甘蔗鞭黑粉菌有性配合及致病相关基因SsDLIC的克隆与功能研究

Sugarcane smut caused by Sporisorium scitamineum occurs worldwide, causing serious loss in both yield and quality. The Basidiomycete fungus S. scitamineum alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen by mating. So mating is not only an important event and a necessary path way for life cycle completion, but also is vital precondition for pathogenicity, even affects the process of fungus infection. In the previous studies, by screening the 30000 transformants gernerated via Agrobacterium tumefaciens-mediated transformation (ATMT), we obtained a T-DNA insertion mutant 131D11, which completely lost pathogenicity, and the T-DNA disrupted gene SsDLIC was proved involved in mating and pathogenicity in S. scitamineum by functional complementation of the mutant 131D11 using the wild-type copy of the gene. However, further study is needed to reveal the roles of SsDLIC in mating and understand its molecular mechanism in the process of fungus infection. This project will study the function of the SsDLIC gene associated with mating and pathogenicity of Sporisorium scitamineum. Gene knocked out and real-time PCR will be used to illuminate the relationship of SsDLIC and the pheromone response factor gene SsPrf1, as well as the relationship of SsDLIC and the mating genes induced by pheromone in a loci (mfa & pra) and b loci (bE & bW), in order to illustrate the functions of SsDLIC in the pheromone signaling and sexual mating. Furthermore, transcriptomics analysis will be used to analyse its molecular mechanism in the process of fungus infection. This project would offer evidence to reveal pathogenicity mechanisms of S. scitamineum and provide theoretical bases and target gene for new control techniques of smut, it could also be helpful for understanding the interaction of pathogen-host at molecular level.

甘蔗鞭黑穗病是世界性病害，造成甘蔗产量质量严重损失。引起该病的甘蔗鞭黑粉菌通过有性配合，从腐生的酵母状细胞转换成具有致病性的双核菌丝，因此其有性配合与致病性密切相关。前期研究中，申请者获得了该菌1株致病力丧失的T-DNA插入突变体，通过基因互补证明突变的相关基因SsDLIC与病菌的有性配合和致病性相关，但其在病菌的有性配合及致病过程中的作用机理尚不清楚。本项目拟开展甘蔗鞭黑粉菌有性配合及致病相关基因SsDLIC的克隆与功能研究，通过基因敲除、荧光定量PCR等方法分析它们与信息素响应基因SsPrf1、交配型基因a位点上信息素基因（mfa）和信息素受体基因（pra）、b位点上信息素诱导基因（bE 和 bW）的关系，探明它们在病菌信息素传输及有性配合中的作用，进一步通过转录组测序研究它们在致病过程中的分子机理。项目完成可为从分子水平上揭示甘蔗鞭黑粉菌的致病机理提供直接的证据。

甘蔗鞭黑穗病是世界性病害，造成甘蔗产量质量严重损失。引起该病的甘蔗鞭黑粉菌通过有性配合，从腐生的酵母状细胞转换成具有致病性的双核菌丝，因此其有性配合与致病性密切相关，甚至影响其侵染过程。本项目研究获得了该菌1株有性配合能力和致病力均丧失的T-DNA插入突变体，通过基因互补证明突变的相关基因SsDLIC与病菌的有性配合和致病性相关。为进一步分析SsDLIC在信号传递和致病性发展中的功能，利用同源重组的方法进行了SsDLIC基因的敲除。敲除突变体JG35Δdlic和JG36Δdlic分别来自一对可亲和的野生型单倍体菌株JG35（a2交配型）和JG36（a1交配型）。和T-DNA插入突变体131D11一样，JG35Δdlic和JG36Δdlic不能与相对性系的交配型JG36或JG35进行有性配合产生白色的绒毛状的菌落，完全丧失了对甘蔗的致病性。荧光定量PCR分析和转录组数据分析结果表明，SsDLIC不仅调控a1位点信息素相关基因SsMFA1和SsPRA1、a2位点信息素相关基因SsMFA2和SsPRA2的基础水平转录，还调控它们信息素诱导的转录。此外，SsDLIC还通过调控细胞色素P450单加氧酶、硫代酸糖基转移酶、ABC转运蛋白等多个基因的表达而参与多个细胞代谢途径。本研究结果表明，SsDLIC在甘蔗鞭黑粉菌的信息素传递和致病过程中起着重要作用，为揭示病菌的致病机制提供了证据，为新的黑穗病防治技术提供了理论依据和靶基因，项目完成还可为从分子水平上揭示病原菌与宿主的相互作用提供直接的证据。.项目还探明了甘蔗鞭黑粉菌a1位点和a2位点交配型基因的组成，明确a位点编码1个信息素基因MFA和1个信息素受体基因PRA，进行基因敲除获得了a1位点MFA1和PRA1的基因缺失突变菌株，并证明敲除突变株的有性配合能力和致病性丧失，为进一步研究甘蔗鞭黑粉菌信息素信号传递和致病机理打下了良好基础。