葡萄霜霉菌保守效应蛋白PvRxLR04951与其靶蛋白互作的分子机制研究

Grape downy mildew, is an old destructive oomycete disease for viticulture. The disease is particularly serious in Guangxi with high temperature and rainy summer. We have completed the whole genome sequencing of P. viticola and the transcriptome sequencing of multiple isolates collected from different regions. A large number of effectors were identified from the genomic and transcriptomic data. A highly conserved effector PvRxLR04951 was found to suppress the tobacco PCD triggered by different elicitors. Furthermore, two candidate host target proteins were identified by Y2H screening. However, the interaction mechanism between effector PvRxLR04951 and these two target proteins is still unknown. This project is to further investigate the molecular mechanism of interaction between them on the basis of our previous work. Firstly, the effector and its targeted proteins will be transiently expressed in N. benthamiana and the interactions will be checked by BiFC and Co-IP. Secondly, the key domains of these proteins contributed to the interactions will be identified by protein truncation and transient expression. Furthermore, the function validation of the two host target proteins will be analyzed using overexpression and RNAi in grapevine. Finally, the PvRXLR04951 will be transformed into downy mildew-resistant grapevine 'R23' and tobacco to demonstrate it can promote pathogen infection or not. Success of this project will provide the molecular basis and theoretical basis for P. viticola pathogenesis and exploring new prevention and control measures for grape downy mildew.

葡萄霜霉病，是危害葡萄生长最严重的卵菌病害之一。广西夏季高温多雨，发病尤其严重。前期我们完成了葡萄霜霉菌全基因组及多个菌株的转录组测序，挖掘出大量的效应蛋白，并筛选到了一个在多菌株中高度保守的，可抑制烟草PCD反应的效应蛋白PvRxLR04951。同时利用酵母双杂技术筛选到它在葡萄体内的2个候选靶蛋白，但其互作的机制尚不清楚。本项目在此基础上，首先利用BiFC和CoIP对该效应蛋白与其靶蛋白的互作进行双重验证；其次利用蛋白截断和烟草瞬时转化寻找两者互作过程中起关键作用的必需结构域；然后利用瞬时RNAi、过表达等转基因手段明确靶蛋白在寄主抵抗病原菌侵染中的作用；最后，将该效应蛋白分别转化葡萄和烟草，验证其是否能够促进病原卵菌的侵染；最终阐明效应蛋白PvRxLR04951与其靶蛋白的互作机制。本研究的完成，将为明确霜霉菌的致病机理和探索新的葡萄霜霉病防治手段提供分子依据和理论基础。

葡萄霜霉病，是危害葡萄生长最严重的卵菌病害之一。广西夏季高温多雨，发病尤其严重。本项目证实了多菌株保守的效应蛋白PvRXLR04951可以显著增强葡萄霜霉菌、辣椒疫霉菌和烟草疫霉菌的致病力。BiFC、GST Pull-down、CoIP实验证实其与VvPLP1在内质网膜上互作。且其WY基序是与VvPLP1互作所必须的，但与抑制坏死无关。靶蛋白VvPLP1的亚细胞定位结果表明其定位于内质网。酶活测定表明VvPLP1具有磷脂酶A活性，可催化甘油磷脂sn-2位的脂肪酸水解。对霜霉菌的抗性功能验证表明VvPLP1在对葡萄霜霉菌的抗性方面发挥重要作用。进一步研究发现，VvPLP1与乙烯合成途径的关键酶VvACO1互作。且在汤姆森无核叶片中瞬时沉默VvPLP1之后，SA、JA途径中关键标记基因的表达量显著上调。这些发现不仅丰富了对葡萄霜霉菌致病机理的认识，还为探索新的葡萄霜霉病防治手段提供了分子依据和理论基础。