Detrimental effects of multiple mutations in position 240 of fusarium graminearum b2-Tubulin

Abstract

Fusarium graminearum causes Fusarium head blight (FHB), a destructive disease of cereal crops worldwide. Carbendazim (methylbenzimidazol-2-ylcarbamate [MBC]) is widely used for controlling FHB. A previous study showed that the F240L mutation in the b2-tubulin of F. graminearum (Fgb2tubulin) confers hypersensitivity to MBC. Whether the substitution of phenylalanine by other amino acids in position 240 of the Fgb2-tubulin gene also confers hypersensitivity to MBC is unknown. Moreover, the biological fitness of these mutants is poorly understood. In this study, we substituted position 240 of Fgb2-tubulin with other amino acids. We found that the F240A, F240E, F240I, and F240Y mutations in Fgb2tubulin could also confer F. graminearum hypersensitivity to MBC, although the effective concentration resulting in 50% inhibition (EC50) differed among the mutations. The F240G mutation, in contrast, decreased the sensitivity to MBC. In addition, a molecular docking assay indicated that the binding affinity between Fgb2-tubulin and MBC were increased by the F240A, F240E, F240I, and F240Y mutations but decreased by the F240G mutation. All mutants had normal conidial morphology, but the growth rates and pathogenicity of the F240A, F240E, F240G, F240I, and F240Y mutants were significantly decreased. Moreover, the F240A and F240G mutants produced twisted hyphae. In addition, microtubules were sparse and rarely observed in b2F240A-EGFP, b2F240E-EGFP, and b2F240G-EGFP. These results indicate that position 240 (phenylalanine) is not only vital to the function of Fgb2-tubulin but also plays an important role in regulating the sensitivity of F. graminearum to MBC. Any mutation in this site would be detrimental to survival.