Molecular Mechanism Of ?1-tubulin In Regulating Growth And Development,and Sexual Reproduction In Fusarium Graminearum

Fusarium head blight(FHB),a destructive disease of cereal crops,is mainly caused by the filamentous ascomycete Fusarium graminearum.In addition to reducing yield,F.graminearum also contaminates the grains with various mycotoxins that are harmful to humans and animals.Microtubules are the key cytoskeletal component and involved in various cellular processes,such as maintenance of cell shape,polar growth,organelle transport,signaling transduction and cell division in eukaryotes.Furthermore,?-tubulin proteins of microtubules are the target of methyl benzimidazole carbamate(MBC)fungicide in controling FHB.F.graminearum contains two ?-tubulin genes(Fg TUB4 and Fg TUB5).Fg TUB4 is essential for vegetative growth and sexual reproduction,whereas Fg TUB5 is dispensable during asexual and sexual development,suggesting that two ?-tubulins are functionally divergent.However,the functional divergence between two ?-tubulin genes is still elusive.In this study,we found that the Fgtub4 mutant was unstable and could produce spontaneous sectors with faster growth rates on V8 plates.The formation of spontaneous sectors is generally due to mutations in genes that have physical or genetic interactions with the knockout gene,thereby facilitating the identification of Fg TUB4-related genes.A total of 22 sectors of Fgtub4 mutant were isolated and then divided into 5 types roughly based on average growth rates.Phenotypic analysis showed that most of 22 sectors were partially recovered the Fgtub4 defects in growth,conidial morphology and plant infection,but not in sexual reproduction.Two sectors(S1 and S10)from type II were identified with stop codon mutations in a microtubule-related gene Fg KAR9 by sequencing analysis.Further characterization showed that deletion of Fg KAR9 under Fgtub4 mutant background could partially recover growth defect of original Fgtub4 mutant.Our research group previously indicated that Fg Kar9 might bind to Fg Tub5 to regulate the interactions between two ?-tubulins and two ?-tubulins,and then promoted the interaction of Fg Tub5 with Fg Tub1.Thus,we speculated that Fg Kar9 could promote Fg Tub5 to combine Fg Tub1 but simultaneously disturb the combinations between Fg Tub5 and Fg Tub2.Based on this hypothesis,we generated triple deletion mutants of Fgtub1Fgtub4 Fgkar9 under Fgtub1 Fgtub4 or Fgtub1 Fgkar9 double-deletion background,respectively.These resulting triple mutants grew as fast as the sectors of type II and had similar colonial morphologies with S1 and S10 sectors,suggesting that Fg Kar9 might disturb the combinations of Fg Tub5 with Fg Tub2 by binding to Fg Tub5.In addition,ten sectors form type III,including sector S18-1 and S24,showed frameshift or stop codon mutations in a transcription factor Fg ACE1 gene.We also demonstrated that mutations in Fg ACE1 could partially restore Fgtub4 growth defect.Phenotypic analysis showed that deletion of Fg ACE1 had no obvious defects in growth,DON production and pathogenicity on wheat,but showed severe defects during sexual reproduction,with smaller perithecia and abortive asci.Although Fg ACE1 only existed in ascomycetous fungi and had similar distribution patterns with Fg TUB5 of two ?-tubulin genes,Fg Ace1 rarely had effects on transcriptional or translational expression levels of Fg TUB5.To analyze the role of Fg Ace1 in regulating Fgtub4 growth defect,48 h aerial hypha samples of PH-1,Fgtub4 mutant and S24 sector were isolated for transcriptome sequencing to dissect differentially expressed genes(DEGs)responsible for Fgtub4 growth defect.RNA-seq analysis showed that 932 genes were down-regulated and995 genes were up-regulated over 2-fold in original Fgtub4 mutant in comparison with that of PH-1.Of them,96 down-regulated and 57 up-regulated genes that altered expression levels in Fgtub4 mutant were recovered to normal expression levels in sector S24 with Fg Ace1 mutation,respectively.Thus,these 153 recovered DEGs might contribute to restore Fgtub4 growth defect in F.graminearum.In this study,we characterized and identified that mutations in either Fg KAR9 or Fg ACE1 gene could partially restore the Fgtub4 growth defect through different regulatory pathways.These results further explored the molecular mechanism of?1-tubulin in regulating growth and development,and sexual reproduction.It also provided a research basis for further determing the biological function and regulatory mechanism of microtubules in F.graminearum.