禾谷孢囊线虫新效应子Ha17370其RxLR基序转运作用和基因功能研究

Cereal cyst nematode (Heterodera avenae) was considered as the economically important pathogen of wheat. In China, H. avenae distributed nearly 80% of the wheat growing areas. Until now, safe and effective management of H. avenae diseases is challenging due to a lack of understanding of their pathogenesis. Functional analysis of H. avenae effectors for new control targets will contribute to developing control strategies. Translocation of effectors to host cells is the key factor to establish parasitism. However, the question of how plant nematode effectors are deposited into plant cells, especially whether RxLR motif functions during the translocation of nematode effectors remains somewhat unclear. Putative RxLR effectors were predicted according to our transcriptome date of H. avenae, among which Ha17370 was cloned. Our results showed that Ha17370 was specifically expressed in phasmid and active in pathogenesis. The study here will use incubation of wheat root tips with purified Ha17370 and its RxLR-mutant protein and immunohistochemistry localization to elucidate the function of RxLR motif in the translocation of nematode effectors. The methods of suppressing cell death, ROS assay and VIGS-HIGS were used to explore the function of Ha17370 in regulating plant immunity and establishing parasitism. Finally, plant genetic transformation, LC-MS and Co-IP will be used to explore the interaction between Ha17370 and the plant. These results will provide important data for the parasitism mechanism of H. avenae, and support to find new control targets for development of new nematode control strategy.

禾谷孢囊线虫是影响小麦安全生产的重要病原，解析其效应子功能并明确致病机理是制定防治新策略的基础。线虫效应子蛋白的转运是建立寄生关系的关键，但植物线虫效应子如何进入寄主细胞、特别是RxLR基序在线虫效应子转运中的作用是亟待解决的重要科学问题。我们根据禾谷孢囊线虫转录组获得了带RxLR基序的效应子，克隆了其中的Ha17370基因，初步结果发现其在线虫尾感器表达并参与致病过程。本项目拟通过对Ha17370的RxLR基序进行突变，采用浸泡小麦根尖并结合免疫组化技术，明确其转运到寄主细胞的途径；通过烟草瞬时表达、ROS测定、BSMV-HIGS等技术体系，明确该效应子在调控免疫反应及寄生致病中的功能；通过分析异源表达效应子拟南芥的蛋白质谱、Co-IP等技术，探讨该效应子与植物的互作机制。研究结果将为解析禾谷孢囊线虫的寄生机理提供重要数据，为寻找防治该线虫的新靶标及制定防治新策略提供理论依据。

禾谷孢囊线虫是影响小麦安全生产的重要病原，解析其效应子功能并明确致病机理是制定防治新策略的基础。线虫效应子蛋白的转运是建立寄生关系的关键，但植物线虫效应子如何进入寄主细胞、特别是RxLR基序在线虫效应子转运中的作用是亟待解决的重要科学问题。我们根据禾谷孢囊线虫转录组获得了带RxLR基序的效应子，克隆了其中的Ha17370基因，发现其在线虫尾感器表达、寄生后三龄表达量最高，并参与致病过程。小麦根尖自主吸收蛋白实验发现Ha17370蛋白可不依赖于线虫存在，独立进行蛋白转运，而RxLR基序突变后丧失了蛋白转运功能。体外干扰Ha17370基因降低禾谷孢囊线虫对小麦根的趋性。异源表达Ha17370增强丁香假单胞菌在烟草体内的生长能力，Ha17370转基因拟南芥对甜菜孢囊线虫更加敏感。基于BSMV介导的寄主体内沉默Ha17370基因后，禾谷孢囊线虫在小麦体内的侵染率及孢囊形成量都显著下降。另外，Ha17370蛋白可抑制植物的防卫反应，如ROS爆发、胼胝质沉积以及植物抗性相关基因表达。利用免疫沉淀及质谱技术、Co-IP实验证明Ha17370与拟南芥、小麦和大麦中AtCBSX3互作，说明禾谷孢囊线虫通过分泌Ha17370蛋白调控寄主体内H2O2水平来达到寄生的目的。研究结果证明带有RxLR基序的Ha17370效应子具有自主转运功能，可抑制植物的防卫反应，促进寄生，这是在植物病原线虫上的首次发现。研究结果为解析禾谷孢囊线虫的寄生机理提供重要数据，为寻找防治该线虫的新靶标及制定防治新策略提供理论依据。