| Fusarium garminearum caused Fusarium head blight(FHB),the most devastating disease in wheat,which decreased the yield and quality of global wheat seriously.Nowadays,control of FHB is mainly depended on the application of chemical fungicides.Carbendazim has been widely applied to control FHB for about 40 years in China.It could bind to tubulin heterodimers and disrupt microtubule dymamics.Previous study showed that carbendazim sensitivity of F.graminearum was much lower than that of Botrytis cinerea,Colletotrichum gloeosporiodies,and Sclerotinia sclerotiorum.The mechanism of this carbendazim selectivity has not been determined.According to sequence alignment of resistance-related β-tubulin isotypes,we found position 240 of F.graminearum β2-tubulin was phenylalanine,which is different from the leucine of other plant pathogenic fungi(position 232 of B.cinerea β-tubulin is equivalent to position 240 of F.graminearumβ2-tubulin).β2-tubulin F240L mutant of F.graminearum(Fgβ2F240L)had normal phenotypes,but showed hypersensitivity to carbendazim.β-tubulin L232F mutant of B.cinerea(BcβL232F)exhibited slight decreased growth rate,but had decreased sensitivity to carbendazim.Moreover,microtubule in Fgβ2F240L mutant was more easily depolymerized by carbendazim than that of wild-type strain.In addition,molecular docking assay indicated that F240L mutation increased the binding affinity between β2-tubulin and carbendazim.Taken together,we concluded that difference in position 240 resulted from differentiation of this site and phenylalanine in position 240 of F.graminearum β2-tubulin is response for the naturally less sensitivity to benzimidazole than other plant pathogenic fungi.After that,we investigated the specificity of position 240 of F.graminearum β2-tubulin We chose alanine(A),glutamic acid(E),Glycine(G),isoleucine(I),Leucine(L),tyrosine(Y)to substitute phenylalanine in position 240.We found that the F240A,F240E,F240I,and F240Y mutations in Fgβ2-tubulin could also confer F.graminearum hypersensitivity to carbendazim,although the EC50 values differed among the mutations.The F240G mutation,in contrast,decreased the sensitivity to MBC.In addition,a molecular docking assay indicated that the binding affinity between Fgβ2-tubulin and MBC were increased by the F240A,F240E,F240I,and F240Y mutations but decreased by the F240G mutation.All mutants had normal conidial morphology,but the growth rates and pathogenicity of the F240A,F240E,F240G,F240I,and F240Y mutants were significantly decreased.Moreover,the F240A and F240G mutants produced twisted hyphae.In addition,microtubules were sparse and rarely observed in β2F240A-EGFP,β2F240E-EGFP,and β2F240G-EGFP.These results indicate that position 240(phenylalanine)is not only vital to the function of Fgβ2-tubulin but also plays an important role in regulating the sensitivity of F.graminearum to carbendazim.Any mutation in this site would be detrimental to survival.F.graminearum contains α1-,α2-,β1-and β2-tubulin isotypes.However,the interaction patterns between these isotypes are not clear and the role of these isotypes in microtubule polymerization of F.graminearum also remains elusive.Moreover,the α1-,α2-,β1-andβ2-tubulin deletion mutants exhibited different sensitivity to carbendazim and growth defects,which have not been well explained.Here,we investigated the interaction patterns among four tubulin isotypes in F.graminearum.Co-localization experiments demonstrated that β1-and β2-tubulin are co-localized.Co-immunoprecipitation experiment suggested thatβ1-tubulin binds to both α1-or α2-tubulin and p2-tubulin binds to α1-tubulin preferentially.However,deletion of α1-tubulin can promote interaction between β2-tubulin and α2-tubulin.Further investigation showed that deletion of α1-tubulin completely disrupted β1-tubulin polymerization and significantly decreased β2-tubulin polymerization,and deletion of α2-,β1-and β2-tubulin have no obvious effect on microtubule polymerization.However,microtubules in α1-and β2-tubulin deletion mutants were easily depolymerized at the presence of carbendazim.Moreover,α1-and β1-tubulin deletion mutants could not produce asci and ascospore.In addition,both β1-tubulin and β2-tubulin were found to bind mitochondrial outer membrane voltage-dependent anion channel(VDAC),indicating they could regulate the function of VDAC.However,whether β1-tubulin and β2-tubulin are functionally divergent in regulating VDAC still needs further investigation.Resistance of F.graminearum to carbendazim was conferred by mutations inβ2-tubulin.Therefore,we focused on the purification of α-/β2-tubulin heterodimers.We tried to purify tubulin from the mycelium of F.graminearum and insect cells.First we usedβ1-tubulin deletion mutant DB6(DB6 contains only α1-β2 and α2-β2 heterodimers)to purify tubulin by DEAE-anion exchange column chromatography based on a previous study of Aspergillus nidulans.We confirmed 300 mM NaCl is enough to elute the α-β2-tubulin from the column.However,because of the binding affinity between α-β2-tubulin and DEAE column is weak,many of α-β2-tubulin heterodimers were lost.So the DEAE-anion exchange column chromatography is not suitable for the first step of purification.Then we labeled β2-tubulin with 6×His and Strep tag.The results indicated that Strep tag could be useful for purification β2-tubulin form mycelium of F.graminearum.However,the purification condition of β2-tubulin-strep still needs to be optimized.We inserted 6×His tag between position 42 and 43 of α1-tubulin and tried to purify α1-tubulin.The results indicated that the internal His-tag could be used for purifying α1-tubulin.We also tried Bac-to-Bac baculovirus expression system at the same time.We successfully expressedβ2-tubulin labeled with EGFP or MBP tag,but we could hardly express α1-tubulin.Therefore the vector and condition of bac-to-bac baculovirus expression system still need to be optimized.Taken together,our results revealed the specificity of position 240 of Fgβ2-tubulin and the functional roles of α-and β-tubulin isotypes in regulation of vegetative growth,microtubule assembly and sexual reproduction.Moreover,we also tried to purifiy α1 andβ2-tubulin.Our results increased the knowledge tubulin in plant-pathogenic fungi and could be useful for investigation and development of new tubulin-binding agents... |
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