Functional Roles Of Alpha(1)-, Alpha(2)-, Beta(1)-, And Beta(2)-Tubulins In Vegetative Growth, Microtubule Assembly, And Sexual Reproduction Of Fusarium Graminearum

The plant pathogen Fusarium graminearum contains two alpha-tubulin isotypes (alpha(1) and alpha(2)) and two beta-tubulin isotypes (beta(1) and beta(2)). The functional roles of these tubulins in microtubule assembly are not clear. Previous studies reported that alpha(1)- and beta(2-)tubulin deletion mutants showed severe growth defects and hypersensitivity to carbendazim, which have not been well explained. Here, we investigated the interaction between alpha- and beta-tubulin of F. graminearum. Colocalization experiments demonstrated that beta(1)- and beta(2)- tubulin are colocalized. Coimmunoprecipitation experiments suggested that beta(1) -tubulin binds to both alpha(1) and alpha(2) f tubulin and that beta(2)-tubulin can also bind to alpha(1)- or alpha(2)-tubulin. Interestingly, deletion of alpha(1)-tubulin increased the interaction between beta(2)-tubulin and alpha(2)-tubulin. Microtubule observation assays showed that deletion of alpha(1)-tubulin completely disrupted beta(1)-tubulin-containing microtubules and significantly decreased beta(2)-tubulin-containing microtubules. Deletion of alpha(2)-, beta(1)-, or beta(2)-tubulin had no obvious effect on the microtubule cytoskeleton. However, microtubules in alpha(1)- and beta(2)-tubulin deletion mutants were easily depolymerized in the presence of carbendazim. The sexual reproduction assay indicates that alpha(1)- and beta(1)-tubulin deletion mutants could not produce asci and ascospores. These results implied that alpha(1)-tubulin may be essential for the microtubule cytoskeleton. However, our Delta alpha(1)-2x alpha(2) mutant (alpha(1)-tubulin deletion mutant containing two copies of alpha(2)-tubulin) exhibited normal microtubule network, growth, and sexual reproduction. Interestingly, the Delta alpha(1)-2x alpha(2) mutant was still hypersensitive to carbendazim. In addition, both beta(1)-tubulin and beta(2-)tubulin were found to bind the mitochondrial outer membrane voltage-dependent anion channel (VDAC), indicating that they could regulate the function of VDAC.IMPORTANCE In this study, we found that F. graminearum contains four different alpha-/beta-tubulin heterodimers (alpha(1)-/beta(1)-, alpha(1)-/beta(2)-, alpha(2)-/beta 1-, and alpha(2)/beta(2)- tubulin heterodimers), and they assemble together into a single microtubule. Moreover, alpha(1)- and alpha(2)-tubulins are functionally interchangeable in microtubule assembly, vegetative growth, and sexual reproduction. These results provide more insights into the functional roles of different tubulins of F. graminearum, which could be helpful for purification of tubulin heterodimers and development of new tubulin-binding agents.