Functional Analyses Of Putative G Rotein-coupled Receptor Genes And AMD1 Gene In Fusarium Graminearium

Fusarium graminearum is an important pathogen of small grains and maize in many areas of the world.Infected grains not only caused yield and epidemics losses severely but also contaminated with mycotoxins harmful to humans and animals.G protein-coupled receptors(GPCRs),mebrane receptor protein,detect and mediate rapid responses to different extracellular signals and play critical roles in regulating growth,development,nutrients sensing,mating,infection and virulence in fungi.The putative GPCRs have been predicted in comparative genomics in Fusarium but the systematic characterization of the GPCRs in Fusarium graminearum has not been reported.Firstly,we generated 85 GPCRs genes deletion mutants in F.graminearum and assayed their defections in growth,conidiation,sexual reproduction,plant infection.Three GPCRs mutants were reduced in wheat infection and virulence and additional 3 GPCRs genes(PRE1,CRL1,CRL2)were responsible for the size of perithecia,perithecia formation and ascus development respectively,however,all the daeletion mutants were normal in growth and conidiation.Then we found the CRL1 gene was responsible for the perithecial initials formation and the late stage of perithecia formation.And the expression level was increased from 1 to 4 days after fertilization then decreased.Further research of crl1 and crl2 indicated that,the deletion of CRL1 had no defect on hyphae fusion,and is male fertility when across with mat1-1-1.Perithecia initials were obserced when inoculated on Wounding carrot agar medium or stem node agar medium.Expressed the ORF of CRL1 in crl1can not restore its defects in sexual development,however,the transformants crl1/CRL1-3'UTR which expressed ORF and its 3'UTR produced normal perithecia and ascospores as the same as wild strain.Normal perithecia but failure development of asciwas observed in crl2 mutants.No complete asci were observed at 8 days post-fertilizaition and GFP of Histone in crl2 mutant which revealed that one and only nuclear in arrested asci?Therefore,CRL2 gene may regulated the further development of the crozier.RNA-seq analysis shows that CRL1 began to express at 1(dpf)days post-fertilization and increased gradually,expressed highest at 4 dpf,the expression pattern is consistent with its defect in perithecia formation.Different with CRL1,CRL2 began to express at 3 dpf,and increased gradually until 8 dpf.Therefore,crl2 restrain perithecia developmentin middle-to lage-stage of sexual production.Whatsmore,in accordance with the importance role in asci development,the GFP signal of Amd1 also began to appearat 4 dpf.Exogenous cAMP did not restore the phenotype of crl1 and crl2 at sexual stage.The deletion of PDE2 could increase c AMP level of intracellular,However,double knock-out mutants crl1 pde2,crl2 pde2 still appeared sexual deficiency as same as crl1 and crl2 mutants.Therefore,the signal transduction mediated by CRL1 ang CRL2that regulate sexual development through cAMP-PKA independent passway.Ascospores are the primary inoculum in the wheat scab fungus Fusarium graminearum that was recently shown to have stage-specific A-to-I RNA editing during sexual reproduction.CRL1 and CRL2 regulated sexual reproduction in early and middle period respectively,however,another gene,AMD1 coding transmembrane protein was specificly expressed in later stage of sexua reproduction.The amd1 deletion mutant was normal in growth and conidiation,but was defective in ascospore discharge,which is consistent with the specific expression of AMD1 at later stages of sexual development.Although they had normal morphology,ascospores became scattered after the breakdown of ascus wall in amd1 perithecia at 8 days post-fertilization(dpf)or longer.Like the Fgkin1 and gea1 mutants,many of these ascospores scattered in perithecia germinated from one end in the amd1 mutant.The expression level of AMD1 was reduced over 5-folds in Fgkin1 mutant perithecia.Furthermore,AMD1 is One of the genes with premature stop codons that require editing to encode full-length functional proteins and encode a protein with a MFS(major facilitator superfamily)transporter domain and 11 transmembrane helixes.In this study,we further characterized the functions of AMD1 and its UAG to UGG editing event.Expression of the wild-type allele but not the uneditable allele of AMD1 rescued the defects of amd1 in ascospore discharge and germination.Overexpression of the fixed/edited allele of AMD1 with a constitutive RP27 promoter had no obvious effect on vegetative growth and asexual or sexual reproduction in F.graminearum.Furthermore,Amd1-GFP localized specifically to the ascus membrane and Amd1 orthologs are only present in ascocarp-forming fungi.Interestingly,deletion of AMD1 results in the up-regulation of a number of genes related to transporter activity and membrane that were normally had no or low expression in 8 dpf perithecia.Taken together,these results indicated that AMD1 may localize to the ascus membrane and play a critical role in maintaining ascus wall integrity during ascospore maturation and A-to-I editing of its transcripts is important for ascospore discharge and auto-inhibition of ascospore germination in F.graminearum.