效应物,hrp G、hrp X正调控XC3176,XC3176与Xcc致病相关。
【Objective】 Type III Secretion System (T3SS) is the most important causative agent in the pathogenesis of Xanthomonas campestris pv. Campestris (Xcc). Type III effectors Translocation directly into host plant cells via the type III secretion system. In this study, candidate genes were obtained from the trait of effector horizontal transfer in order to identify a new effector that is dependent on type III secretion. 【Method】 Avr Bs1 with 58 amino acids N-terminal deletion was used as the reporter to construct the effector identification plasmid p LJB3176 which was introduced into Δavr Bs1 and Δhrc V to identify XC3176 by detecting the anaphylactic reaction of the reported strain on pepper ECW-10R Is it a Type III effector? The XC3176 fusion GUS reporter plasmid p LGUS3176 was constructed and introduced into wild strain Xcc 8004, Δhrp G and Δhrp X. The effect of hrp X on XC3176 was detected by measuring the GUS activity. The XC3176 deletion mutant and the complementary strain were constructed, and the effect of XC3176 on the pathogenicity of Xcc 8004 was detected by shearing the leaves. 【Results】 XC3176 fusion Avr Bs1 reporter strain could induce anaphylactoid reaction on non-host pepper ECW-10R. GUS activity assay showed that the enzyme activity of Δhrp G / p LGUS3176, Δhrp X / p LGUS3176 was significantly lower than that of 8004 / p LGUS3176, The pathogenicity test showed that the mutant Δ3176 significantly reduced the lesion length on the host carrot compared to the wild type Xcc 8004, and the complement length of the mutant C3176 could be restored to the wild type. 【Conclusion】 XC3176 is a type III effector dependent on hrc V secretion. Hrp G and hrp X positively regulate XC3176. XC3176 is associated with pathogenicity of Xcc.