西瓜食酸菌Ⅲ型效应蛋白AopAI的鉴定及其功能初步分析

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

摘要:

【背景】西瓜食酸菌（Acidovorax citrulli，Ac）引起的细菌性果斑病是葫芦科植物重要的病害之一，通过Ⅲ型分泌系统（type Ⅲ secreted system，T3SS）分泌至植物体内的Ⅲ型效应蛋白（type Ⅲ effector，T3E）是该菌重要的致病因子，目前对Ac T3E的认识仍然非常有限。【目的】鉴定西瓜食酸菌候选的T3E Acidovorax outer protein AI （AopAI），分析其对Ac致病力的影响和干扰植物免疫的方式。【方法】利用生物信息学方法分析AopAI序列特征、AvrBs1无毒报告系统验证蛋白转运功能；通过荧光定量PCR技术分析aopAI基因表达的调控及其对植物病原相关分子模式（pathogen-associated molecular pattern，PAMP）激发的免疫反应（PAMP-triggered immunity，PTI）信号通路标记基因表达的影响；利用基因插入突变和基因功能互补方法，检测菌的致病力、植物组织过氧化氢和胼胝质积累量的变化；运用瞬时表达技术分析AopAI亚细胞定位和其抑制激发子诱导细胞死亡的能力。【结果】AopAI蛋白序列中不含跨膜螺旋区和信号肽，含有二磷酸腺苷（adenosine diphosphate，ADP）核糖基转移酶保守结构域；在T3SS核心基因hrpG和hrpX突变体中aopAI基因表达量显著降低；表达AopAI及AvrBs1功能区（59-445 aa）的avrBs1突变体可诱导ECW-10R辣椒叶发生过敏性坏死反应，表明AopAI具有转运功能；aopAI基因突变体在黄瓜子叶上的致病力减弱，但与其互作的黄瓜子叶组织中过氧化氢和胼胝质的含量均显著增加；AopAI在本氏烟叶瞬时表达后，显示其定位于细胞膜和细胞核，还表现抑制激发子NIP诱导的叶细胞死亡，导致叶细胞的PTI信号通路标记基因GRAS2和ACRE31的表达量显著降低。【结论】在西瓜食酸菌中具有一个定位于细胞核和细胞膜、有ADP核糖基转移酶结构域的T3E蛋白AopAI，该T3E是能够抑制NIP诱导的细胞死亡的毒性蛋白，通过抑制ACRE31调节的免疫途径降低植物过氧化氢和胼胝质的积累，以抑制植物PTI防御反应机制。

Abstract:

[Background] Bacterial fruit blotch caused by Acidovorax citrulli is a prevalent disease in cucurbitaceous crops, and the type III effectors (T3Es) secreted by the type III system into plants are the major pathogenic factors of A. citrulli. However, the knowledge about the T3Es in A. citrulli is limited. [Objective] To identify a novel T3E, Acidovorax outer protein AI (AopAI), in A. citrulli, and investigate its impact on the bacterial pathogenicity and interference with plant immunity. [Methods] Bioinformatics tools were used to analyze the sequence characteristics of AopAI, and then the AvrBs1 as an avirulent reporter was used to verify the transport function of AopAI. Fluorescence quantitative polymerase chain reaction (PCR) was carried out to determine the expression of aopAI and the effect of AopAI on the expression of marker genes of the PAMP-triggered immunity (PTI) signaling pathway in plants. Furthermore, the gene insertion mutation and functional complementation methods were employed to examine the pathogenicity of the pathogen and the accumulation of hydrogen peroxide and callose in plants. Finally, the transient expression method was used to predict the subcellular localization and the ability of AopAI to inhibit elicitor-induced cell death. [Results] AopAI had a conserved domain of adenosine diphosphate (ADP) ribosyltransferase and no transmembrane helix region or signal peptide. The expression of aopAI was significantly down-regulated in the mutants of hrpX and hrpG (core regulatory genes of T3SS). The co-expression of aopAI and the AvrBs1 functional region (59-445 aa) induced the hypersensitive reaction in the leaves of 'ECW-10R' pepper, which indicated that AopAI was a transporter protein. The aopAI mutant presented declined pathogenicity to cucumber cotyledons, while it increased the accumulation of hydrogen peroxide and callose. After transient expression of AopAI in Nicotiana benthamiana, the protein was localized in the cell membrane and nucleus. Additionally, AopAI inhibited the elicitor NIP-induced leaf cell death and down-regulated the expression of the PTI marker genes GRAS2 and ACRE31. [Conclusion] We identified a typical T3E, AopAI in A. citrulli, which was localized in the nucleus and cell membrane and had an ADP ribosyltransferase domain. AopAI functioned as a virulent protein inhibiting NIP-induced cell death and reduced hydrogen peroxide and callose by inhibiting the ACRE31-regulated immune pathway, thus suppressing the PTI defense mechanism in plants.