梨果生炭疽菌中CfMK1激酶下游因子鉴定及其调控附着胞形成的机制研究

Recently, pear anthracnose caused by Colletotrichum fructicola has become more and more serious, which has become one of the important restrictive factors affecting the yield and quality of pears in China. C. fructicola successfully infects leaves and fruits by forming appressorium to break through the host's defense. In previous work, the applicant found that CfMK1, the terminal kinase of the MAPK signaling pathway in C. fructicola, was essential for the appressorium formation and host invasion. However the molecular mechanism of CfMK1 kinase in regulating appressorium formation is still unclear. To this end, the project intends to use CfMK1 kinase as the entry point, comprehensively using the combination of affinity purification and mass spectrometry, phosphorylation proteomics technology, CO-IP technology to screen and identify the downstream factors of CfMK1. The function of downstream factor and the effects of phosphorylation of downstream factors on its localization and function will be further studied to clarify the molecular mechanism by which CfMK1 regulates the formation of appressorium. The results will help to reveal the infection mechanism of C. fructicola, and provide a new strategy and theoretical basis for the prevention and control of pear anthracnose by blocking the formation of appressorium.

近年来，由果生炭疽菌（Colletotrichum fructicola）引起的梨炭疽病危害日益加重，成为影响我国梨产量和品质的重要制约因素之一。果生炭疽菌通过形成附着胞来突破寄主的防御成功侵染叶片和果实。在前期工作中，申请者发现果生炭疽菌MAPK信号通路的末端CfMK1激酶是附着胞形成和侵染植物所必需的，但是CfMK1下游参与调控附着胞形成的因子尚不明确。为此，本项目拟以CfMK1激酶为切入点，综合运用亲和纯化与质谱相结合的技术、磷酸化蛋白质组学技术、CO-IP技术来筛选和鉴定CfMK1下游参与调控附着胞形成的因子，并进一步研究下游因子的功能以及CfMK1催化的磷酸化修饰对下游因子的定位和功能的影响，从而明确CfMK1通过下游因子调控附着胞形成的分子机理。研究结果将有助于揭示果生炭疽菌的侵染机制，同时为通过阻断附着胞形成途径实现梨炭疽病的防控提供新的策略和理论依据。

近年来，由果生炭疽菌（Colletotrichum fructicola）引起的梨炭疽病危害日益加重，已成为影响我国梨产量和品质的重要制约因素之一。为了解析梨炭疽菌侵染寄主的分子机理，本项目以果生炭疽菌MAPK通路的末端激酶CfMK1为切入点主要开展了以下工作：（1）构建了CfMK1基因缺失突变体ΔCfmk1和回补菌株ΔCfmk1/CfMK1，并对它们开展了致病性测定、附着胞形成、植物表皮穿透等系统的表型分析，解析了CfMK1在果生炭疽菌中的分子生物学功能，明确了CfMK1是附着胞形成和侵染植物所必需的；（2）构建了CfMK1-GFP和H1-mCherry（标记细胞核）共表达菌株，使用共聚焦荧光显微镜观察了CfMK1-GFP在分生孢子、营养生长菌丝和侵染型菌丝阶段的定位和表达情况，明确了CfMK1在各个生长阶段主要定位于细胞质中，但是在营养生长和侵染型生长阶段表达量升高并且会进入细胞核中富集表达；（3）开展了ΔCfmk1和野生型菌株的转录组分析，得到了1886个上调和1554个下调的差异表达基因，GO分析发现差异基因主要富集于细胞壁和致病相关条目，揭示了CfMK1参与调控不同亚群基因的表达；（4）构建了CfMK1激酶活性丧失的等位基因CfMK1(K52R)-Flag的表达菌株，使用该菌株作为底物诱捕菌株开展了蛋白质亲和纯化与质谱分析，鉴定得到了27个候选互作蛋白；（5）基于本项目的组学分析结果和文献报道，筛选了靶向真菌鞘脂合成途径来扰乱附着胞形成，可用于防治梨炭疽病的生防次生代谢产物Dihydromaltophilin。本项目初步揭示了CfMK1调控果生炭疽菌附着胞形成的分子机制，为通过阻断附着胞形成途径实现梨炭疽病的防控提供了理论依据。项目执行期内发表标注SCI论文2篇、中文核心期刊1篇，参与制定江苏省地方标准1项，完成了项目规定的考核指标。