The conserved Dis1/Stu2/XMAP215 microtubule association proteins (MAPs) family plays an important role in microtubule dynamics, nucleation, and kinetochore-microtubule attachments. However, function of Dis1/Stu2/XMAP215 homolog in plant pathogenic fungi has not been determined. Here, we identified and investigated the Dis1/Stu2/XMAP215 homolog (FGSG_10528) in Fusarium graminearum (FgStu2p). Co-localization experiment and co-immunoprecipitation (Co-IP) assay demonstrated that FgStu2p is a microtubule associated protein. Besides, FgStu2 could also interact with Fgγ-tubulin and presumed FgNdc80, which suggested that the FgStu2 gene might associate with microtubule nucleation and kinetochore-microtubule attachments like Dis1/Stu2/XMAP215 homologs in other species. Moreover, the FgStu2 promoter replacement mutants (FgStu2-Si mutants) produced twisted hyphae and decreased growth rate. Microscope examination further showed that the microtubule polymerization was reduced in FgStu2-Si mutants, which could account for the aberrant morphology. Although the microtubule polymerization was affected in FgStu2-Si mutants, the FgStu2-Si mutants didn’t show highly increased sensitivity to anti-microtubule fungicide carbendazim (methyl benzimidazol-2-ylcarbamate [MBC]). In addition, the FgStu2-Si mutants exhibited curved conidia, decreased number of conidial production, blocked ability of perithecia production, decreased pathogenicity and deoxynivalenol (DON) production. Taken together, these results indicate that the FgStu2 gene plays a crucial role in vegetative growth, morphology, sexual reproduction, asexual reproduction, virulence and deoxynivalenol (DON) production of F. graminearum, which brings new insights into the functions of Dis1/Stu2/XMAP215 homolog in plant pathogenic fungi.
The plant pathogen Fusarium graminearum contains two α-tubulin (α 1 and α 2 ) isotypes and two β-tubulin isotypes (β 1 and β 2 ). The functional roles of these tubulins in microtubule assembly are not clear. Previous studies showed that α 1 - and β 2 -tubulin deletion mutants showed severe growth defects and hypersensitivity to carbendazim, which have not been well explained. Here, we investigated the interaction between α- and β-tubulin of F. graminearum . Co-localization experiments demonstrated that β 1 - and β 2 -tubulin are co-localized. Co-immunoprecipitation experiment suggested that β 1 -tubulin binds to both α 1 - and α 2 -tubulin and β 2 -tubulin can also bind to α 1 - or α 2 -tubulin. Interestingly, deletion of α 1 -tubulin increased the interaction between β 2 -tubulin and α 2 -tubulin. Microtubule observation assays showed that deletion of α 1 -tubulin completely disrupted β 1 -tubulin-containing microtubules and significantly decreased β 2 -tubulin-containing microtubules. Deletion of α 2 -, β 1 - or β 2 -tubulin respectively had no obvious effect on the microtubule cytoskeleton. However, microtubules in α 1 - and β 2 -tubulin deletion mutants were easily depolymerized in the presence of carbendazim. The sexual reproduction assay indicates that α 1 - and β 1 -tubulin deletion mutants could not produce asci and ascospores. These results implied that α 1 -tubulin may be essential for the microtubule cytoskeleton. However, our Δα 1 -2×α 2 mutant (α 1 -tubulin deletion mutant containing two copies of α 2 -tubulin) exhibited a normal microtubule network, growth and sexual reproduction. Interestingly, the Δα 1 -2×α 2 mutant was still hypersensitive to carbendazim. In addition, both β 1 -tubulin and β 2 -tubulin were found to bind the mitochondrial outer membrane voltage-dependent anion channel (VDAC), indicating they could regulate the function of VDAC. Importance: In this study, we found that F. graminearum contains four different α-/β-tubulin heterodimers (α 1 -β 1 , α 1 -β 2 , α 2 -β 1 and α 2 -β 2 ) and they assemble together into a single microtubule. Moreover, α 1 -, α 2 -tubulins are functionally interchangeable in microtubule assembly, vegetative growth and sexual reproduction. These results provide more insights into functional roles of different tubulins of F. graminearum which could be helpful for purification of tubulin heterodimers and developing new tubulin-binding agents.
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