| Maize ear rot is a kind of fungal disease which is common in maize planting areas all over the world.The research reports are mainly caused by Fusarium,among which Fusarium verticillioides is the main pathogen of ear rot of maize.Oxysterol binding protein(OSBP)and its related protein homologues(ORPs),as lipid binding/transferring proteins,widely exist in eukaryotes,and their main functions are involved in cell lipid metabolism,vesicle transport and signal transduction.However,at present,the research of ORPs is mainly concentrated in mammals and Saccharomyces cerevisiae,and the biological function of ORPs in most filamentous fungi is still unclear,especially in F.verticillioides.In this study,the biological function of F.verticillioides ORPs was studied,and the main results were as follows:1.Through bioinformatics analysis,seven ORPs members were identified in F.verticillioides,and their full-length genes ranged from 1282 bp to 3654 bp.Their corresponding genes were named as FvoshA,FvoshB,FvoshC,FvoshD,FvoshE,FvoshF and FvoshG respectively.The proteins of seven members of ORPs family of F.verticillioides were obtained by prokaryotic expression and protein chromatography,and it was determined by fluorescence titration that only FvoshC could specifically bind ergosterol.FvoshC gene knockout mutant ΔFvoshC was obtained by PEG-mediated homologous recombination technology,and then the restoration and overexpression vector of FvoshC was constructed.The restoration C-ΔFvoshC and overexpression mutant OEΔFvoshC were obtained by protoplast transformation.2.By observing the ultrastructure of each strain,it was found that ΔFvoshC hyphae were obviously small and shriveled.The content of fumonisin in WT,ΔFvoshC,CΔFvoshC and OE-ΔFvoshC was determined by HPLC,and it was found that the content of FB1 in ΔFvoshC decreased significantly.Q-PCR was used to determine the expression level of each member of fumonisin synthesis gene cluster,which showed that all members were down-regulated.Pathogenicity test showed that the deletion of FvoshC gene resulted in weak pathogenicity,and the above results showed that FvoshC was positively correlated with fumonisin B1 biosynthesis and pathogenicity of the pathogen.The lipid metabolomics of WT and ΔFvoshC mycelium were analyzed.Compared with the control WT,the upregulated metabolites of ΔFvoshC in treatment group were sphingosine(SPH),phosphatidylcholine(PC),ceramide(Cer)and lysophosphatidic acid(LPA),while the down-regulated metabolites were triglyceride(TG),diglyceride(CL)and monogalactose diglyceride(MGDG).KEGG functional pathway analysis shows that the metabolic pathways are Lipid metabolism,Glycine biosynthesis and metabolism,Transport and catabolism,and Signal transduction.The above results indicate that FvoshC gene can regulate lipid metabolism and signal transduction of F.verticillioides.3.FUM8,as a member of fumonisin synthesis gene cluster,is located in vesicle.The content of FBI in ΔFvoshC decreased significantly,and the expression of FUM8 decreased significantly.Further observation of the number of vesicles in ΔFvoshC showed that the number of vesicles decreased significantly,and the expression of genes related to vesicle formation and transport was down-regulated by Q-PCR.Autophagy also occurs in the process of vesicle transport and fusion,and the expression of autophagy-related genes is down-regulated by Q-PCR.These results indicate that FvoshC is involved in the regulation of vesicle formation,transportation and autophagy.FvoshC interacting protein was studied.The prediction results of STRING online software showed that there was an interaction between FvoshC and FvSec14.The interaction between FvoshC and FvSec14 was proved by yeast two-hybrid,and it was speculated that FvoshC interacted with FvSec14 to further function.4.With FvoshC as the target,Xanthatin,a natural product inhibitor with high activity,was obtained by computer high-throughput screening.The biological activity test showed that Xanthatin could significantly inhibit the growth of F.verticillioides.The results of HPLC showed that FvoshC could effectively inhibit the production of FB1,which indicated that FvoshC had a positive effect on FB1 resistance and disease control.To sum up,this study found that FvoshC can regulate inositol transport and vesicle formation by interacting with FvSec14,and then regulate the pathogenicity of bacteria and toxin biosynthesis.The results of this study will provide a theoretical basis for the prevention and control of corn ear rot and mycotoxin pollution,and protect the quality and safety of agricultural products. |
