黄栌枯萎病菌微菌核特异表达转录因子bZIP1的生物学功能与调控机制研究

Verticillium dahliae is a ubiquitous soil-borne fungus and the causal agent of smoke-tree vascular wilt, which presents a major threat to the health of smoke-trees and the “red-leaf” scenery of the Fragrant Hills Park in Beijing, China. V. dahliae produces microsclerotia, which function as primary inoculum of wilt disease and are long-term survival structures. Microsclerotia represent a significant and pivotal conundrum for the control of Verticillium wilt and a key and academic issue to reveal the mechanism of Verticillium disease. In previous NSFC project (31000302), we have studied the gene expression profiles and transcriptomes of microsclerotia formation of smoke-tree wilt fungus, screened the genes differentially expressed during microsclerotia formation of the fungus, and obtained a significantly expressed transcription factor bZIP1. Based on these results, in this proposal, we will delete bZIP1 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 bZIP1 will be established using RNA-Seq technology. Finally, in order to elucidate the cis-acting elements and regulatory networks of transcription factor bZIP1, the promoter sequences of bZIP1-regulated genes are to be identified and compared, and numbers of important genes regulated by bZIP1 are to be disrupted and characterized as well. The data will not only further shed light on elucidating molecular mechanism of microsclerotia formation in V. dahliae, helping to find specific target to microsclerotia development to develop and produce novel fungicides, providing new views and theoretic direction to design integrated control strategy and practice to Verticillium wilt of smoke-tree Cotinus coggygria; but also suggest highly valuable references to control Verticillium wilt on other host plants.

大丽轮枝菌侵染黄栌引起的枯萎病已成为影响黄栌林健康和红叶景观的重要生物灾害；该病原产生的微菌核，作为病害的主要侵染源具有很强的抗逆能力，是控制枯萎病的难点，亦是揭示病害发生机制的重要科学问题。基于青年基金对黄栌枯萎病菌微菌核形成过程特异表达基因筛选及转录组学的研究，已明确了该过程的基因表达谱，获得了1个显著上调表达的转录因子bZIP1。本项目拟通过遗传转化方法研究bZIP1在黄栌枯萎病菌微菌核形成和致病过程中的生物学功能；然后采用RNA-Seq技术建立bZIP1突变体转录组数据库，鉴定其调控的下游靶标基因；通过开展重要下游基因的功能及启动子序列的分析，阐明bZIP1的调控机制和顺式作用元件。研究结果将有助于进一步揭示黄栌枯萎病菌微菌核形成的分子机制，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。

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。大丽轮枝菌产生的微菌核作为病害循环的主要初侵染源和帮助病原菌度过不良环境的休眠结构，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。黄栌枯萎病菌形成微菌核是一个十分复杂的生物学过程，揭示其分子机制需要通过系统地筛选微菌核形成过程中相关的基因，进而开展重要差异表达基因的功能。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。为了阐明重要转录因子家族在黄栌枯萎病菌微菌核形成和致病过程中的功能及其调控网络。本项目围绕黄栌枯萎病菌微菌核特异表达的bZIP转录因子、Crz1转录因子和HOG信号途径组分开展了功能研究，采用遗传学和功能基因学方法，获得了黄栌枯萎病菌bZIP、VdCrz1、VdPbs2、VdHog1和VdMsn2等基因的突变体，明确了黄栌枯萎病菌2个在微菌核形成过程中差异表达的bZIP转录因子不参与微菌核形成但协同调控活性氧应答，阐明了VdPbs2-VdHog1信号通路控制黄栌枯萎病菌微菌核形成和参与了逆境胁迫的应答并在致病过程发挥着关键作用，揭示了HOG信号途径下游转录因子VdMsn2负调控微菌核形成，鉴定了轮枝菌C2H2锌指转录因子家族，分析了其在微菌核形成过程中表达规律，并发现了含同型结构域的C2H2锌指转录因子亚家族基因数目在轮枝菌中显著扩张现象，阐述了Ca2+信号关键转录因子VdCrz1参与调控微菌核形成和致病性。此外，本项目还研究了枯草芽孢杆菌抗真菌代谢物抑制黄栌枯萎病菌微菌核形成的转录机制和病菌不同营养条件下基因表达谱，为开展黄栌枯萎病控制以及解析黄栌枯萎病菌炭和氮代谢网络奠定了基础。研究结果在理论上将有助于揭示大丽轮枝菌生长发育及致病过程的规律，在实践中将有助于针对微菌核特异的新型农药靶标基因的筛选、病害管理策略的设计，从而有效控制轮枝菌枯萎病, 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。