SsFKH1转录因子调控核盘菌菌核形成与侵染垫发育的机制研究

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogenetic fungus with a broad host range and a worldwide distribution. The sclerotia and cushion of S. sclerotiorum will be induced and produced into multicellular structures from the aggregation of hyphae during the vegetative development stages respectively under ambient pH, cAMP and light. Sclerotia are requried for fruiting body. And the cushion play a central role in the life and infection cycles of this pathogen.On the basis of some research previously, SsFKH1, forkhead (FKH)-box-containing protein, is an important transcriptional factor involved in formation and development of sclerotia and cushion. Transcriptional factor SsFKH1 regulate sclerotia formation and cushion development according to our results. Therefore our focusing work is as follows. (1) to verify the function of SsFKH1 responsing to development of sclerotia and cushion, clarify properties of biology and pathogenicity of SsFKH1 further. (2)to identify candidate genes and target genes regulated by SsFKH1 during formation of sclerotia and cushion through comparing expression profiles between SsFKH1 mutant and wild type.(3) Find out and confirm proteins interacted with SsFKH1 upstream and downstream during regulating sclerotia and cushion, and reveal the whole regulation pathway and network. (4) The objective of this project aim at and open on the regulation mechanism of SsFKH1 to formation of sclerotia and cushion in S. sclerotiorum. The project will lay the theoretic foundations for deeply understanding growth, development,life cycle andprovide a novel ideas for sustainable control diseases of crops of S. sclerotiorum in the field.

核盘菌（Sclerotinia sclerotiorum (lib.) de Bary）是一种死体营养型植物病真菌。环境pH、cAMP、光诱导菌丝将发育成菌核与侵染垫。菌核是该菌无性繁殖与有性生殖的转换器。侵染垫是菌丝侵染与致病的特殊构造，决定其致病性。前期研究结果表明，SsFKH1是影响核盘菌菌核形成与致病性的关键转录因子，发现SsFKH1参与调控核盘菌菌核形成途径，以及侵染垫发育。为此，本项目拟：（1）进一步验证SsFKH1与菌核形成及侵染垫发育的关系，揭示其生物学功能；（2）鉴定SsFKH1在菌核形成和侵染垫发育中所调控的候选基因，明确其靶标基因及主要功能；（3）探索与Ss-FKH1转录因子上、下游调控的互作蛋白及其功能，阐明SsFKH1的调控途径与调控网络;(4) 解析核盘菌转录因子SsFKH1对菌核形成与侵染垫发育的调控机制，为全面认识核盘菌生长发育与有效防控菌核病奠定理论基础。

SsFkh1是影响核盘菌菌核形成与致病性的关键转录因子，参与调控核盘菌菌核形成途径以及侵染垫发育。为此，本项目进一步验证了SsFkh1与菌核形成及侵染垫发育的关系。通过基因敲除与回补，获得SsFkh1基因缺失突变体与基因回补突变体，将敲除突变体、野生型与回补突变体菌株进行比较， 发现SsFkh1的缺失导致菌核与侵染垫发育出现异常。.为探索转录因子SsFkh1在调控核盘菌菌核与侵染垫发育过程中的相关机制，课题组利用转录组测序探究其差异表达基因发现，SsFkh1基因缺失突变体与野生型存在差异表达基因2005个，进一步进行GO注释与KEGG分析发现SsFkh1的缺失导致MAPK介导的细胞壁完整性通路大量富集。随后对MAPK介导的细胞壁完整性通路基因研究发现，SsMkk1、SsSmk3、SsBck1和SsPkc1的缺失影响核盘菌的生长发育及细胞壁完整性通路、侵染垫发育与毒力。此外，SsMkk1与SsFkh1能够相互作用。表明SsFkh1可能参与到MAPK通路中调控核盘菌菌核和侵染垫的生长发育。.通过酵母双杂交及酵母单杂交寻找与SsFkh1共同参与调控核盘菌菌核形成的相关因子以及能够调控SsFkh1转录表达的相关靶标蛋白。本课题组寻找到SsGCN1、SsMCA1等9个候选蛋白与 SsFkh1存在互作关系，SsCH1、Ssyak1等7个候选蛋白可能与SsFkh1启动子序列结合来调控SsFkh1的转录表达。通过以上研究结果对SsFkh1转录因子调控核盘菌菌核与侵染垫形成机制有了宏观脉络，本项目研究揭示了SsFkh1转录因子可能参与MAPK介导的细胞壁完整性通路影响，此外通过参与调控SsFkh1的候选蛋白以及与SsFkh1共同行使功能的候选蛋白，解析SsFkh1调控菌核形成与侵染垫发育机制。研究成果在Molecular Plant Pathology，Phytopathology发表论文3篇。