Toxicological Mechanism Of Validamycin Regulatingthe Interaction Between Trehalase And Regulator Protein 14-3-3 In Fusarium Graminearum

Fusarium graminearum is an important plant pathogenic fungus,which can not only reduce the yield and quality of agricultural products,but also secrete mycotoxins to contaminate agricultural products and threaten food safety.Validamycin is a kind of efficient and safe biological-source fungicide,being applied to prevent and control Rhizoctonia in the field for nearly half a century,and yet it has not found resistant strains to validamycin.And recently our lab found that validamycin inhibits deoxynivalenol DON biosynthesis of F.graminearum,which demonstrates the application value of controlling of Fusarium head blight(FHB).Therefore,the study of the toxicological mechanism of this fungicide displays values both on theoretical and practical aspects.It has been known so far that validamycin inhibits the trehalase activity of fungal and induces the host plants to produce defense response.It has been found that in Saccharomyces cerevisiae,trehalase interacts with the regulatory 14-3-3 protein Bmh1 subtype,together regulating the anti-stress responses.The14-3-3 proteins,widely distributed in eukaryotic cells and highly conserved evolutionarily,mainly bind to the target proteins through two sites and then regulate different life progresses.In this study,through sequence alignment,it was found that the phosphorylation sites of trehalase S60 and S83 in F.graminearum were consistent with those in S.cerevisiae;and via homologous comparison,we found that there exist two Bmh proteins in F.graminearum,named as Fg Bmh1 and Fg Bmh2 respectively.Fg Nth,Fg Bmh1 and Fg Bmh2 were localized in the same cell structures at different growth stages by fluorescence labeling,and Co-IP results further diaplayed that neutral trehalase Fg Nth interacts with both Fg Bmh1 and Fg Bmh2.When treating mycelia with 1,10 and 100 μg/m L for 12 h,we found that validamycin enhanced the interaction between Fg Nth and Fg Bmh1 in an rising trend with the increase of the concentration,but validamycin had no effects on the interaction between Fg Nth and Fg Bmh2.Treat all strains with four stresses,including validamycin,Na Cl,Sorbitol and high temperature(37C)for 5min to observe whether they effect the interaction between Fg Nth and Fg Bmh1.The results showed that both Na Cl and validamycin significantly enhanced the interaction between Fg Nth and Fg Bmh1.It can be concluded that the treatment time of validamycin,whether 12 h or 5min,both significantly enhanced the interaction between Fg Nth and Fg Bmh1,and validamycin could enhance the interaction between them in a short period of time.It is speculated that validamycin plays a role in activating some pathways rapidly like osmotic stresses.It is known that Bmh1 and Bmh2 probably physically interact with MAPK kinase Pbs2 and heat shock protein Hsp70 in S.cerevisiae,regulating the stress responses to osmotic and high temperature stresses.We determined Fg Pbs2(FGSG＿08691)and Fg Hsp70(FGSG＿00838)in F.graminearum.via homologous comparison Co-immunoprecipitation assay showed that neither Fg Bmh1 nor Fg Bmh2 could interact with Fg Pbs2,suggesting that neither Fg Bmh1 nor Fg Bmh2 function as a MAPKK kinase to activate MAPKK Fg Pbs2.We also confirmed that Fg Bmh2 can bind to Fg Hsp70 but not to Fg Bmh1 in F.graminearum.Previous laboratory studies have shown that the sensitivity of Fg Nth deletion mutant of F.graminearum to validamycin decreased compared with the wild-type strain.In this study,the sensitivity of Fg Bmh1 and Fg Bmh2 deletion mutants to validamycin also significantly decreased through gene knockout strategy.We speculated that Fg Bmh1 and Fg Bmh2 probably are involved in the regulation of validamycin sensitivity.Under Na Cl,KCl,Sorbitol and high temperature stresses conditions,the tolerance of Fg Nth,Fg Bmh1 and Fg Bmh2 deletion mutants increased.Compared with wild-type strain,the DON production and pathogenicity of Fg Nth,Fg Bmh1 and Fg Bmh2 deletion mutants were significantly decreased.,which indicates that validamycin inhibited the DON biosynthesis and pathogenicity of F.graminearum and was related to the interaction between Fg Nth and Fg Bmh1.These novel findings in this study provide reference values for further understanding the toxicological mechanism of validamycin inhibiting trehalase activity.