Regulatory Mechanism Of Lipid Droplet Synthesis In Fusarium Graminearum

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 AFgSit4 and △FgPpgl strains,while the content of LDs in the hyphae of △FgSch9 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 FgPahl interacts with phosphatase complex FgNeml/Spo7.Phos-tag assays demonstrated that rapamycin treatment resulted in dephosphorylation of FgPahl,and the dephosphorylation of FgPahl was prevented in FgNEMl 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 AFgCakl 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,FgSPO7 or FgPAHl 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-FgPahl cascade plays critical roles in fungal development,pathogenicity,and biotic stress response in F.graminearum.