盾壳霉感应酸性pH信号的分子机制及其生防功能研究

Coniothyrium minitans is a mycoparasite and an important biocontrol agent of Sclerotinia sclerotiorum. At the early interaction stage of C. minitans-S. sclerotiorum, S. sclerotiorum usually creates an acidic pH environment by secretion of oxalic acid (OA). The acidic pH signal induces C. minitans to regulate expression of related genes for degradation of OA and production of antifungal substances. At the later stage, degradation of OA by C. minitans can increase ambient pH, thus creating an environment with weakly acidic and neutral pH, which significantly enhances the production and activity of cell wall degrading enzymes related to mycoparasitism of C. minitans. However, the molecular mechanisms for acidic pH sensing/response and its biocontrol function of C. minitans are poorly understood. In this project, we proposed to identify the key genes related to acidic pH sensing/response using the transcriptome profiling, to clone the key genes, and to characterize their function, expression patterns and the subcellular localization. Meanwhile, the regulatory mechanisms to their downstream genes and to CmpacC will be elucidated using the methods of transcription analysis and gel shift assay (EMSA). In theory, the results will advance our knowledge on molecular mechanisms for acidic pH sensing/response and its biocontrol function in C. minitans, and will broaden our understanding of the molecular mechanisms involved in the interaction between C. minitans and S. sclerotiorum. In practice, the results will be useful for initiation of new strategies to genetically modify the wild type C. minitans, aiming at improving the biocontrol efficacy of C. minitans against S. sclerotiorum.

生防菌盾壳霉是核盘菌的重寄生真菌。在盾壳霉与核盘菌互作早期，盾壳霉感应核盘菌分泌草酸所营造的酸性pH信号来调控相关基因的表达，从而发挥降解草酸作用和抗生作用；在互作后期，由于草酸被降解后pH升高，盾壳霉通过CmpacC信号途径来调控重寄生相关基因的表达，从而发挥重寄生作用。但是，目前对盾壳霉感应酸性pH信号的分子机制及其生防功能尚不清楚。本项目提出通过对盾壳霉野生型和CmpacC突变体感应酸性pH信号的转录组分析，鉴定感应酸性pH信号的关键基因，并验证其功能；研究这些基因的表达模式及其蛋白的亚细胞定位，通过转录功能分析和凝胶阻滞试验研究关键基因与下游基因的调控关系，以及CmpacC与酸性pH信号基因的调控关系；最后构建和完善盾壳霉与核盘菌互作的pH调控模型。项目的完成将明确盾壳霉感应酸性pH信号的分子机制及其生防功能，有助于认识盾壳霉与核盘菌互作的分子机理，为盾壳霉遗传改良提供新思路。

生防菌盾壳霉是核盘菌的重寄生真菌。核盘菌分泌的草酸在盾壳霉与核盘菌互作过程中起重要作用。本项目系统研究了盾壳霉响应草酸的分子机制，取得了以下研究进展：(1)针对草酸具有酸性、还原性、螯合钙离子这三重特性，通过盾壳霉响应草酸转录组分析，筛选出四类响应草酸胁迫关键基因，分别是草酸脱羧酶CmOxdc1/CmOxdc3、Ca2+信号通路转录因子CmCRZ、bZIP转录因子和组氨酸激酶CmHisKAs；(2)分析了CmOxdc3与CmOxdc1的表达模式和基因功能，发现CmOxdc3仅受草酸、丙二酸、盐酸诱导表达，而CmOxdc1受多种酸诱导表达，CmOxdc3在降解高浓度草酸时发挥关键作用，而CmOxdc1在降解低浓度草酸时起主要作用，CmOxdc3主要在液泡降解草酸，而CmOxdc1分泌到胞外降解草酸；(3)明确了CmCRZ的功能，发现CmCRZ敲除突变体生长缓慢、产孢量显著降低、重寄生能力显著下降、对草酸胁迫更耐受，而对碱性环境更敏感、菌丝胞内H2O2水平显著升高，对氧化剂耐受性增强，且胞外氧化性物质分泌显著增多、抗生能力增强，证实CmCRZ基因参与调控了盾壳霉的生长、产孢、氧化还原平衡、pH感应、Ca2+信号、以及抗生和重寄生等过程；(4)对4个差异表达的bZIP基因进行了敲除，其中3个CmbZIP基因（CmbZIP07、-09和-13）的敲除对盾壳霉的生长、产孢和响应草酸胁迫均没有显著影响。而CmbZIP16的敲除显著增强了盾壳霉对H2O2的敏感性，说明CmbZIP16在响应氧化胁迫时发挥重要作用；(5)对6个CmHisKA基因（CmHisKA01、-03、-07、-09、-13和-20）进行了敲除，这6个基因的敲除对盾壳霉响应草酸胁迫没有影响，而CmHisKA09超表达转化子对草酸敏感，说明CmHisKA09可能参与感应草酸信号。项目取得的结果明确了CmOxdc3的功能，发现其主要起降解胞内草酸的作用；明确了CmCRZ基因的功能，发现CmCRZ在盾壳霉与核盘菌互作过程中扮演重要角色；发现CmHisKA09可能是感应草酸信号的受体蛋白。本项目加深了对盾壳霉响应草酸分子机制的认识，为全面揭示盾壳霉-核盘菌互作分子机制奠定了基础。项目实施4年来已发表论文4篇，其中SCI论文1篇，核心期刊论文2篇，国内会议论文1篇，培养了2名博士研究生，3名硕士研究生。