由镰刀菌复合种(Fusarium complex)引起的赤霉病是谷类作物上的一种毁灭性病害。该病害流行时不仅造成粮食大幅减产，并且病原菌在受侵染的籽粒中还会产生霉菌毒素，严重危害人畜健康。当前由于缺乏抗病的小麦品种，化学防治仍是防治小麦赤霉病的主要手段。在我国主要使用多菌灵、戊唑醇和氰烯菌酯等药剂防治赤霉病。但由于长期频繁使用杀菌剂，在田间已经出现严重的多菌灵抗性问题。因此，开发新型防病抑毒杀菌剂对防治赤霉病有重要的理论和现实意义。 脂滴(lipid droplet，LD)在真核生物的脂质代谢平衡中具有重要作用，但目前人们对脂滴在丝状真菌中的合成机制以及生物学意义知之甚少。我们课题组前期研究初步发现，雷帕霉素(rapamycin)可以诱导禾谷镰刀菌体内积累大量脂滴，表明TOR(target of rapamycin)信号途径可能参与调控脂滴合成。本文在此基础上深入解析禾谷镰刀菌中TOR途径对脂滴合成的调控机制，并研究了脂滴在禾谷镰刀菌中的生物学功能，取得的主要结果如下： 1)利用BODIPY和尼罗红染色发现，EC₉₀浓度的雷帕霉素能诱导野生型菌株中大量积累脂滴，而EC₉₀浓度的氰烯菌酯、多菌灵和戊唑醇等药剂没有这种作用。 TOR下游的激酶Sch9缺失后不影响脂滴积累，而磷酸酶FgSit4或FgPpg1缺失后均严重阻碍了菌体中的脂滴积累。这些结果表明，TOR途径通过FgSit4／FgPpg1支路调控禾谷镰刀菌中的脂滴合成。 2)TOR失活后诱导积累的脂滴主要依赖于三酰基甘油(triacylglycerols，TAG)，而非甾醇酯(sterol esters，SE)的合成。脂滴内中性脂的主要成分包括TAG和SE。TAG含量检测发现，雷帕霉素处理后，野生型菌丝中TAG含量显著上升；将禾谷镰刀菌中SE合成基因FgARE1和FgARE2单独或同时敲除后，均不影响雷帕霉素对脂滴合成的诱导。 3)亲和捕获及酵母双杂实验表明，磷脂酸磷酸酶FgPah1与磷酸酶复合体VgNem1／Spo7互作。Phos-tag实验证明：雷帕霉素处理后，FgNem1／Spo7对FgPah1进行去磷酸化。 4)通过BODIPY染色以及TAG含量测定发现，蛋白激酶FgCAK1的敲除突变体(△FgCak1)中脂滴合成及TAG含量减少。Co-IP及酵母双杂实验表明，FgCak1与复合体FgNem1／Spo7互作。Phos-tag检测发现，雷帕霉素处理引起FgNem1发生磷酸化修饰。FgCak1缺失后，FgNem1的磷酸化水平不再明显上升。这些结果表明，TOR失活后，蛋白激酶FgCak1对FgNem1进行磷酸化修饰。 5)表型分析发现，△FgNem1，△FgSpo7或△FgPah1生长速率减慢，有性发育受损，DON产量下降，对氧化压力的敏感度上升，致病力明显降低。此外，△FgNem1， △FgSpo7和△FgPah1对胆碱及细菌胁迫的敏感度明显上升。 本研究结果表明，TOR信号途径通过FgNem1／Spo7-FgPah1介导调控的脂滴合成对禾谷镰刀菌的生长发育，DON合成，致病力以及生物压力响应具有重要作用。 关键词：禾谷镰刀菌；雷帕霉素；TOR途径；脂滴；磷酸化；致病性

 Fusarium head blight (FHB) caused by Fusarium complex is one of the most devastating diseases of cereal crops worldwide. In addition to severe yield losses, FHB leads to mycotoxin contamination in infested grains that poses a serious threat to human and animal health. Currently, chemical fungicide control is still the most effective strategy for controlling FHB due to the unavailability of FHB resistant wheat cultivars. Carbendazim, tebuconazole and phenamacril are widely used for suppression of FHB in China. Unfortunately, carbendazim-resistant F. graminearum strains have been detected in the field after long-term intensive application. Therefore, applying a novel antifungal and anti-mycotoxin fungicide is urgently needed for sustained management of FHB. Lipid droplets (LDs) play an important role in the lipid metabolism of eukaryotic cells. However, the biogenesis regulation and biological functions of LDs are largely unknown in filamentous fungi. Our previous study showed that rapamycin treatment resulted in a significant increase of LDs biogenesis in the plant pathogenic fungus F. graminearum, indicating the TOR (target of rapamycin) signaling pathway may be involved in LDs biosynthesis. In this study, we deeply analyzes the regulation mechanism of TOR signaling pathway in LDs biogenesis and the biological function of LDs in Fusarium graminearum. Results in our study showed that: 1)The wild-type hyphae treated with rapamycin, phenamacril, carbendazim and tebuconazole at the EC₉₀ concentration, respectively for determination of LDs biogenesis by BODIPY and Nile Red staining assays. Results showed that only rapamycin treatment could specially induce LDs accumulation. LDs staining assays and transmission electron microscopy (TEM) examination showed that LDs biogenesis was not induced in the ΔFgSit4 and ΔFgPpgl strains, while the content of LDs in the hyphae of ΔFgSch9 was similar to that of the wild-type after the rapamycin treatment. These results indicate that FgTOR inhibition by rapamycin induces LD biogenesis through the FgPpgl/Sit4 signaling branch in F. graminearum. 2)LDs biogenesis upon TOR inhibition mainly depends on biosynthesis of triacylglycerols (TAGs) but not sterol esters (SEs) in F. graminearum. TAG and SE are the most predominant neutral lipids in LDs. TAG content quantification showed that TAGs in the rapamycin treated hyphae was obviously increased in comparison with that of the non-treatment control. On the other hand, deletion of SE synthesis genes FgAREl and FgARE2 did not influence the LDs induction upon rapamycin treatment. 3)Affinity capture and yeast two hybrid assays showed that phosphatidate phosphatase FgPah1 interacts with phosphatase complex FgNem1/Spo7. Phos-tag assays demonstrated that rapamycin treatment resulted in dephosphorylation of FgPah1, and the dephosphorylation of FgPah1 was prevented in FgNEM1 deletion mutant. 4)BODIPY staining and TAG quantification assays revealed that deletion of kinase FgCAKl resulted in serious defects in TAG and LDs synthesis upon rapamycin treatment. Co-IP and yeast two hybrid assays showed that protein kinase FgCakl interacted with FgNeml/Spo7 complex. More importantly, we observed that rapamycin treatment resulted in phosphorylation of FgNeml, and the phosphorylation level of FgNeml was no longer obviously increased in ΔFgCakl by Phos-tag assays. These results indicate that kinase FgCakl targets FgNeml and regulates the phosphorylation of FgNeml upon TOR inhibition. 5)Phenotypic analysis revealed that deletion of FgNEM1, FgSP07 or FgPAH1 leads to serious defects in vegetative growth, sexual development, DON production, ROS defense, virulence, sensitivity to choline and competition of F. graminearum with a bacterium. Results in this study indicate that LD biogenesis regulated by TOR through the FgNeml/Spo7-FgPah1 cascade plays critical roles in fungal development, pathogenicity, and biotic stress response in F. graminearum. keywords : Fusarium graminearum; rapamycin; TOR pathway; lipid droplets; phosphorylation; pathogenicity