叶枯型果生炭疽菌关键致病因子鉴定及其致病功能研究

Glomerella leaf spot (GLS) caused by Colletotrichum fructicola is a devastating disease on apple. We have identified 32 candidate GLS pathogenicity genes via omics analysis, among which 20 are highly expressed during pathogenesis and being GLS genome-specific,12 are Colletotrichum-specific small secreted proteins (Candidate Effector Proteins, CEPs) which can induce or suppress hypersensitive responses upon being transiently expressed in tobacco leaves. We postulate that these candidate genes contain key factors regulating GLS pathogenicity. We propose to screen for key regulators by knocking out these 32 genes, and characterizing the growth, sporulation, morphological and pathogenic traits of obtained mutants, to determine the expressional and subcellular localization patterns of key genes based on qRT-PCR and GFP reporter gene. For functionally-important CEPs, we will validate their secretory natures with yeast expression system, identify their host targets based on protein-protein interaction techniques. We will also investigate the presence polymorphism and sequence diversity patterns of these genes among Colletotrichum isolates based on PCR and Southern blot. The proposed research will unravel the GLS pathogenic mechanisms, and lay a theoretical foundation for resistance breeding and disease control.

果生炭疽菌（Colletotrichum fructicola）引起的炭疽叶枯病是苹果上的一种毁灭性病害。前期利用组学分析获得了叶枯型果生炭疽菌候选致病基因32个，其中20个为致病菌株所特有，12个可激活或抑制过敏性坏死反应。推测其中存在炭疽叶枯病菌的关键致病因子。本项目拟利用基因敲除技术突变32个候选基因，测定其在营养生长、产孢、形态等方面的贡献及其在致病性中的作用；利用qRT-PCR和GFP基因分析关键致病基因表达特征、亚细胞定位；利用酵母表达系统测定效应蛋白的分泌性,利用蛋白-蛋白互作技术揭示其在苹果中的互作因子；利用 Southern杂交、PCR等技术分析关键致病基因的保守性和多样性。该项目将揭示苹果炭疽叶枯病菌致病机理，为苹果炭疽叶枯病的抗性育种研究和防治提供理论依据。

炭疽叶枯病是我国苹果生产上的重大威胁病害，主要由果生炭疽菌种内的叶枯致病型（GLS Pathotype）菌株侵染引起，推测这些菌株含有特异的炭疽叶枯致病因子。本项目结合运用基因组比较、基因敲除、和蛋白互作技术手段，对候选效应蛋白和叶枯致病菌株特有基因进行了深入挖掘和功能验证，鉴定到了多个炭疽叶枯致病关键基因，并揭示了部分效应蛋白的致病作用机制，取得如下主要结果：1）基于烟草瞬时表达体系筛选到了12个能激活坏死或抑制Bax诱导坏死的候选效应蛋白，并通过敲除和致病性分析获得了CfCE11、CfCE12、CfCE28、CfCE92四个关键致病因子，其中CfCE11和CfCE28突变体完全丧失致病能力，CfCE12突变体致病力下降大约30%，CfCE92突变体下降约50%。2）侵染组织学分析显示，CfCE12，CfCE28和CfCE92不影响病菌侵染结构分化，但会抑制侵染点周围的寄主免疫，CfCE11则可影响病菌的附着胞发育，进而影响对寄主的侵染能力。3）通过酵母双杂、BIFC和免疫共沉淀试验（CoIP）鉴定到CfCE12、CfCE28、CfCE92三个效应蛋白的寄主靶标，证明CfCE28与苹果莽草酸途径相关的MdDAHPS1互作，定位于叶绿体上；CfCE12与苹果水杨酸信号相关的MdNIMIN2互作，定位于细胞核和细胞膜上；CfCE92靶向苹果NDPK2及Gpx64，定位于细胞核及细胞膜。4）选取来自苹果、梨、猕猴桃、辣椒等不同寄主的果生炭疽菌菌株进行全基因组测序和比较分析，鉴定到2个叶枯致病型特有的基因组区域和93个叶枯致病型特有基因，进一步批量基因敲除鉴定到NRPS、氧化还原酶和功能未知蛋白3个炭疽叶枯菌特有关键致病因子，这些基因的敲除突变体几乎完全丧失致病力。综上，本项目成功挖掘到7个炭疽叶枯致病关键基因，并进一步研究明确了三个效应蛋白的寄主靶标和毒性作用机制，研究结果对于揭示炭疽叶枯病菌的致病机理和进一步抗病品种培育具有重要理论和现实意义。