大丽轮枝菌DNA甲基化调控机制及其对致病性的影响

Verticillium dahliae is a devastating filamentous soil fungus which causes crop wilting and results in tremendous financial loss. V. dahliae can initiate a dormancy structure-microsclerotia to survive in soil for dozens of years, which caused difficulty in maintaining. Therefore, crop breeding for high resistance against V. dahliae is desperately needed. However, the poor knowledge in molecular mechanism during V. dahliae-plant interaction leads to limited use of candidate gene for breeding. DNA cytosine methylation is an essential epigenetic regulation for many important biological events in eukaryote. Understanding the potential virulence gene expression controlled by DNA cytosine methylation in fungi during its interaction with plant will provide novel view in fungi-plant interaction knowledge. Previously, the genomic DNA sequence of the high pathogenicity V592 strain isolated from wilting cotton in Xinjiang province was sequenced and the VdDIM2 gene which functions as DNA cytosine methyltransferase sequence was found. Knock-out of VdDIM2 gene mutant VdΔdim2 strain showed 5 times less in pathogenicity in Arabidopsis compared to V592 strain. While what kinds of DNA cytosine methylation type and virulence gene expression regulated by VdDIM2 gene was still unclear. Thus unveil these molecular mechanism regulated by VdDIM2 gene could bring benefits in understanding V. dahliae-plant interaction and providing potential genes for crop breeding.

大丽轮枝菌是植物黄萎病的主要致病菌并造成巨大经济损失。该病菌抗逆性强，因此对高抗品种培育靶标基因的需求尤为迫切。然而大丽轮枝菌-植物互作分子调控机制仍不甚明朗，因此可利用抗病育种基因资源较少。DNA胞嘧啶甲基化是广泛存在于真核生物中的重要表观遗传学调控机制，但是对于DNA甲基化在植物-病原互作中的作用人们的了解还很少。本课题组前期对新疆地区棉花黄萎病高致病菌株V592进行全基因组测序，并成功分离获得其DNA甲基转移酶编码基因VdDIM2，敲除该基因菌株VdΔdim2与V592相比对拟南芥致病力减弱达5倍。解析VdDIM2基因调控何种DNA胞嘧啶甲基化方式，同时调控何种致病相关基因转录水平表达，将为理解大丽轮枝菌-植物互作机制提供新的思路，也为抗黄萎病育种提供参考。

大丽轮枝菌（Verticillium dahliae Kleb.）是植物黄萎病的主要致病菌，其寄主广泛，包括重要农作物如棉花，番茄，马铃薯等 400 多种，并造成巨大的经济损失。该病菌抗逆性强且防治难度极大，对高抗品种培育靶标基因的需求尤为迫切。DNA 胞嘧啶甲基化是广泛存在于真核生物中的重要表观遗传学调控机制。然而真菌 DNA 胞嘧啶甲基化能否调控致病力相关研究极少。因此，解析真菌DNA胞嘧啶甲基化调控机制及其对致病性的影响，将对病原-植物互作研究提供新思路。也为植物抗黄萎病育种提供参考。本项目主要研究三个方面：大丽轮枝菌DNA甲基化调控图谱，DNA甲基化调控大丽轮枝菌致病性的分子机制以及转VdDIM2i株系的抗病性检测。取得4个重要结果：1.大丽轮枝菌JR2菌株DNA甲基转移酶Dim2发挥甲基转移酶功能调控全基因组DNA甲基化水平；2. Dim2蛋白能通过DNA甲基化抑制钾离子通道蛋白ß亚基蛋白积累，并提高大丽轮枝菌对逆境的响应与适应性，从而满足定殖宿主所需侵染结构形成；3.DIM5-HP1-DIM2复合体也能够调控钾离子通道蛋白ß亚基蛋白的表达与积累4.Dim2i转基因拟南芥与番茄植株能够大幅提高对JR2菌株的抗病性。本研究所发现DNA甲基转移酶Dim2能正向调节大丽轮枝菌的致病力，Dim2功能的丧失使大丽轮枝菌转变为无毒菌株，是植物抗病育种理想的靶位点。目前宿主介导的RNA沉默技术（HIGS）在棉花抗病性育种中已取得理想的效果。本课题组也证明Dim2i转基因番茄在实验室条件下对JR2菌株有理想的抗病性。