禾谷孢囊线虫新效应子HA34609寄生致病功能和对合胞体形成机制的影响研究

The cereal cyst nematode (Heterodera avenae) is an important threathen disease on wheat, it caused serious yield losses of wheat and potential threaten to wheat production and food security in China. The effectors secreted by cyst nematodes have been shown to play a role in the pathogenicity and the feeding site development. In our previous study, a novel effector, named Ha34609, targeted to tonoplastc and play a role in the development and growth of the nematodes was isolated from Heterodera avenae, but the function and mechanism of action is not clear. In this study, we investigate the action site of Ha34609 in host cell by immunofluorescence localization, the developmental expression of Ha34609 at different life stages of H. avenae by Real Time PCR, and the copy number of this gene by Southern blot. The function of Ha34609 during the plant parasitism and syncytium development using RNAi and over-expression. Identify the physically interact proteins of Ha34609 by yeast two-hybrid system and confirm the interaction between host interact proteins and Ha34609 by BIFC and GST Pull Down, determine the molecular mechanism of host interact proteins via VIGS and over-expression. The results will supply evidences to understand the interaction molecular mechanism between cereal cyst nematodes and host，supply the new evidences for the regulatory mechanisms of the syncytium development by plant nematode effector and it is very important beneficial to make decision control strategies.

小麦禾谷孢囊线虫是小麦生产上最重要线虫病害，严重威胁我国粮食安全。孢囊线虫效应子在寄生致病和合胞体形成和发育中发挥着关键作用。前期的研究中申请人从禾谷孢囊线虫中鉴定出一个新效应子Ha34609，作用于液泡膜和影响线虫发育，但生物学功能和作用机理国内外尚无研究。本研究拟采用免疫组织定位研究其在寄主内的作用位点，进行发育表达分析和拷贝数分析；通过RNAi和过表达技术解析该效应子在致病和取食位点形成的生物学功能；用酵母双杂交技术鉴定该效应子的互作蛋白，用BiFC和GST Pull Down技术进行互作验证，采用VIGS技术明确其互作机制，从而系统明确该效应子在禾谷孢囊线虫致病过程的功能和调控合胞体形成和发育分子机制。研究结果将为探究孢囊线虫致病分子机理和调控取食位点形成机制提供新的证据，为制定小麦孢囊线虫的绿色防控策略提供新靶标和技术支撑，对保障我国粮食安全具有十分重要的理论和实用意义。

小麦禾谷孢囊线虫是小麦生产上最重要线虫病害，严重威胁我国粮食安全。孢囊线虫效应子在寄生致病和合胞体形成和发育中发挥着关键作用.在前期研究鉴定出一个新效应子Ha34609基础上，经过五年研究，取得以下结果：Southern 杂交表明禾谷孢囊线虫Ha34609单拷贝，在二龄线虫的亚腹食道腺中特异性表达，亚细胞定位于液泡膜上。免疫组化表明，Ha34609是一种分泌蛋白，在禾谷孢囊线虫和雌虫头部周围的植物组织中能明显的看到荧光信号，RNAi干扰Ha34609后，二龄幼虫侵染率和孢囊数分别减少76.5%和52.8%；在大麦中超表达Ha34609增加了对线虫的敏感性，侵染率和孢囊数分别增加了43.3 %和70.1%。组织病理学切片表明，过表达植物取食位点的合胞体的面积均明显变大，7天时合胞体面积增加了29.6%；RNAi植物的合胞体面积均明显变小，21天时合胞体面积减小了72.3%。酵母双杂交技术筛选到水通道蛋白（Hordeum vulgare tonoplast intrinsic protein，Hv-tip2;3)。通过BIFC体内和GST pull-down体外等实验证明Ha34609可与大麦Hv-tip2;3发生特异性互作。缺失该蛋白的大麦突变体降低了对线虫敏感性，线虫数量和孢囊数分别减少74%和62.2%，突变体7天后的合胞体的面积减小了54%。证实S15A是互作蛋白Hv-tip2;3的潜在磷酸化位点。与野生型大麦相比，过表达植物中抑制了由flg22处理触发的胼胝质沉积，且抑制了6种防御基因的表达。DAB染色法发现过表达植株中H2O2的积累水平明显增加，植株能明显的看到棕色的斑点。在烟草中表达Ha34609能抑制Bax诱导产生的坏死反应。研究结果为探究孢囊线虫调控取食位点形成机制提供新的证据，为小麦孢囊线虫的防控提供新靶标，对保障我国粮食安全具有十分重要的理论和实用意义。