F240 of beta(2)-Tubulin Explains why Fusarium graminearum is Less Sensitive to Carbendazim than Botrytis cinerea

beta-Tubulin is the target of benzimidazole fungicides, the most widely used of which is carbendazim (methyl benzimidazol-2-ylcarbamate [MBC]). MBC sensitivity is determined by the differential affinity of MBC for beta-tubulins. However, the mechanism of less sensitivity of Fusarium graminearum to MBC compared with other fungi, including Botrytis cinerea, Colletotrichum gloeosporioides, and Sclerotinia sclerotiorum, remains exclusive. Alignment of beta-tubulin amino acid sequences showed that position 240 of beta-tubulins is leucine (L) in most pathogenic fungi but is phenylalanine (F) in the Fg beta(2)-tubulin of the F. graminearum wild type. The effective concentration resulting in 50% inhibition (EC50) value of MBC against the Fg beta(2)F240L mutant of F. graminearum is 0.047 mu g/ml, which was 10-fold lower than that of wild-type strain 2021. Moreover, The EC50 value of MBC against the Bc beta L 240 F (actually position 232) mutant of Botrytis cinerea was 0.44 mu g/ml, which was ninefold higher than that of B. cinerea wild-type strain Bt4-1. In response to MBC treatment (0.15 mu g/ml), microtubules were clearly visible in Fg beta(2)-enhanced green fluorescent protein (EGFP) but not in Fg beta(2)F240L-EGFP. Moreover, a molecular docking assay indicated that F240L mutation created a pi-pi interaction between Fg beta(2)-tubulin and MBC and increased the binding affinity of Fg beta(2)-tubulin to MBC. Our results suggest that F240 is responsible for the naturally less MBC sensitivity in F. graminearum compared with B. cinerea, C. gloeosporioides, and S. sclerotiorum by decreasing the binding affinity between Fgb 2-tubulin and MBC.