Functional Characterization Of SR Proteins FgSrp1 And FgSrp2 In Fusarium Graminearum

In eukaryotic organisms,SR（serine and arginine-rich）proteins play important roles in pre-m RNA splicing and efficiency of gene expression.The versatile functions of SR（serine/arginine-rich）proteins in pre-m RNA splicing and processing are modulated by reversible phosphorylation.SR protein Fg Srp1 is phosphorylated at five conserved sites in Fusarium graminearum,a causal agent of wheat head blight and producer of deoxynivalenol（DON）mycotoxins.In this study,we generated the knock-out mutants F.graminearumSRP1 gene via Split-PCR method,and the positive transformants were confirmed via PCR and southern blot.We showed that Fg SRP1 is not essential for vegetative growth.On PDA plates,the Fgsrp1 deletion mutant was reduced approximately 22% in growth rate.We also showed that the Fgsrp1 deletion mutant rarely produced conidia and caused only limited symptoms on wheat heads and corn silks.Deletion of Fg SRP1 also reduced ascospore ejection and deoxynivalenol（DON）production,Interestingly,FgSRP1 had two transcript isoforms due to alternative splicing and both of them were required for its normal functions in growth and DON biosynthesis.Although isoform A encoding the full-length protein was the predominant transcript,isoform B encoded a small peptide and had an increased expression level in DON producing cultures.Fg Srp1 localized to the nucleus and nuclear localization of Fg Srp1 is consistent with its likely functions as a SR protein involved in RNA processing.When the nuclear localization signal sequence in Fg SRP1 gene was deleted,GFP signals of the resulting Fgsrp1/Fg SRP1^?NLS-GFPtransformant were observed in both the nucleus andcytoplasm.We also determined the functions of Fg SRP11–90-GFP（containing the RBD domain）and Fg SRP^191–195-GFP（containing the SR-rich regions）transformants,both Fg SRP11–90and Fg SRP^191–195transformants had similar defects with Fgsrp1 in growth,conidiation,sexual reproduction,plant infection and DONproduction.prp4 encoding the only proteins kinase of the splicesome components is an essential gene in S.pombe,deletion of the Fg PRP4 kinase gene is not lethal in F.graminearum and Fg Prp4 plays an important role in intron splicing.In this study,we verified that Fg Srp1 interacts with Fg Prp4 in Bi FC and co-immunoprecipitation（co-IP）assays.Fg Srp1 wasphosphorylated at the S112,S114,S147,S149 and S183 residues,and S112,S114,S147,S149 four phosphorylation sites were well conserved.Deletion of all four conserved phosphorylation sites but not individual ones affected the Fg SRP1 function,suggesting their overlapping functions.In comparison with PH-1,RNA-seq analysis showed that 1072 and 1708 genes were down-and up-regulated over twofold respectively,in the Fgsrp1 mutant.We then analyzed the effect of Fg SRP1 deletion onalternative splicing.Although deletion of Fg SRP1 had nogenome-wide effects on alternative splicing,a total of 145 differential alternative splicing events from 139 genes weredetected in the Fgsrp1 mutant in comparison with the wild-type strain PH-1.Except for Fg SRP1,we also determined the functions of Fg SRP2.Although srp2 is essential for growth in S.pombe,Fgsrp2 deletion mutant was normal in conidia,plant infection and sexual reproduction.Fg Srp2 localized to the nucleus and interacted with Fg Srp2 in vivo.The study also showed that the Fgsrp1srp2 double deletion mutant was reduced approximately 30% in growth rate,rarelyproduced conidia,caused only limited symptoms on wheat heads and affected perithecium melanization.Taken together,FgSRP1 is important for conidiation,pathogenesis and alternative splicing.The Fg Srp1 SR protein is likely important for pre-m RNA processing or splicing of various genes in different developmental and infection processes.Fg Srp1 and Fg Srp2 have overlapping function in F.graminearum.