The plant pathogenic fungus, Fusarium graminearum, is known to have two β-tubulin genes (named Fg-β1tub and Fg-β2tub). Mutations in Fg-β2tub rather than in Fg-β1tub have been shown to confer resistance to carbendazim (MBC), even though Fg-β1tub has higher homology than Fg-β2tub to the β-tubulin isotypes related to benzimidazole resistance in other fungi. However, sequence alignment of β-tubulin isotypes related to benzimidazole resistance showed that the number and position of introns in Fg-β2tub are more consistent than Fg-β1tub to those in other β-tubulin genes. In detail, Fg-β1tub lacks three introns, i.e., intron i3, i4, and i6 corresponding to positions in Fg-β2tub of F. graminearum. To investigate the effects of the divergence introns on the function of β-tubulins in F. graminearum, a strategy of intron deletion and insertion was used. Our results showed that deletion of the second intron from Fg-β1tub gene increased Fg-β1tub expression levels leading to increased sensitivity to MBC. Besides, inserting the divergence introns into Fg-β1tub can increase Fg-β1tub expression leading to increased sensitivity to MBC. In addition, intron-mediated Fg-β1tub gene expression requires a splicing-competent intron within the body of the host gene. Furthermore, the insertion and deletion of introns in Fg-β1tub gene have no significant effect on hyphal growth, conidiation and virulence in F. graminearum. Thus, we proposed that introns may be among the factors contributing to the evolution and functional divergence of two β-tubulin genes and also significantly regulate the expression of β-tubulin genes, which, in turn, affects sensitivity to MBC fungicides in F. graminearum.
Keywords: Evolution; Fungicide sensitivity; Fusarium graminearum; Gene expression; Intron; β-tubulin.