葡萄霜霉菌效应蛋白RxLR53与中国野生葡萄互作因子的鉴定和功能研究

The effector proteins play a key role in inhibiting or evading host defense responses. On the basis of genome sequencing of Plasmopara viticola，The function of tobacco leaf cell death induced by RxLR53 was identified, and RxLR53 was used as bait protein to screening the yeast expression library of wild grape 'Liuba-8' inoculated with P. viticola. The interaction protein GATA26 transcription factor and effector protein RxLR53 was obtained. The interaction between RxLR53 and GATA26 transcription factor protein and RxLR53 functional domain were verified by Y2H, BiFC,CoIP. To understand the effection of RxLR53 on subcellular localization of GATA26 protein, we generated transgenic N. benthamina plants overexpressing GATA26(35S-GATA26) and transgenic N. benthamiana plants carrying a hairpin-structure GATA26 expression cassette (GATA26-RNAi), and to investigate on the biological role of interaction between RxLR53 and GATA26. GATA26-silenced N. benthamiana plants，35S-GATA26 N. benthamiana plants and WT plants were then mechanically inoculated with genetic modification of Phytophthora capsici L. The development of symptom appearance will be evaluated as compared with WT, meanwhile, the pathogenicity of RxLR53 gene was further analyzed in the transferred European grapevine plants inoculated by P. viticola. The goals are to identify the pathogenicity of effectors from the Oomycete P. viticola and the mechanism of defense response in host plant at the molecular level. This study facilitates further investigation on translocation of effector and its virulence mechanisms in host plant cells and will facilitate the development of novel disease control strategies.

效应蛋白在抑制或逃避寄主防御反应中起关键作用。本研究在葡萄霜霉菌基因组测序基础上，筛选鉴定葡萄霜霉菌效应蛋白RxLR53诱导烟草叶片细胞死亡功能，以RxLR53为诱饵蛋白，筛选接种霜霉菌的野生葡萄‘留坝-8’叶片酵母表达文库，获得GATA26转录因子与效应蛋白RxLR53互作。利用Y2H、BiFC、CoIP验证RxLR53与GATA26转录因子蛋白互作及RxLR53功能结构域。利用转基因和RNAi技术获得GATA26过表达和表达下调本氏烟草植株；鉴定过表达和表达下调GATA26转基因烟草植株接种转RxLR53效应因子基因辣椒疫霉菌侵染效率，分析RxRL53与烟草GATA26互作生物学功能。同时将葡萄GATA26基因过表达和RNAi干扰载体转化欧洲葡萄‘无核白’获得转基因植株，接种霜霉菌分析RXRL53效应因子与葡萄GATA26蛋白互作功能，揭示葡萄霜霉菌效应因子致病性和寄主的防卫反应机理。

葡萄霜霉病是由葡萄霜霉菌引起的，严重影响葡萄的品质和产量。因此研究葡萄霜霉菌效应因子致病机理，探究葡萄-霜霉菌互作的分子机制对抗病育种具有重要的意义。通过葡萄愈伤细胞原生质体瞬时转化体系验证了PvRxRL53与VvImpα/α4在葡萄细胞核中互作，PvRxRL53在葡萄中亚细胞定位依靠VvImpα/α4。利用拟南芥的同源蛋白AtGATA26突变体植株进行功能研究，结果显示在gata26突变体中下调SA和JA信号通路maker 基因的表达，突变体相比较于拟南芥野生型表现出对于辣椒疫霉更加的感病。利用重组质粒Pcambia2300-VvGATA26-GFP通过农杆菌介导的次生原胚团遗传转化体系，获得了转VvGATA26基因植株。葡萄霜霉菌侵染欧洲葡萄“黑比诺”和中国野生葡萄“留坝-8”接种葡萄霜霉病后，葡萄VvGATA26基因在感病葡萄“黑比诺”接种葡萄霜霉菌后48 h和72h 高表达。葡萄霜霉菌效应因子PvRxRL53体外抑制葡萄VvGATA26体外降解。通过葡萄‘无核白’茎段愈伤悬浮细胞体系转化CRISPR/Cas9介导的GATA26沉默载体，结果发现基因编辑后的愈伤细胞SA激素含量显著提高。本研究结果表明，葡萄VvGATA26转录因子，通过调控抑制SA的合成，促进霜霉菌的侵染。而葡萄霜霉菌侵染后葡萄VvImpα/α4因子促进PvRxRL53效应蛋白在葡萄细胞核中定位，与VvGATA26转录因子互作，抑制其蛋白降解，促进葡萄霜霉菌侵染的分子机制。另外，本研究鉴定到一个候选葡萄霜霉菌RxLR效应因子的无毒同系物，命名为PvAvh77。瞬时转化试验发现，PvAvh77能够在河岸葡萄、本氏烟草叶片触发非特异性细胞死亡，构建一系列缺失突变体，在葡萄和烟草叶片瞬时表达发现，PvAvh77-M2能够更快速和强烈诱导叶片细胞死亡。利用农杆菌介导的遗传转化将PvAvh77在无核白葡萄叶片中瞬时表达增加了葡萄霜霉菌的定殖，并且其抑制植物免疫反应依赖细胞核定位。进一步研究发现带有信号肽的PvAvh77（PvAvh77+SP）能够在植物质外体部分激发植物免疫反应。纯化的PvAvh77-M2蛋白预处理无核白葡萄叶片能够明显激发葡萄的基础免疫反应，包括诱导葡萄抗病相关基因（VvEDS1、VvEDS5、VvPR1、VvPR2）的表达，抑制葡萄霜霉菌的侵染。