Characterization Of MRNA Splicing Related SNU66 And Purine Nucleotide Metabolism Pathway Genes In Fusarium Graminearum

Wheat(Triticum aestivum L.)is one of the major cereal crops in China,when infested by Fusarium graminearum will result in Fusarium head blight(FHB),and the yield will reduce or even give rise to totally lost.DON and other fungal toxins produced by this pathogen affect the quality of wheat kernel and threaten the health of human and livestock.This study was focused on mRNA splicing and purine nucleotides metabolism to explore the mechanism of development and pathogenicity in Fusarium graminearum and was expected that results can shed light to theoretical basis for the prevention and control of FHB.Protein kinase plays critical roles in regulating growth and development and responses to environmental stresses in fungi.Deletion of Prp4,the only kinase among spliceosome components,is not lethal in F.graminearum but Fgprp4 mutants have severe growth defects and produced spontaneous suppressors.To identify novel suppressor mutations of Fgprp4,we sequenced the genome of suppressor S37 that was normal in growth and fully recovered for sexual reproduction and identified a tandem duplication of 9-aa in the tri-snRNP component FgSNU66.The 9-aa tandem duplication of FgSNU66 was verified to recover the defect of Fgprp4 mutant directly.Meanwhile,S37 was found to partially rescue the intron retention level of Fgprp4.Among the 19 additional suppressor strains found to have mutations in FgSNU66(out of 260 screened),five had the same 9-aa duplication event with S37 and another five had the R477H/C mutation.The rest had nonsense or G-to-D mutations in the C-terminal 27-aa(CT27)region of FgSnu66,which is absent in its yeast ortholog.Truncation of this C-terminal region reduced the interaction of FgSnu66 with FgHub1 but increased its interaction with FgPrp8 and FgPrp6.Five phosphorylation sites were identified in FgSnu66 by phosphoproteomic analysis and the T418A-S420A-S422 A mutation was shown to reduce virulence.Overall,our results showed that mutations in FgSNU66 can suppress deletion of Fgprp4,which has not been reported in other organisms,and the C-terminal tail of FgSnu66 plays a role in its interaction with key tri-snRNP components during spliceosome activation.Purine nucleotides play important roles in nucleic acids constitutes,energy metabolism,signal transduction,and coenzyme factor.This study made an in-depth analysis of purine nucletides metabolism.Deletion of ACD16 which catalyzes purine de novo synthesis resulted in defects of growth,development and pathogenicity in Fusarium graminearum.ADE12 catalyzing IMP to AMP shows the same phenotypes as ADE16 when deleted.These results indicate that purine de novo synthesis pathway is critical for growth,development and pathogenicity in Fusarium graminearum.The genes AAH1,GUD1,XPT1 and APT1 which are involved in purine salvage pathways have no phenotypes when deleted whether in all fungal developmental stages or in plant infection period.The purine salvage pathway which is absent of importance further emphasizes the irreplaceable of the de novo purine synthesis in F.graminearum.Interestingly,ACD1(AMD1)which catalyzes reaction of AMP to IMP is dispensible for growth but important for asexual and sexual reproduction.Deletion of ACD1 resulted in that the conidiation was significantly reduced,abnormal morphology of conidia,smaller perithecia,and no asci and ascospores were produced when cracking the perithecia in comparison with the wild type.In addition,acd1 mutant was normal in formation of infection cushion but defective in spreading of infectious hyphae in plant cells.Exogenous IMP and adenine can recover the sexual defect of acd1.RNA-seq data and qRT-PCR result showed that the expression of purine de novo genes in sexual stage were reduced compared with that in hyphae stage,especially IMD1-4 which catalyzing IMP to GMP.Addition of GMP can directly rescue the sexual defect of acd1,and deletion of IMD1-4 resulted in the same sexual defect with acd1.These results suggest that the sexual defect of acd1 is due to depletion of GMP and disrupts the homeostasis of purine metabolism.Overall,this study is focused on two conserved life process of pre-mRNA splicing and purine nucleotides metabolism,the results showed that mutation in Snu66 could rescue the defects of prp4 kinase mutant,and ACD1 mediated purine homeostasis plays specific roles in reproduction and pathogenicity in Fusarium graminearum.