西瓜食酸菌Ⅲ型分泌效应物基因aopW功能初步分析

Preliminary functional analysis of the type Ⅲ secreted effector gene aopW in Acidovorax citrulli

摘要:

【背景】细菌性果斑病(bacterial fruit blotch, BFB)是葫芦科植物上一种严重的检疫性病害，其病原菌为西瓜食酸菌(Acidovorax citrulli)。目前已知Ⅲ型分泌效应物(type Ⅲ secreted effectors, T3SEs)是该病菌的关键致病因子，但对其效应物功能和作用机制的认识非常有限。【目的】鉴定西瓜食酸菌Ⅲ型分泌效应物基因aopW，分析其编码蛋白质影响植物免疫的方式，为更深入地认识该基因在西瓜食酸菌致病机制中的作用奠定基础。【方法】利用生物信息学分析其序列特征；借助荧光定量PCR技术分析aopW的表达调控及其与抗病相关基因表达间的关系；利用基因突变及基因功能互补手段，通过分析致病性、寄主活性氧积累量等解析基因功能；采用瞬时表达技术了解AopW诱导非寄主hypersensitive response (HR)能力及其亚细胞定位情况。【结果】aopW基因启动子区存在Ⅲ型分泌系统(type Ⅲ secretion system, T3SS)核心基因结合位点，其编码的蛋白不存在信号肽和跨膜螺旋区，与Ⅲ型分泌效应物harpin蛋白同源；T3SS核心基因hrpG/hrpX突变体中aopW基因的表达量显著降低；缺失aopW基因后突变体对寄主的致病力降低，但其互作的寄主活性氧积累量增加；AopW定位于烟草细胞质和细胞质膜，可以诱导本氏烟草发生HR，影响病原相关分子模式(pathogen-associated molecular pattern, PAMP)触发的免疫(PAMP-triggered immunity, PTI)信号通路及激素信号通路相关基因的表达。【结论】西瓜食酸菌AopW为Ⅲ型分泌效应物harpin蛋白，其在与寄主黄瓜互作中发挥毒性因子功能，与烟草互作中可能以PAMP触发植物PTI和激素抗病信号通路并引发细胞坏死。

Abstract:

[Background] Bacterial fruit blotch (BFB) is a serious quarantine disease caused by Acidovorax citrulli on the plants of Cucurbitaceae. Type Ⅲ secreted effectors (T3SEs) are known as the key pathogenic factors in the pathogen, while little is known about their roles and functioning mechanisms. [Objective] To identify the T3SE gene aopW of A. citrulli and analyze the way that the protein affects plant immunity, so as to lay a foundation for further understanding the role of aopW in the pathogenic mechanism of A. citrulli. [Methods] Bioinformatics tools were used to analyze the sequence of the gene. Fluorescence quantitative PCR was performed to determine the expression of aopW and the relationship in the expression between aopW and disease resistance genes. Gene mutagenesis and gene function complementation were employed to explore the gene functions through the analysis of pathogen pathogenicity and host accumulation of reactive oxygen species. Hypersensitive response (HR) induced by AopW and the subcellular localization of AopW in the non-host plant were investigated by transient expression technique. [Results] There was a binding site with the type Ⅲ secretion system (T3SS) core gene in the promoter region of aopW gene, and the protein encoded by aopW had no signal peptide or transmembrane domain. AopW was homologous to harpin. The expression of aopW in the mutants of T3SS core genes hrpG/hrpX was significantly down-regulated. After aopW was deleted, the pathogenicity of the mutant decreased, while the accumulation of reactive oxygen species in the host increased. AopW was mainly located in the cytoplasm and cytoplasmic membrane and induced HR in Nicotiana benthamiana. Moreover, AopW affected the pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) signaling pathway and the expression of the genes involved in hormone signaling pathways.[Conclusion] The AopW of A. citrulli is a harpin of the T3SE family, which plays a role as a toxic factor in the interaction of A. citrulli with the host cucumber. It may be involved in the PAMP-triggerred plant PTI and hormone signaling pathways and induce cell necrosis in N. benthamiana.