The Mechanism Of Validamycin Inhibiting The Synthesis Of Fumaroxin

Fusarium verticillioides is one of the main pathogens causing maize ear rot,which has caused huge economic and production losses to maize planting.In addition to severely affecting maize yields,it also produces secondary metabolites,fumonotoxin,which contaminate grain and cause disease in both humans and animals.At present,the main methods used to control maize ear rot are maize resistant plants and chemical control,but some chemical agents can’t effectively inhibit the synthesis of fumonitins while controlling the occurrence of the disease,and even stimulate the synthesis of fumonitins.validamycin is a biogenic antibiotic independently developed in China,which can produce validoxylamine under the action of β-D-glycosidase and inhibit trehalase activity.In the early stage of this experiment,it was accidentally found that validamycin could not only control maize big spot disease and maize sheath wilt,but also reduce the level of fumonin pollution,but its mechanism of toxin inhibition was not clear.Therefore,this study focused on the molecular mechanism of validamycin blocking fumarine toxin synthesis,which mainly included the following three aspects.1.Firstly,the synthesis ability of fumarins under different concentrations of validamycin was verified in the laboratory,and validamycin was found to stably inhibit fumarin synthesis.The mycelial growth rate method was used to observe the inhibitory effect of validamycin on the mycelial growth of F.verticillioides,but it could inhibit spore germination.Transmission electron microscopy was used to further observe the subcellular structure of F.verticillioides treated with the agent.It was found that the mitochondrial structure of the pathogen was damaged,and this was verified by ATPase activity.At the same time,the expression of FUM family genes related to fumarin synthesis showed a decreasing trend,which was consistent with the results of fumarin synthesis.It was demonstrated that wellgangamycin could inhibit fumarin synthesis by regulating the fumarin synthesis gene cluster.2.Candidate targets for wellgangamycin were identified by biopHysics and 3D printing technology,including 11 candidate target proteins such as trehalase,β-glucosidase,ATP-dependent serine protease and heat shock protein.This project focused on the role of candidate target trehalase in the process of fumarine toxin synthesis.A trehalase deletion mutant was constructed using the wild-type strain F.verticillioides as the starting strain.By analyzing the amount of fusarine toxin synthesis and expression of the FUM family gene,trehalase was positively correlated with the growth,pathogenicity and ability of fusarine pseudopHyllosa to synthesize fumarine toxin.The contents of glucose,pyruvate and ATPase in wild-type and mutant strains were determined.The results showed that the above indexes in mutants were consistent with those treated with validamycin.3.Computational chemistry was used to verify the interaction ability of F.verticillioides with trehalase.The binding activity of validamycin and trehalase was analyzed by molecular interlocking.ASP286,TRP285,ASN322,GLY450,GLN333,ASP452,GLN606,GLN398,TYR28,ARG447 and LYS190 were identified.These mutant sites are identified by computer point mutations that play an important role in receptor and ligand binding.TRP285,ASP286 and GLN606 were the main amino acids,and their ability to interact was reduced after mutation.Conclusion:validamycin regulates growth and development,pathogenicity and fumarine toxin synthesis by inhibiting trehalase activity in F.verticillioides.This result reveals the mechanism by which validamycin inhibits fumaratoxin synthesis,providing guidance for the rational use of chemical agents to prevent and control fumaratoxin pollution.