白粉菌诱导下广谱抗病基因RPW8.2在草莓上特异定位表达及利用研究

Powdery mildew of strawberry (Fragaria ×ananassa Duch.), caused by the obligate parasite Podosphaera aphanis syn., is one of the major fungal diseases of this crop worldwide, and resulted yield losses in strawberry may reach 60% in certain cultivars. The progress in heritability and utilization of powdery mildew resistance in strawberry is very slow due to the lack of effectiveness resistance powdery mildew gene(s) in the last two decades. The Arabidopsis resistance protein RPW8.2 is the first protein that is induced and specifically targeted to the extra-haustorial membrane (EHM) during haustorium biogenesis of powdery mildew fungi so far. In this proposed project, we want to test the expression profile of RPW8.2 pre-and post-inoculation by powdery mildew in RPW8.2 transgenic strawberry cv 'Zaohong'. Moreover, we try to create a system of RPW8.2 specifically targeting to EHM in the transgenic strawberry post inoculation by powdery mildew, and test a speculation that RPW8.2 can be used as a delivery vehicle to target generic antimicrobial cargo proteins to the host-pathogen interface. Also, we try to identity and characterize some components of disease resistance between pathogen and plants interaction and to reveal how and why the broad-spectrum protein RPW8.2 specifically targeting to the EHM post-inoculation by powdery mildew in transgenic strawberry. Finally, the aims of this project are designed to understand the novel defense mechanism activated by RPW8.2 in strawberry using a combination of molecular biological, cell biological, molecular phytopathological approaches and promise a new clue for studying molecular mechanism of between powdery mildew and plants interactions, also to give more evidences for RPW8.2 as a candidate gene for engineering resistance to powdery mildew stresses in agriculturally important crops particularly strawberry.

白粉病是危害设施草莓的重要病害之一，在生产上造成了巨大损失。因为缺少公认的有效抗病基因，国内外在草莓白粉病抗性遗传及利用方面进展缓慢。本项目拟通过研究植物源广谱抗病基因RPW8.2（该基因编码的蛋白2009年底被证明，是在所有病原菌和寄主植物互作过程中发现的、第一个特异锚定到寄主与病原菌互作界面的蛋白），在转基因感病草莓品种"早红"叶片接种白粉菌前后的表达模式，建立白粉菌诱导下RPW8.2基因在草莓上特异定位表达系统，并进一步探索利用该基因这种特异定位能力运载其他抗(病)菌肽的可能性，鉴定和分析白粉菌与转基因草莓植株互作过程中一些抗病信号分子，认识白粉菌诱导下该基因在草莓中的特异定位表达规律，从分子生物学、细胞生物学和分子植物病理学等方面综合分析该基因在草莓中的抗性功能，初步探讨RPW8.2基因在草莓中的抗病分子机理，为白粉菌和植物互作分子机理研究提供新线索，为生产上应用该基因提供参考依据

白粉病是危害设施草莓的重要病害之一，在生产上造成了巨大损失。因为缺少公认的有效抗病基因，国内外在草莓白粉病抗性遗传及利用方面进展缓慢。拟南芥广谱抗病蛋白RPW8.2在2009年底被证明，是在所有病原菌和寄主植物互作过程中发现的、第一个特异锚定到寄主与病原菌互作界面的蛋白，是一个很好的植物源广谱抗病蛋白。本项目从拟南芥抗病材料MS-0中克隆了广谱抗病基因RPW8.2并构建了相关的植物表达载体；分离了一个高致病性的草莓白粉菌菌株，命名为NWAU1，并将其序列登录了GenBank（登录号KU207048），以便后续很好开展抗病性及草莓与白粉菌互作研究；利用基因枪法，在 “straight”黄瓜子叶中瞬时表达RPW8.2基因，接种拟南芥白粉菌UCSCI后，能明显观察到RPW8.2基因在菌丝周围表达；获得RPW8.2转基因烟草22株，接种拟南芥白粉菌UCSC1后，RPW8.2-YFP融合蛋白聚集在萌发的附着孢周围，并伴随着细胞坏死，说明在白粉菌诱导下RPW8.2基因可以在烟草中特异定位表达；比较了“早红”、“红颜”和“N-17”草莓叶片的再生体系，发现“N-17”草莓叶片不定芽诱导率高，适合进行遗传转化；采用根癌农杆菌介导法，获得了经Southern Blot和RT-PCR证实的“N-17”草莓RPW8.2转基因植株42株；用白粉菌NWAU1接种转基因草莓叶片，RPW8.2-YFP能在转基因草莓中特异表达，与拟南芥等转基因植物中的结果相似，可以特异定位到白粉菌菌丝周围；进一步观察转基因草莓叶片白粉菌菌丝生长，转基因株系菌丝生长量小且伴随H2O2积累，而非转基因草莓叶片上菌丝生长量大，H2O2积累较少；RPW8.2在草莓中抗病性与SA和MeJA信号途径有关，而与ABA无关。这些结果初步说明，在白粉菌诱导下，RPW8.2是通过特异定位表达在菌丝周围，激发H2O2积累，抑制草莓白粉菌菌丝生长，从而增强转基因草莓的抗病性。这些结果为最终利用RPW8.2开展草莓抗病遗传改良研究提供理论依据和技术支撑。参加学术会议3次，发表SCI收录论文2篇，中文核心期刊1篇，会议摘要1篇，正在培养研究生4人。