Function And Regulation Of TUB1 Gene In Sexual Reproduction And Carbendazim Resistance In Fusarium Graminearum

Fusarium head blight?FHB?,mainly caused by Fusarium graminearum,is one of the most important fungal diseases in wheat.Ascospores produced by F.graminearum are the main primary infection sources of FHB.Currently,due to the lack of FHB resistant wheat cultivars,chemical fungicides are the most efficient approach to control FHB.However,long-term application of chemical classes of fungicides results in drug resistance of pathogens.Carbendazim resistance mutations were identified in beta-tubulins before.Although the mutations in F.graminearum Tub2 widely existed in field,mutations in Tub1 were rarely found.Our previous study revealed that TUB2 played a major role in vegetative growth,whereas TUB1 is more likely to affect sexual development.In this study,we performed functional characterization of TUB1 gene to explore the roles of TUB1 during sexual reproduction and its relationship with carbendazim-resistance.The main results are as follows:1.The tub1 mutant produced smaller perithecia in comparison with the wild type strain.Perithecia formed by the tub1 mutant were not able to develop asci and ascospores but only had ostioles and limited ascogenous hyphae.The tub1 mutant was female sterile,suggesting an important role of TUB1 in protoperithecium development.Silencing of TUB1 by MSUD?meiotic silencing of unpaired DNA?also led to defects in ascosporogenesis,when the tub1mutant was used as female to outcross with mat1-1 mutant.This result indicated that TUB1 is required for ascospore development.In addition,five amino acid residues?T23,G34,V119,V180 and D197?were identified as potential functional residues of TUB1 by RIP?repeat induced point mutation?.2.In the complement assay of tub1 mutant,allele lacking of the 3'-UTR regions of TUB1 could only rescued the defect of tub1 mutant in vegetative growth,but not in sexual reproduction.Only allele containing 3'-UTR regions could fully rescued the defects of tub1mutant in both vegetative growth and sexual reproduction.Therefore,3'-UTR is essential for the function of TUB1 in sexual reproduction,but not in vegetative growth.RNA-seq analysis in F.graminearum indicated that long 3'-UTR transcripts were preferred by genes in sexual development stages in comparison with those in vegetative growth.To investigate the regulatory mechanism,a total of 7 protein factors involving in alternative polyadenylation?APA?were identified in F.graminearum.Three of them are essential genes and their deletion mutants were lethal.The other 4 genes deletion mutants?hrp1,fip1,rna15 and cdc73?were generated.When assayed for vegetative growth and sexual reproduction,both hrp1 and rna15 mutants were significantly reduced in growth rate and their colonies were abnormal.Moreover,perithecium formation was blocked both mutants.In contrast,the fip1 mutant was normal in growth but defective in ascospores discharge.The cdc73 mutant had a 25%reduction in growth rate and produced 2-cell ascospores?the wild type produces 4-cell ascospores?.RNA-seq analysis indicated that genes in those 4 gene deletion mutants were likely to expressed long 3'-UTR transcripts,and the preference was associated with phenotypes.Nevertheless,we failed to found differences in 3'-UTR length of TUB1transcripts either between sexual and vegetative stage,or wild type and mutants.These results indicate that APA plays an important role in sexual reproduction,but independent for the sexual-specific functions of TUB1 in F.graminearum.3.Three A-to-I RNA editing sites were identified in TUB1 transcripts,which resulted in amino acids changes?N347D,T362A,and I368V?.To investigate the role of A-to-I RNA editing on the functions of TUB1 gene,edited allele and non-editable allele of TUB1 gene were generated for each site,respectively.The non-editable allele of all sites and the edited allele of T362A and I368V were able to recover the defects of tub1 mutant in sexual reproduction.In contrast,the edited allele of N347D could not fully complement the defects in sexual development of tub1 mutant.4.The reported carbendazim-resistance site?F167Y?in TUB2 was introduced into TUB1,which resulted in increased carbendazim-resistance of the tub1/TUB1^F167Y transformants.In addition,the tub1/TUB1^F167Y transformants were defective in ascospore morphology.Therefore,the possible reason that few carbendazim-resistance mutations of TUB1 was identified in field was that the TUB1 mutation affected the sexual development of F.graminearum.Overall,TUB1 plays critical roles in protoperithecium development and ascospores formation.The stage-specific function of TUB1 is regulated by both 3'-UTR and A-to-I RNA editing.Similar to Tub2,Tub1 is also involved in carbendazim-resistance,but the carbendazim-resistance mutation in TUB1 leads to defects in sexual development.