| Wheat gibberellic disease,caused by Fusarium graminearum,not only severely reduces productivity of wheat and causes enormously economic losses,but also produces mycotoxins that pollute wheat grain and pose a huge threat to the health of human and livestock.mRNA alternative splicing widely exists in higher eukaryotes where a gene encodes pre-mRNA that can generate various transcription products by different splicing forms,and then translate into various functionally different proteins.In animal cells,SR protein phosphorylation by SR protein kinase(Srpkl)is involved in the regulation of alternative splicing and plays an important role in a variety of process,such as tissue differentiation and organ development.The phenomenon of mRNA alternative splicing also exists in filamentous fungi,but the regulatory mechanism of the splicing events is still a blank.In this study,the protein kinase gene was identified as a research object in F.graminearum.The Fgsrpk1 gene deletion cassette was constructed by split maker approach and transformed into the protoplasts of the wild type(PH-1)strain to generate the FgSrpk1 deletion mutants.Testing phenotype with a series of assays,compared with the wild type,we found that the growth rate of Fgsrpk1 mutant was significantly reduced,and aerial hyphae became thinner.Fgsrpk1 mutant was completely defective in sexual reproduction and didn’t form perithecium completely.The mutant was completely non-pathogenic to wheat heads and corn silks.Further tests established that DON production of Fgsrpk1 mutant at inoculation point of wheat grain was only 1.4%of the wild type.We also observed the subcellular localization of FgSRPK1 gene in Fluorescent microscopy show that FgSrpkl-GFP fusion protein is mainly localized to cytoplasm in conidia and young germinating hyphae.However,in small proportion of the hyphae,stronger GFP signals were observed in the nucleus,implying that the FgSrpkl may be functionally similar to Srpkl protein kinase in animal cells in regulating cell cycle signaling.During G2/M phase,FgSrpkl is translocated into the nucleus.In both conidia and young hyphae,interestingly,FgSrpkl-GFP fusion proteins were specially localized to septa1 pores,indicating that FgSrpkl may be involved in other additional functions in filamentous fungi.In order to determine the biofunction of domains and ATP-binding sites in SRPK1,we deleted the domain and point-mutated the ATP-binding site of SRPK1.The growth rate and morphology of colony of C-terminal deletion mutant were not affected,but the growth rate of colony of N-terminal,K1,K2 deletion mutants and point mutation of ATP-binding site were similar to Fgsrpk1 mutant.These indicated that the N-terminal,K1,K2 and ATP-binding site were required for the function of FgSrpk1.It is interesting that the growth rate of Spc domain deletion mutant was less than wild-type PH-1 but more than Fgsrpk1 mutant.We also found that the subcellular localization of FgSrpkl was from cytoplasm to nucleus due to the deletion of Spc domain.It was demonstrated that FgSrpklis localized to cytoplasm through its spacer domain and anchoring to various protein complexes.We also found that FgSrpk1△spc can interact with the SR proteins(FgNp13,FgHrb1 and FgSrp1)in nucleus by BiFC assays,and these three SR proteins may be the substrate of FgSrpk1,but this hypothesis needs further investigation.In this study,we have carried out a preliminary exploration of the function of SRPK1 in F.graminearum,then,we also will further identify and characterize the function of target genes whose mRNA splicing are regulated by FgSRPK1 kinase in vegetative growth,sexual reproduction and plant infection processes.The completion of this study will enable us understand the multiple roles of FgSrpkl kinase in vegetative growth,sexual reproduction and pathogenicity and provide reference for the study of SR protein kinase in other filamentous fungi as well as providing theoretical basis for new strategy of wheat gibberellic disease management and control. |
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