一个新的C2H2锌指蛋白Rcm1调控甘蔗鞭黑粉菌有性配合的机理研究

Cyclic adenosine monophosphate (cAMP) as an intracellular second messenger plays an important role in the regulation of Sexual mating process, a critical step required for pathogenicity in Ustilago. However, what other type of regulator involved cAMP signaling transduction and how they work remains elusive so far, except for the key factor G-protein. In previous work, we established a sexual mating defective mutant library by T-DNA insertion mutagenesis in Sporisorium scitamineum. Screening the library with exogenous cAMP, the mutant 8X79 was obtained by regaining mating ability. The mutant gene was identified as an unknown functional gene which encodes a C2H2 type of zinc finger peptide and was designated rcm1 (regulator of cAMP-mating 1). To annotate the function of novel gene Rcm1 in sexual mating process and investigate the underlying mechanism of Rcm1 in regulating cAMP-sexual mating signaling pathway in S.scitamineum, we seek to study Rcm1 functional domains/ motifs, downstream regulated genes and interaction proteins by expression profiling, yeast two-hybrid screening, immunoprecipitation, histochemical approaches and CRISPR-Cas9 genome-editing method for S. scitamineum. Our work will not only enrich our current knowledge on the function of C2H2 type of zinc finger proteins, but will also bring us a step closer to the completion of the cAMP-Sexual mating model. Eventually, our study aims to shed lights to the mechanisms of S. scitamineum pathogenicity to provide effective application of control measures.

有性配合是黑粉菌侵染致病的关键环节，环磷酸腺苷（cAMP）是调控有性配合过程的重要信号分子，目前除G蛋白外，有关cAMP-有性配合信号通路的调控因子仍然知之甚少。前期研究以甘蔗鞭黑粉菌为材料建立了一个有性配合缺陷突变体库，通过cAMP复筛获得一个有性配合恢复的突变体8X79，基因鉴定结果表明8X79编码一个功能未知的C2H2锌指蛋白，命名为cAMP-有性配合调控因子Rcm1。本项目将通过表达谱分析，酵母双杂交筛选，免疫共沉淀，荧光组织化学结合CRISPR/Cas9基因编辑技术对Rcm1的亚细胞定位、调控元和互作网络进行鉴定解析。研究将明确：1）新型锌指蛋白Rcm1对cAMP-有性配合信号传导的作用机制；2）Rcm1在甘蔗鞭黑粉菌有性配合中的功能。预期结果将丰富人们对C2H2锌指蛋白功能的认识，完善cAMP信号对有性配合的调控模型；为探索新型的甘蔗黑穗病防控技术提供理论依据。

甘蔗鞭黑粉菌 Sporisorium scitamineum 引起的甘蔗黑粉病是甘蔗生产的主要病害，有性配合是其侵染致病的关键环节。本课题围绕环磷酸腺苷（cAMP）信号对有性配合的调控以及锌指结构蛋白SsCrz1在有性配合发生过程中的作用展开。研究首先建立了一套基于甘蔗鞭黑粉菌原生质体转化DNA 双片段的基因敲除技术体系，该方法简单高效，极大地提高甘蔗鞭黑粉菌的基因敲除效率，为后续研究奠定了坚实的基础。在此基础上，我们对甘蔗鞭黑粉菌法尼基转移酶ß亚基基因进行了敲除研究，发现该突变体有性配合不能发生，致病力近乎丧失，揭示了有性配合信号分子的异戊二烯化修饰是发生有性配合及侵染致病的关键。我们进一步的研究分析了cAMP信号通路相关基因对有性配合的调控作用，发现cAMP信号正调控胞内的ROS的水平，因此病原菌能利于寄主产生的ROS促进有性配合的发生。我们的前期研究鉴定了一个有性配合增强的锌指结构蛋白基因突变体sscrz1，究其原因发现该基因突变影响了细胞周期蛋白基因SsCLN1和SsCLB1的表达，导致细胞周期停滞从而促进了有性配合相关基因的表达。最后，我们针对甘蔗鞭黑粉菌有性配合过程开展了生防应用研究，取得了明显的室内和田间防效。我们的研究明确了甘蔗鞭黑粉菌有性配合过程的cAMP信号调控作用及锌指结构蛋白SsCrz1的功能，初步展示了针对甘蔗鞭黑粉菌有性配合这一关键阶段开展的新型的病害防控技术具有较好前景。