Functional Roles of α ₁ -, α ₂ -, β ₁ -, and β ₂ -Tubulins in Vegetative Growth, Microtubule Assembly, and Sexual Reproduction of Fusarium graminearum

Abstract

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.

FgPfn participates in vegetative growth, sexual reproduction, pathogenicity, and fungicides sensitivity via affecting both microtubules and actin in the filamentous fungus Fusarium graminearum

“…The mutants of PH-1::LifeAct-GFP, PH-1::FgMyo5-GFP, and Δ FgPfn ::LifeAct-GFP, Δ FgPfn ::FgMyo5-GFP were cultured in YEPD for 36h, and then the GFP fluorescence signals were observed with TCS SP8 confocal microscope (Leica). The mutants of PH-1::Fgβ 2 -GFP and Δ FgPfn ::Fgβ 2 -GFP were observed as previously described [ 63 ]. The mutants of PH-1::Tri1-GFP and Δ FgPfn ::Tri1-GFP were cultured in TBI for 36h, and then the GFP fluorescence signals were observed with TCS SP8 confocal microscope (Leica).…”

“…observed with TCS SP8 confocal microscope (Leica). The mutants of PH-1::Fgβ 2 -GFP and ΔFgPfn::Fgβ 2 -GFP were observed as previously described [63]. The mutants of PH-1:: Tri1-GFP and ΔFgPfn::Tri1-GFP were cultured in TBI for 36h, and then the GFP fluorescence signals were observed with TCS SP8 confocal microscope (Leica).…”