Potential Roles Of β-tubulins In DON Export In Fusarium Graminearum

Microtubulins are major components of the cytoskeleton and play an important role in maintaining cell morphology,cell division,signal transduction,and substance transport.Previous studies have shown that Actin filaments,the same as microtubulins which are important components of the cytoskeleton as well,play an important role in DON transportation.When the cells were treated with latrunculin A,an inhibitor of Actin polymerization,the fluorescently labeled motile vesicles with Tri12-GFP(a trichothecene efflux pump)were not observed.And our lab has identified two β-tubulins(FgTUB1,FgTUB2)of Fusarium graminearum before,which had functional differentiation on both sexual and asexual reproduction.Based on previous research,we want to explore whether these two β-tubulins also have roles in the DON transport.At present,the results of this study show that the knockout of FgTUB1 gene does not significantly affect the DON production in TBI medium and rice medium,the pathogenicity in flowering wheat head is comparable to PH-1,while it was reduced in corn silk.The knockout of FgTUB2 gene resulted in significant defects in the DON production and infection of wheat and corn silk.However,QRT-PCR showed that the knockout of FgTUB1 or FgTUB2 gene could increase the expression of TRI genes(TRI5,TRI6,TRI12)in comparison with PH-1,indicating that the reduction in the DON production is not due to the decrease of TRI gene expression and there possibly has functional differentiation between two β-tubulins,FgTUB2 is more important for DON transportation than FgTUB1.Compared with PH-1,the expression of TRI5 in Fgtub1 increased 2-fold,but DON production just increased 1.2-fold,indicating that FgTUB1 may be involved in DON transportation.Therefore,to avoid negative feedback control because of gene deletion,we treated the PH-1 with 40 μg/ml Nocodazole which can disrupt FgTub1 but not significantly affect FgTub2.The results showed that Nocodazole significantly reduced the DON production and the TRI genes expression.Fewer DON production in Fgtub1 with 80 μg/ml Nocodazol than Fgtub1 which can further certify Fgtub2 is involved in DON transportation.To further clarify the function of FgTUB1 and FgTUB2 in DON transport,we measured the DON production in vivo.The results showed that intracellular DON accumulation inFgtub1,Fgtub2 mutant and 40 μg/ml Nocodazole treated PH-1 increased 2.6-fold,9.5-fold and 7-fold respectively compared with PH-1.The expression of Tri1 and Tri12 in Fgtub1 and Fgtub2 mutants,and 200μg/ml Nocodazol treated PH-1 which can disrupt FgTub1 and FgTub2,are observed to character the function of FgTUB1 and FgTUB2 in DON transportation.We found that knockout FgTUB1 or FgTUB2 does not affect the expression of Tri1 and Tri12,but 200 μg/ml Nocodazole treated PH-1 was defective in Tri12 movement,indicating that FgTUB1 and FgTUB2 are involved in DON transport.Moreover,we found the sensitivity of FgTUB2 to Nocodazole is different between cytoplasm and cell nucleus,FgTUB2 is less sensitive in cell nucleus than that in cytoplasm,indicating that FgTUB2 may be related to nuclear division.To sum up,FgTUB1 and FgTUB2 are involved in DON transportation,but FgTUB2 is more important for DON transport and nuclear division.