真菌特有的丝氨酸/苏氨酸磷酸酶PPZ1调控杉木炭疽菌致病分子机制研究

Chinese fir (Cunninghamia lanceolata) is an important fast-growing trees species in China. Both of its afforestation area and timber volume are ranked in the first place among afforestation trees species. Anthracnose is widespread in the plantation area of Chinese fir and becomes one of the major threats to the Chinese fir industry. However, the pathogenic mechanism of this disease is still not clear. Dephosphorylation is an important way to regulate the function of target proteins, and serine/threonine (Ser/Thr) mediated dephosphorylation has a crucial role in regulating the biological processes of eukaryotes. PPZ1 is one type of Ser/Thr phosphatases unique to fungi, but the function of this enzyme and its regulatory mechanism in phytopathogenic fungi are still unclear. A PPZ1 homologue, CgPPZ1, was identified from Colletotrichum gloeosporioides. It was found that this gene is involved in appressorium development and virulence of C. gloeosporioides. This project will analyze the potential biological functions of CgPPZ1 in growth, stress resistance and pathogenicity, and identify 2 to 3 regulatory proteins, and analyze the interaction mechanism between regulatory proteins and CgPPZ1, and systematically reveal the function and regulation of CgPpz1. These results will help to further reveal the pathogenic mechanism of C. gloeosporioides on Chinese fir, and provide an important theoretical basis for screening of novel fungicides. At the same time, it will supply important reference value for analyzing pathogenic mechanism of other woody pathogenic fungi.

杉木是我国重要的用材树种，其造林面积和材积量均位列主要造林树种首位。杉木炭疽病是我国杉木产业健康发展的主要威胁之一，但目前该病的致病机制还不是很清楚。去磷酸化作用是蛋白功能调控的重要方式，丝氨酸/苏氨酸(Ser/Thr)磷酸酶介导的去磷酸化作用对真核生物的生物学过程具有重要调节作用。PPZ1是真菌特有的一类Ser/Thr磷酸酶，该基因在植物病原真菌中的功能及其调控机制目前还不清楚。项目前期从杉木炭疽菌中鉴定到一个PPZ1蛋白同源物CgPPZ1，发现该基因的缺失突变体致病力显著降低。本项目将在此基础上，全面解析CgPPZ1在杉木炭疽菌生长、胁迫抗性及致病等方面的功能；鉴定与其互作的调控蛋白2-3个，并解析调控蛋白与CgPPZ1的互作机制，从而系统揭示CgPPZ1的生物学功能及其调控机制。研究结果将有助于揭示杉木炭疽病菌的致病分子机制，并为筛选新型的杀菌剂作用靶标提供重要理论基础。

1.杉木是我国重要的速生用材树种，炭疽病在杉木栽培区普遍发生，该病已成为我国杉木产业健康发展的主要威胁之一。杉木炭疽菌（Colletotrichum gloeosporioides）属半活体营养型真菌，尽管已经鉴定了部分致病相关基因，但关于该菌致病的分子机制目前还不完全清楚。PPZ磷酸酶是真菌所特有的一类丝氨酸/苏氨酸磷酸酶(Ser/Thr)，但关于该基因在植物病原真菌中的功能及其调控机制的研究还十分有限。.2.本研究在杉木炭疽基因组中鉴定了一个磷酸酶基因CgPPZ1，该基因在附着胞发育与侵染阶段上调表达。该基因敲除突变显著抑制病菌的生长、产孢、孢子萌发和附着胞发育。该基因负调控杉木炭疽菌的钾离子吸收，并通过调节渗透胁迫应答信号通路核心组分CgHog1的磷酸化水平来响应胞外渗透胁迫；该基因缺失突变体也表现出明显细胞壁完整性缺陷。与寄主互作过程中，该基因敲除突变体清除寄主活性氧的能力减弱，致病力显著下降。为深入揭示CgPpz1调控杉木炭疽菌细胞壁完整性与致病力的分子机制，本项目采用亲和层析和质谱分析的方法鉴定了342个CgPpz1潜在的结合蛋白，开展了部分结合蛋白与CgPpz1互作机制的研究。首次鉴定了CgMkk1与CgPpz1的互作关系，发现CgPpz1通过调节CWI-MAPK通路核心组分CgMkk1的磷酸化水平来调控病原菌的细胞壁完整性和致病力。发现转录因子CgCrzA是激酶CgMkk1的底物蛋白，CgMkk1磷酸化CgCrzA是杉木炭疽菌响应胞外细胞壁阻抑剂CFW胁迫所必需的，CgMkk1磷酸化CgCrzA的关键氨基酸位点为S280，该位点的突变会影响到杉木炭疽菌的细胞壁完整性。采用CHIP-seq方法鉴定到了2,944个转录因子CgCrzA潜在的下游靶基因，试验证实了磷酸化的CgCrzA能从胞质转移至胞核，并与几丁质合酶基因CgCHS5和CgCHS6启动子区域结合启动它们的转录表达来促进几丁质合成，从而抵抗细胞壁阻抑剂的胁迫。通过以上研究，系统揭示CgPPZ1调控杉木炭疽菌应答胞外胁迫与致病等方面的生物学功能及其分子调控机制，结果有助于深入揭示杉木炭疽病菌的致病机理，并为评价PPZ1及其调控蛋白作为杀菌剂靶标的潜能提供重要理论依据。.3.本项目发表论文7篇，其中SCIE论文6篇，中文核心期刊论文1篇；培养研究生6人。