西瓜食酸菌Ⅲ型效应蛋白AopBF1的鉴定与功能分析

Identification and functional analysis of the type III effector AopBF1 in Acidovorax citrulli

摘要:

【背景】细菌性果斑病（bacterial fruit blotch，BFB）是一种发生在葫芦科作物上的检疫性病害，其病原菌为西瓜食酸菌（Acidovorax citrulli），西瓜食酸菌通过Ⅲ型分泌系统（type Ⅲ secretion system，T3SS）将重要的致病因子Ⅲ型效应蛋白（type Ⅲ effector，T3E）转运到植物体内，从而致病。目前，对于T3E致病机制的认识非常有限。课题组前期已鉴定到西瓜食酸菌FC440菌株中的一些候选T3E。【目的】明确西瓜食酸菌FC440菌株候选T3E中AopBF1的序列特征、转运特性及其在病原菌致病过程中的作用，可以为深入解析病原菌致病机制奠定理论基础。【方法】利用生物信息学手段，预测分析AopBF1的T3E序列特征；通过RT-qPCR、无毒蛋白报告系统检测AopBF1所受调控及其转运特性；观察aopBF1突变体（插入突变）及过表达时西瓜食酸菌的致病力表型，分析AopBF1对西瓜食酸菌致病性的贡献。【结果】AopBF1具有T3E的序列特征、不含保守结构域，具有蛋白激酶序列特征；AopBF1在T3SS核心调控基因hrpX、hrpG突变株中的表达量显著降低；当aopBF1基因与AvrBs1功能区（59-445 aa）片段同时于avrBs1突变株中表达时，能够诱发含Bs1蛋白的ECW-10R辣椒叶片发生过敏性坏死反应；aopBF1突变株对寄主黄瓜的致病力显著减弱，而同时黄瓜组织中过氧化氢、超氧阴离子自由基及胼胝质的积累量表现为显著增加；AopBF1过表达菌株对寄主的致病力显著增强，其在诱导本氏烟的hypersensitive response （HR）反应发生上表现延迟；AopBF1瞬时表达时，显示定位于本氏烟细胞的细胞质、细胞核和细胞膜，可诱发本氏烟发生HR反应，促进PAMP-triggered immunity （PTI）及激素通路相关基因的表达。【结论】AopBF1是西瓜食酸菌的一个具有蛋白激酶特征的T3E，抑制寄主活性氧和胼胝质积累等PTI免疫反应以促进西瓜食酸菌的致病，激发含R抗性蛋白的本氏烟的PTI和激素相关抗病免疫反应。

Abstract:

[Background] Bacterial fruit blotch is a prevalent quarantine disease caused by Acidovorax citrulli in cucurbit crops. A. citrulli secretes the key pathogenic factor, known as type III effectors (T3Es), into plant cells via the type III secretion system (T3SS). However, the pathogenesis of T3Es remains elusive. In previous studies, we have identified several candidate T3Es in A. citrulli FC440. [Objective] To reveal the sequence features, transport characteristics, and the role in the pathogenic process of the candidate T3E AopBF1 from A. citrulli FC440 strain, so as to lay a theoretical foundation for dissecting the mechanism of pathogenesis. [Methods] Bioinformatic tools were used to predict and analyze the T3E sequence characteristics of AopBF1. Then, RT-qPCR and an avirulent protein reporter system analysis were conducted to reveal the regulation and transport characteristics of AopBF1. The pathogenicity of aopBF1 mutant (insertion mutation) and overexpressing strain was investigated to unveil the role of AopBF1 in pathogenicity. [Results] AopBF1 had the typical T3E characteristics, possessing a protein kinase domain and no other conserved domain. The expression of AopBF1 was significantly down-regulated in the mutants of the T3SS core regulatory genes hrpX and hrpG. The co-expression of aopBF1 and the AvrBs1 functional region (59-445 aa) in the avrBs1 mutant induced hypersensitive reactions (HRs) in the leaves of ECW-10R pepper containing the Bs1 protein. The pathogenicity of the aopBF1 mutant in cucumber was significantly reduced, while the accumulation of hydrogen peroxide, superoxide anion radical, and callose significantly increased. The overexpression of aopBF1 significantly enhanced the pathogenicity and delayed HRs in Nicotiana benthamiana. The AopBF1 transiently expressed in N. benthamiana was localized in the cytoplasm, nucleus, and cell membrane, and then induced HRs and up-regulated the expression of PTI and hormone pathway-related genes. [Conclusion] AopBF1 is demonstrated to be a T3E with a protein kinase domain in A. citrulli. It can inhibit PTI immune responses such as host reactive oxygen species and callose accumulation to enhance the pathogenicity of A. citrulli and can trigger PTI and hormone-related immune responses in N. benthamiana containing R proteins.