禾谷镰孢菌TOR信号途径对致病和毒素合成调控的分子机制

In eukaryotic cells, the target of rapamycin (TOR) signaling pathway plays important roles in nutrient metabolism, ribosome biogenesis, and extension of lifespan. However, little is known about the function of this pathway in filamentous fungi. Our previous studies showed that the TOR pathway is essential for virulence and DON biosynthesis in Fusarium graminearum. Additionally, we found that the methylation level of lysines 4 on histone (H3K4) was dramatically reduced by the treatment of 0.36 g/ml rapamycin for 6 h, and identified a methyltransferase FgSet1-3, a key component of COMPASS complex regulating H3K4 methylation. Deletion of SET1-3 resulted in decreased methylation of H3K4. The FgSet1-3 mutant was nonpathogenic and unable to produce DON. Moreover, we found that FgSet1-3 interacted with the type 2A protein phosphatase FgPpg1, a core component of TOR pathway, and with the subunit of RNA polymerase II FgRpb1. The results we have obtained showed that the TOR pathway may modulate the expression of pathogenecity-related and DON biosynthetic genes through regulating H3K4 methylation. Based on these preliminary data, the objectives of this project are to i) identify the residues on COMPASS complex dephosphorylated by the type 2A phosphatases, ii) explore the biological functions of each component of COMPASS complex that catalyzes H3K4 methylation, iii) elucidate the molecular mechanism of transcriptional activation of pathogenecity-related and DON biosynthetic genes modulated by the COMPASS complex. Expected results of this project are useful for further understanding the biological function of the TOR pathway, and also for the exploitation of a drug target in the TOR pathway.

真核生物中TOR信号途径在营养代谢、核糖体合成和延长生命等方面起重要作用。本团队前期研究发现禾谷镰孢菌中TOR途径对病菌致病和DON毒素合成起关键作用。此外，雷帕霉素处理后，组蛋白H3上第四位赖氨酸（H3K4）的甲基化水平显著下降；同时还鉴定到催化H3K4甲基化的甲基转移酶复合体COMPASS中的核心元件Set1-3，其敲除突变体的H3K4甲基化水平显著降低，不致病且不产毒素；还发现Set1-3与TOR下游PP2A磷酸酶Ppg1、RNA聚合酶II的Rpb1亚基互作。表明TOR途径可能通过调控H3K4甲基化进而调控致病和毒素合成基因的表达。在此基础上，本项目将进一步鉴定PP2A对COMPASS复合体磷酸化作用的具体位点，明确COMPASS复合体中各个组分的功能，解析该复合体调控致病和DON毒素合成基因转录激活的机理。预期结果为深入解析TOR途径的生物学功能，发掘该途径上的新药靶奠定坚实基础。

本研究通过基因功能研究、组蛋白修饰检测、表达谱分析、ChIP-qPCR、蛋白互作技术解析了TOR信号途径通过H2B泛素化和H3K4甲基化调控DON毒素合成的分子机制，结果如下：1、禾谷镰孢菌H3K4甲基化（H3K4 me3）受COMPASS复合体调控，该复合体包含7个元件：FgSet1、FgSwd1、FgSwd2、FgSwd3、FgSdc1、FgBre2、FgSpp1。其中FgSet1是甲基转移酶，其缺失造成H3K4甲基化受阻，菌丝生长减慢，DON 毒素和色素合成能力、致病力下降，细胞壁胁迫抗性增强。FgSet1在COMPASS复合体中，直接与FgBre2、FgSdc1、FgSwd2、FgSpp1互作，其中ΔFgBre2、ΔFgSdc1与ΔFgSet1的表型相似。此外，FgSet1可调控FgAURF和FgAURJ的转录影响菌体产色素能力，同时，FgSet1正调控FgMSG5表达，从而负调控FgMgv1磷酸化水平与细胞壁胁迫抗性。2、TOR信号途径上关键元件PP2A磷酸酶FgPpg1敲除导致H2B泛素化（H2B ub1）水平下降。敲除H2B ub1的泛素结合酶FgRad6，泛素连接酶FgBre1，发现H3K4 me3受H2B ub1正调控，进一步发现，FgRad6敲除会影响FgBre2在靶基因染色体区段的富集，进而影响H3K4 me3修饰的发生。3、与ΔFgPpg1、ΔFgSet1类似，ΔFgRad6和ΔFgBre1产DON能力下降。ChIP-qPCR发现H3K4 me3在FgTRI基因簇5’端的富集水平与FgTRI基因转录水平正相关。此外，Co-IP证明FgSet1与RNA聚合酶II互作，共同参与基因转录调控。总之，本研究发现，TOR途径中PP2A磷酸酶调控H2B ub1发生，H2B ub1调控H3K4 me3在FgTRI基因的富集，进而调控DON合成。研究结果揭示了真核生物中H2B ub1调控H3K4 me3的新机制，加深了TOR信号途径调控次生代谢的认识，为小麦赤霉病新药靶和新型杀菌剂的研究奠定了基础。