十字花科黑腐病菌一个新的III型效应物XC3176的鉴定

Identification of a new type III effector XC3176 in Xanthomonas campestris pv．campestris

摘要:

摘要:【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AvrBs1作为报告系统构建效应物鉴定报告质粒pLJB3176，导入ΔavrBs1和ΔhrcV，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pLGUS3176，导入野生菌株Xcc 8004、ΔhrpG和ΔhrpX，通过测定菌株GUS活性检测hrpG、hrpX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AvrBs1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ΔhrpG/pLGUS3176、ΔhrpX/pLGUS3176比8004/pLGUS3176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hrcV分泌的III型效应物，hrpG、hrpX正调控XC3176，XC3176与Xcc致病相关。

Abstract:

Abstract:［Objective］Type III secretion system (T3SS) is essential for many phytopathogenic bacteria to cause disease in susceptible host plants and to elicit a hypersensitive response in resistant host and non-host plants．Xanthomonas campestris pv． campestris (Xcc) uses T3SS to deliver T3SS effectors (T3SEs) directly into host cells，where they play important roles in pathogenesis．The aim of this study was to identify a new T3SE in Xcc．［Methods］ To validate if XC3176 is a T3S effector translocated into plant cells，the promoter and signal region of XC3176 were fused to the plasmid pLJB of harboring HＲ-inducing AvrBs1 C-terminal domain lack of 58 N-terminal amino acid residues． The recombinant plasmid pLJB3176 was introduced by triparental conjugation into ΔavrBs1 and ΔhrcV． Hypersensitive response induced by the obtained strains ΔavrBs1/pLJB3176 and ΔhrcV/pLJB3176 were examined on the pepper ECW-10R． To determine transcription of XC3176，GUS fusion report strains were constructed． The virulence of Xcc strains was investigated on the Chinese radish by the leaf-clipping method．［Results］Hypersensitive response was elicited on the pepper ECW-10R by the strain ΔavrBs1/pLJB3176，but not ΔhrcV/pLJB3176．The GUS activities in the mutant strains ΔhrpX and ΔhrpG were significantly lower than that in the wild type Xcc strain．The mutant of XC3176 reduced virulence significantly and the complementary strain C3176 could restore the virulence as the wild-type strain．［Conclusion］XC3176 is a T3SSdependent effector of Xanthomonas campestris pv． campestris． The expression of XC3176 is regulated by hrpG and hrpX．XC3176 is required for the full virulence of Xcc 8004.