黄栌枯萎病菌C2H2-Homeobox转录因子家族扩张的生物学意义和调控微菌核形成与致病性的功能解析

Verticillium dahliae is a ubiquitous soilborne fungus and the causal agent of smoke tree vascular wilt, which presents a major threat to the famous “red-leaf” scenery of the Fragrant Hills Park in Beijing. V. dahliae produces multicelled and infect a variety of hosts, melanized resting bodies, also known as microsclerotia (MS) that can survive for years in the soil. The MS are the primary source of infection of the Verticillium disease cycle. Thus, MS formation marks an important event in the disease cycle of V. dahliae. Transcription factors, as one of the largest families of trans-acting molecules, play crucial roles in transcriptional regulation of gene expression, by mode of activation or repression. Transcription factors are usually termed the master regulators of gene expression. By biding to the DNA, they tightly control where and when the target genes are expressed. Research of transcription factors is of great significance for expounding mechanisms of pathogens’ growth and development and infection. We identified a total of 79 typical C2H2 zinc finger genes in V. dahliae. Comparative analysis revealed that four plant pathogenic fungi, V. dahliae, Fusarium oxysporum, Magnaporthe oryzae, and Botrytis cinerea, have comparable numbers of predicted C2H2 zinc finger genes with similar characteristics. Phylogenetic analysis identified a containing seven genes with similar gene structures. Based on these results, in this proposal, we will study the biological significance of the expansion of C2H2-Homeobox subfamily in V. dahliae, will systematically delete this family using homologous recombination mediated by PEG genetic transformation method and characterizes the biological function in microsclerotia formation and pathogenesis in detail. Moreover, the transcriptome and differential expression profiles of genes regulated by an important C2H2-Homeobox transcription factor will be established using RNA-Seq and Chip-Seq techniques. Finally, the promoter sequences of genes regulated by C2H2-Homeobox transcription factor are to be identified and compared, and numbers of important genes regulated by the transcription factor are to be disrupted and characterized as well. Therefore, the cis-acting elements and regulatory networks of C2H2-Homeobox transcription factor will be elucidated. The data will not only further shed light on elucidating molecular mechanism of microsclerotia formation, growth and development, stress response and pathogenesis in V. dahliae, helping to find V. dahliae-specific novel fungicides targets, providing new insights and theoretic guide to design integrated control strategy and practice to Verticillium wilt of smoke-tree; but also suggest highly valuable references to control Verticillium wilt on other host plants.

黄栌枯萎病已成为影响黄栌健康和红叶景观的重要生物灾害，其病原是大丽轮枝菌。形成微菌核和寄主范围广是轮枝菌病害难以有效控制的根本原因，微菌核具有很强的抗逆性，是控制病害的关键和难点，亦是揭示病害发生机制的重要科学问题。转录因子操控着生命活动中的基因表达调控，是阐明生物学表型内在基因调控网络及互作的基础。前期研究中我们鉴定并发现了含Homeobox的C2H2锌指转录因子家族在轮枝菌中显著扩张。本项目拟通过遗传学和功能基因组学方法揭示该基因家族扩张的生物学意义和进化机制，明确其调控黄栌枯萎病菌微菌核形成、生长发育、逆境胁迫和致病过程的生物学功能和作用机理；然后采用RNA-Seq和Chip-Seq技术鉴定关键转录因子调控的下游靶标基因及DNA结合位点，绘制转录因子-DNA相互作用图谱，阐明该基因家族的调控机制和顺式作用元件。研究结果将深入阐释该基因扩张的生物学意义和微菌核形成与致病性的调控机制。

微菌核、寄主范围广和寄生维管束是轮枝菌病害难以有效控制的根本原因，微菌核具有很强的抗逆性，是控制病害的关键和难点，亦是揭示病害发生机制的重要科学问题。转录因子操控着生命活动中的基因表达调控，是阐明生物学表型内在基因调控网络及互作的基础。本项目采用林木病理学、遗传学、分子生物学、组学和生物信息学等多种技术与方法研究了C2H2-Homeobox、VdAtf1、VdHapX、VdCrm1和MAPK等基因的生物学功能，研究结果初步明确了大丽轮枝菌基因组C2H2-Homeobox转录因子亚家族显著扩张现象并阐明了其调控微菌核形成和毒性的机理，阐述了转录因子VdAtf1通过调控NO胁迫应答，营养缺乏的耐受性和侵入钉形成而影响致病性的分子调控网络且与已知氮代谢调控因子VdNut1存在着反向反馈调控关系，揭示了转录因子VdMcm1控制菌丝生长、微菌核形成和致病性的作用方式以及次级代谢的转录调控机制，解析了转录因子VdHapX调控轮枝菌如何维持铁平衡和毒性的分子基础，明确了转录因子VdCmr1调控黑色素生物合成，但并不影响微菌核形成的遗传基础，在转录组和蛋白质学水平阐明了VdCmr1响应高温与紫外胁迫的应答机制，解析了黄栌枯萎病菌信号传导途径基因VdSsk2-VdPbs2-VdHog1级联、VdSkn7和VdSsk1调控黄栌枯萎病菌微菌核形成，控制病原菌黑色素合成和侵染结构的作用机制，揭示了枯草芽孢杆菌脂肽抑制微菌核形成的分子基础。研究结果先后共发表了9篇SCI论文，培养了4名研究生。本项目的实施丰富人们对轮枝菌转录因子家族和信号分子的研究，研究结果进一步揭示黄栌枯萎病菌微菌核形成、病原菌逆境胁迫和致病过程的分子机制，为寻找微菌核形成的特异性分子靶标，开发新型杀菌剂，设计新的黄栌枯萎病控制策略提供理论指导；并且对其他植物轮枝菌枯萎病的控制亦有极高的参考价值。