Regulation Of FgCYP51s On The Sensitivity To EBI Fungicides And DON Biosynthesis In Fusarium Graminearum

Fusarium head blight（FHB）predominately caused by Fusarium graminearum is a destructive fungal disease in the wheat region of the world,causing yield losses and grain quality reduction.In infected grains,F.graminearum produces trichothecene mycotoxins that are deadly hazardous to human and animals.Due to the lack of wheat varieties with high resistance to FHB,chemical control is still the main strategy for the control of FHB.Currently,ergosterol biosynthesis inhibitor（EBI）has become the main fungicides for the control of FHB.The target of EBI is sterol 14α-demethylase（CYP51）,which has specific mutations,leading to resistance of plant pathogens to EBI fungicides.Field resistance mechanism of many plant pathogenic fungi to EBI fungicides has been extensively investigated,but no information about resistance mechanism of F.graminearum to EBI fungicides is available.In recent years,food safety caused by mycotoxins in the diseased wheat grains has become a severe society issue.Studies have shown that EBI fungicides can promote the biosynthesis of deoxynivalenol（DON）in F.graminearum,but its regulatory mechanism is still unclear.In 2017,resistant strains of F.graminearum to tebuconazole were isolated at Xuyi Sanhe Farm in Jiangsu,and found the G443S substitution in target protein CYP51A in EBI-resistant strains of F.graminearum.In order to reveal the resistant molecular mechanism of FgCYP51A-G443S of F.graminearum to EBI fungicide,we constructed the point mutants FgCYP51A-G443S to verify the correlation of EBI resistance with G443S substitution via homologous replacement with site-directed mutation method.Five kinds of EBI fungicides were used to determine the effective inhibitory medium concentration(EC₅₀)and survival fitness of the G443S mutants in F.graminearum.The results showed that the G443S mutants exhibited a high-level resistance to five EBI fungicides compared to the wild strain PH-1.In the absence of fungicides,the virulence and sexual reproduction decreased significantly,and sporulation increased significantly.But there was no significant difference in the growth rate.Moreover,the relative expression level of the G443S mutants was determined by q RT-PCR.The results showed that the expression level of FgCYP51s gene of the G443S mutants were significantly decreased compared with the wild strain after EBI fungicide treatment.In order to explore the effect of the G443S point mutation of FgCYP51A on the biosynthesis of DON,the extracellular DON content and TRI5 gene expression of the G443S mutants were determined.The results showed that compared with the wild strain PH-1,DON content was stimulated and the TRI5 expression was upregulated in the G443S mutants,which will contribute to the strong adaptability of F.graminearum populations conferring G443S substitution in the field.Farnesyl pyrophosphate（FPP）,the final product of the isoprene biosynthetic pathway of F.graminearum,is not only the precursor substance of DON biosynthesis,but also an important source of the ergosterol biosynthetic pathway,but the mutual regulation of ergosterol biosynthesis pathway and DON biosynthesis pathway is not clear.In order to further reveal the effects of genetic differentiation of FgCYP51s on the DON biosynthesis.The DON content and expression of TRI5 gene results showed that compared with the wild strain PH-1,the DON content and TRI5 gene expression of the FgCYP51A and FgCYP51B deletion mutants were significantly increased.The"toxisomes"are special spherical cell structures formed around the nucleus by the remodeling of the endoplasmic reticulum of F.graminearum,which is an important site for fungal toxin synthesis.In this study,Fg Tri1-GFP::FgCyp51s-RFP double-tagged strain was constructed by a fluorescent protein fusion expression vector,and the subcellular localization of the FgCYP51s gene was determined.The results showed that FgCYP51s co-localized with Fg TRI1 in the toxisomes,speculating that the toxisomes might be a secondary metabolites.In order to explore the regulation of FgCYP51s on the information of toxisomes,the Fg TRI1-GFP protein was expressed in theΔFgCyp51s deletion mutants,and the morphology of the toxisomes was observed under the confocal microscope.The results showed that compared with wild strain PH-1,the number of toxisomes had increased significantly and the fluorescence signal intensity was also significantly enhanced.The expression level of Fg TRI1 protein in theΔFgCyp51s deletion mutants was verified by Western blot.The results showed that the expression level of Fg TRI1 protein in theΔFgCyp51a andΔFgCyp51b deletion mutants was significantly increased compared with the control.Studies have shown that EBI fungicides can promote the biosynthesis of DON.In this study,three EBI fungicides,epoxiconazole,tebuconazole and prochloraz,were used to determine the effects on the DON content of F.graminearum.The results showed that compared to the no fungicide treatment,the DON content of F.graminearum was significantly higher after the EC₅₀-dose treatment of the three EBI fungicides,while the DON content was significantly lower after all the 10×EC₅₀-dose treatments.To further reveal the effect of toxisomes as affected by EBI fungicides,the strain PH-1::Tri1-GFP was treated with EC₅₀ and 10×EC₅₀ doses of tebuconazole and epoxiconazole,respectively.Western blot results confirmed that EBI fungicides can significantly increase the expression level of the Fg TRI1 protein in DON biosynthesis compared with the control,In summary,the G443S point mutation of FgCYP51A regulates the resistance of F.graminearum to EBI fungicides.Moreover,we have identified the target gene FgCYP51s of EBI fungicide involved in the structure formation of toxisomes and discovered new functions of F.graminearum toxisomes in regulating the biosynthesis of secondary metabolites such as ergosterol.