大丽轮枝菌水平转移获得VdDfs基因簇调控寄主落叶性状的分子机制研究

Ever science the defoliating (D) phenotype of V. dahliae was first found in cotton in early 1960s, the genetic basis and regulatory mechanism about this differentiation are unclear. In the previous research of this project, the genomic region VdDfs, horizontally transferred from Fusarium, was only detected in the D pathotype strains of V. dahliae. The genomic region VdDfs shows the characteristic of the secondary metabolite gene cluster. Knock-out of either the whole genomic region of VdDfs or only the genes encoding polyketide synthase (PKS) in VdDfs led to the lost of the D function. However, the question on whether VdDfs gene cluster could cause the host plant defoliation by participating in the synthesis of toxic secondary metabolites and the mechanism underlying it is unclear. In this project, the functional gene(s) in VdDfs gene cluster will be further identified by double PKS genes knock-out experiment in Vd991, complementary experiment in the ΔDfs mutants, and heterogeneous complementary experiments in the non-defoliating strains. We will also construct the gene network that participated in the defoliating secondary metabolites synthesis and identify the toxic secondary metabolites, by RNA-seq and the secondary metabolites analysis. The defoliating function of the toxic compounds will be illustrated via quantitative analysis of the toxic compounds in defoliating and non-defoliating strains, and their defoliating activity and pathogenicity analysis on cotton plants. We intend to clarify the relationship between the natural selection of VdDfs and the defoliating function by comparing the VdDfs genetic variation before and after the horizontal gene transfer, and also the influence of this kind of genetic variation on the defoliating function. In conclusion, this program will help us to get insight to the molecular mechanism of V. dahliae regulating the defoliating phenotype via the synthesis of toxic compounds using the gene cluster VdDfs, which was horizontally transferred from other fungi.

自上世纪60年代发现大丽轮枝菌引起寄主落叶性状至今，其分化的遗传基础和调控机制一直不清。本项目前期发现大丽轮枝菌从棉花枯萎病菌水平转移获得VdDfs片段仅存在于落叶型菌株；VdDfs具有次级代谢基因簇特征，整体或者其中两个编码聚酮合酶基因敲除均丧失了落叶功能。然而，大丽轮枝菌是否通过VdDfs基因簇合成毒性产物引起落叶的机制尚不清楚。因此，本项目拟：分析聚酮合酶共敲除、回补和异源表达对落叶性状的影响，确证VdDfs基因簇中功能基因；通过基因表达谱和次级代谢产物分析，构建VdDfs基因簇调控寄主落叶性状的基因网络，明确合成的毒性产物；分析毒性产物含量、落叶活性和细胞毒性，阐明毒性产物引起寄主落叶性状的功能；比较分析VdDfs水平转移前后的遗传变异及其对落叶功能的影响，阐明VdDfs选择进化与落叶功能的关系。最终揭示大丽轮枝菌通过水平转移获得VdDfs基因簇合成毒性产物引起落叶性状的分子机制。

自上世纪60年代发现大丽轮枝菌引起寄主落叶性状以来，科研工作者围绕病原落叶性状表型鉴定、分子鉴定方法、种群分化特征等开展了系列研究，为解析大丽轮枝菌落叶性状特征提供了良好的基础。然而，引起寄主落叶性状的遗传基础一直不清楚，制约了人们对大丽轮枝菌引起寄主落叶性状的致病分子机制的理解和防控技术的研发。本项目前期通过来源棉花大丽轮枝菌Vd991基因组测序发现，大丽轮枝菌从棉花枯萎病菌水平转移获得的一个基因簇VdDfs缺失后大丽轮枝菌Vd991丧失了引起棉花落叶的功能，其中VdDf5和VdDf6可能是关键功能基因编码聚酮合酶。在此基础上，本项目通过基因共敲除、异源回补等遗传学实验确证VdDf5和VdDf6为调控寄主落叶性状的关键功能基因；通过分析VdDfs基因簇水平转移前后基因变异与落叶功能关系，阐明了该基因簇水平转移后发生的选择进化与其引起寄主落叶的功能高度相关；通过VdDfs基因簇参与的次级代谢网络分析、靶标产物筛选和定量，鉴定了VdDfs基因簇调控的毒性代谢产物为N-酰基乙醇胺（NAE12:0）；通过毒性代谢产物引起落叶体外验证、落叶和非落叶型菌株群体毒性代谢产物定量比较分析，明确了落叶型大丽轮枝菌产生的过量NAE12:0是引起落叶的关键；过量的NAE12:0会干扰植物体内磷脂代谢通路，显著诱导植物脂肪酸酰胺水解酶（FAAHs）基因的表达，从而抑制体内抗性相关基因表达。该研究首次揭示落叶型大丽轮枝菌通过产生过量的NAE12:0转运到寄主后通过干扰体内磷脂代谢通路而降低植物抗性，同时NAE12:0也与脱落酸作用类似，进一步了引起了寄主内源激素紊乱，最终在大丽轮枝菌毒力功能的协作下引起了寄主叶片脱落。上述研究结果首次阐明大丽轮枝菌水平转移获得的VdDfs基因簇通过介导毒性化合物的合成引起寄主落叶的致病分子机制，为大丽轮枝菌落叶/非落叶种群及致病力分化研究提供了理论依据。