胶孢炭疽菌依赖HOG MAGK信号途径抗咯菌腈调控基因的鉴定

In filamentous fungus, the high osmolarity glycerol(HOG) response pathway consisting of the Ssk2/Ssk22-Pbs2-Hog1 MAP kinase (MAPK) cascade is important for survival under hyperosmotic conditions. And a large number of studies have shown that the HOG pathway plays a role in resistance to phenylpyrrole fungicides. But it is unclear how pathogens depend on the HOG MAPK pathway to regulate it’s on fludioxonil resistance. Despite the fact that Colletotrichum gloesporioides is an important plant pathogenic fungus that causes huge economic losses to the natural rubber production in most rubber growing countries, little is known about the fungal molecular characters that are important for pathogenicity and fungicidal resistance of it. In our early study, we cloned the CgPBS2 gene and obtained its deletion mutants, and confirmed that CgPBS2 gene is involve in fungus resistance to the fungicide fludioxonil. And we also obtained the different expressed gene between wild type strain and null mutant under the condition with or without 50µg/L fludioxonil by the transcriptional analysis method. In this study we will isolate the interacting proteins of HOG MAPK proteins by using GST pull-down approach followed by mass spectrometry analysis. And then after analyzed the transcriptome data and protein isolated results, we will identify and functional analyze its regulatory genes on fludioxonil resistance based on HOG MAPK pathway of C. gloeosporioide by gene deletion/ complementation. The research results will help us to elucidate a number of novel characteristics of the HOG pathway and fludioxonil resistance-related genes, as well as the Pbs2-dependent genes. Hence this study provides an excellent opportunity to develop a novel therapeutic approach to treat the serious plant anthracnose disease caused by C. gloeosporioides.

HOG MAPK途径是真菌对渗透压等各种胁迫反应所必须的。已有研究表明HOG MAPK信号途径参与调控病原菌对咯菌腈等吡咯类杀菌剂的敏感性。但尚不清楚病原菌是如何依赖HOG MAPK途径来调控病原菌对咯菌腈的敏感性。胶孢炭疽菌是橡胶树生产中一种重要的病原菌，致病机理和抗药机制研究基础薄弱。项目组前期证实了胶孢炭疽菌HOG MAPK关键基因Pbs2与病原菌对咯菌腈的敏感性有关，并分别测定了野生型和突变体在有/无咯菌腈压力下各菌株的转录组。在已有基础上，本项目采用GST Pull-Down技术钓取与HOG MAPK途径关键蛋白互作的蛋白，结合转录组数据，分离鉴定依赖HOG信号途径的抗咯菌腈关键调控基因，利用敲除/互补平台鉴定其功能。本项目的完成，可丰富HOG MAPK信号网络成员，深入了解该途径是如何介导调控菌体抗药性，以期为进一步挖掘病菌防治新靶标提供依据。

研究表明HOG MAPK信号途径参与调控病原菌对咯菌腈等吡咯类杀菌剂的敏感性，但调控机理不清。本项目围绕炭疽菌HOG MAPK途径是如何调控病原菌对咯菌腈的敏感性的问题，从该途径成员的互作蛋白和下游转录因子及靶基因两个角度开展研究。主要研究结果有：. （1）利用酵母双杂技术，从炭疽菌cDNA酵母文库中，钓取了CsSsk1、CsPbs2、CsHog1和CsAtf1候选互作蛋白，合计63个。并重点分析了含细胞色素b5结构域的脂肪酸羟化酶CsSCS7和一个细胞色素P450（CsCytP450 4F5）蛋白和CsPbs2的互作能力，以及证实了CsCytP450 4F5、CsCSC7与HOG MAPK途径关键成员基因表达具有协同性。. （2）首次证实CsSCS7基因是炭疽菌的重要生长因子，参与生长发育、致病性、咯菌腈和咪鲜胺药剂敏感性调控等功能，该基因可能影响了部分中长链脂肪酸和短链脂肪酸的合成。. （3）首次证实了HOG MAPK下游转录因子CsAtf1参与对咯菌腈敏感性调控。该基因缺失时，咯菌腈处理可提高分生孢子萌发能力从而增强对咯菌腈的抗性，同时证实该转录因子缺失可导致附着胞形成缺陷，降低致病功能。. （4）分析了CsAtf1调控的靶基因，证实该转录因子调控细胞色素氧化酶家族相关基因CsCyp51、CsErg5、CsSCS7等表达。过表达CsErg5基因能提高炭疽菌对咯菌腈的抗性，证实Erg5基因参与炭疽菌对咯菌腈敏感性调控功能。. 本研究结果表明HOG MAPK途径与细胞色素氧化酶家族成员有着密切的关系，一方面，HOG MAPK途径可通过下游转录因子CsAtf1调控细胞色素氧化酶相关基因（CsCyp51和CsErg5等）的表达，影响菌体对咯菌腈的敏感性。另一方面，该途径关键成员CsPbs2可与生长重要因子CsCSC7（含细胞色素b5结构域）等互作，但互作的生物学意义仍需进一步研究。