杨树炭疽病菌附着胞及相关侵入结构形成的关键基因鉴定与功能解析

Populus are good woody plants and play important roles in landscape and ecological construction. Poplar anthracnose is one of main diseases, causing severe economic losses and potential threat. Forming appressorium and related infection structre are vital for suceessful infection for this fungus, however, which key genes are invovled in these preocess and what function are much unknown. This proposal, we will take advantage of methods and strategies of forward genetics to genome-wide insertional mutagenesis and functional characterization of infection-related morphogenesis associated genes. Using Agrobacterium tumefaciens-mediated transformation method, genome-wide T-DNA-tagged insertional mutants will be obtained. Through TAIL-PCR analysis, T-DNA integration analysis will be conducted in the genome of poplar anthracnose fungus. At the same time, after making phenotype screen, we will identify infection-efficiency mutants and discover many new pathogenicity genes. Finally, functional characterization of candidate genes involved in infection-morphogenesis is to be done, which will contribute to elucidating molecular mechanism and genetic pathway during morphogenesis of infection. Not only these result will uncover the molecular basis of infection-morphogenesis in poplar anthracnose fungus at systematic level, understanding T-DNA integration into the chromosome, and providing data for finding specific gene expressed during infection.; but also it will help to establish molecular pathological system of poplar tree-fungus, and refer to study of other woody plant diseases.

杨树是我国用材林、防护林和园林绿化的重要造林树种，随着栽培面积的不断扩大，杨树炭疽病作为重要的生物灾害，已造成了严重的经济损失，并具有潜在的生态威胁。其病害防治的关键是抗病育种和精准化防技术，但病原菌附着胞及侵染结构的形成是其成功侵染的关键，有哪些关键基因参与调控以及这些基因的作用机理，知之甚少，尚无系统深入的认识，从而制约高效防治技术的建立。本项目拟通过正向与反向遗传学相结合的研究思路，采用功能基因组学研究技术，系统筛选杨树炭疽病菌附着胞及侵染结构形成的突变体，鉴定相关基因，找出其关键基因，分析关键基因产物的亚细胞定位和功能结构域，并通过表达谱阐明关键基因的调控网络及遗传途径，揭示杨树炭疽病菌附着胞及侵染结构形成的分子机理。本项目将有助于获得可作为杨树抗病育种、设计杨树炭疽病菌新型杀菌剂候选靶标的关键致病基因及其调控网络和遗传途径，还对其它林木病害分子植物病理学的研究具有很高的参考价值。

本项目利用基因敲除手段结合分子生物学表型分析方法，研究了我国引起杨树炭疽病的胶孢炭疽菌（Colletotrichum gloeosporioides）附着胞的产生以及其侵染的分子机制。本研究对真菌内信号转导关键基因进行了鉴定与敲除，通过同源分析鉴定和Split-marker方法先后敲除了胶孢炭疽菌跨膜蛋白CgMsb2，小G蛋白CgRhoB，CgCdc42，PAK激酶CgSte20，锚定蛋白CgSte50以及Pmk1 MAPK的CgSte11和CgPmk1，转录因子CgAp1，CgPig1。研究结果揭示了胶孢炭疽菌孢子萌发后对于外界疏水刺激信号的响应，萌发后芽管的发育，以及附着胞形成后黑色素的积累等对于胶孢炭疽菌附着胞形成发育的关键影响因素；此外还首次发现在分别敲除Pmk1 MAPK的CgSte11，CgPmk1以及CgSte50后，突变体完全无法形成附着胞，在完整寄主以及创伤寄主上均丧失致病力。本研究以附着胞和致病性为切入点，结果为以后附着胞形成以及侵染的分子机制应用在其它以附着胞为侵染结构的病原真菌研究奠定了基础。本研究在我国林木病原真菌分子生物学研究中处于领先位置，加速了我国杨树炭疽病的防控以及农药靶标位点选取等研究工作进展，并为我国培养了植物病原真菌分子生物学的相关专业人才。