Functional Analysis Of G Protein-Coupled Receptors Gip1/Gia1 During Sexual Reproduction In Fusarium Graminearum

Sexual reproduction in fungi involves sexual structural differentiation,mate recognition,karyogamy,and meiosis.Receptor-mediated signal recognition is a decisive factor in determining the ordered switching of the different stages of sexual reproduction.As a typical GPCR,the pheromone receptor is widely present in fungi,but its function is only before karyogamy.Sexual structure differentiation and meiosis are also essential for the formation of perithecia,asci,and ascospores.However,the signal pathways and the upstream receptors are still largely unknown.We functionally characterized the 18 GPCR genes that are specifically expressed or highly up-regulated in mating cultures and found that two non-pheromone receptors,Gip1 and Gia1,play specific roles during perithecia and asci/ascospores formation in Fusarium graminearum.In this study,scanning electron microscopy,section staining,and Hoechst 33258 nuclear staining were used to observe,and it was found that the gip1 mutant was unable to produce protoperithecium and ascogenous hyphae,and therefore perithecium.We also found that the gia1 mutant was normal in perithecium development,crozier formation,and karyogamy,but failed to undergo meiosis and form ascospores in developing asci.These results suggest that Gip1 and Gia1specifically mediate signal sensing during sexual structure differentiation and meiosis,respectively,in F.graminearum.The orthologs of GIP1 and GIA1 in Fusarium verticillioides were able to complement defects in gip1 and gia1 mutants,respectively.In addition,FvGIP1 and FvGIA1 play a similar role to their F.graminearum orthologs in regulating the formation of perithecia and asci,indicating that GIP1/GIA1 orthologs genes were functionally conserved.Phylogenetic analysis revealed that the GIA1 orthologs gene evolved only in Sordariomycetes,and that the GIP1 orthologs gene was mainly present in Hypocreales.The defects of gip1 mutant and gia1 mutant were partially rescued by Nc GPR-1（GIA1 orthologs gene）,indicating that GIP1was a paralog of GIA1 generated by gene duplication in Sordariomycetes fungi.GIP1 and GIA1 were different in expression patterns,downstream signaling pathways,and ligand recognitions.Although both GIP1 and GIA1 were specifically expressed during sexual reproduction.GIP1 expression was rapidly increased upon fertilization and became detectable at 1-dpf.Its transcript abundance peaked at 4-dpf and decreased rapidly afterwards.In contrast,GIA1 transcripts were rare before 3-dpf but rapidly increased after4-dpf.The expression level of GIA1 kept on increasing and remained high even at 8-dpf.Both Gip1 and Gia1 interacted with the G protein Gpa1,but the signaling pathways downstream of G protein were different,as found by overactivation of G protein and MAP kinases.It was found that Gip1 regulates the formation of protoperithecia via the Hog1signaling pathway and cAMP-PKA signaling pathway,and regulates the maturation of perithecia via the Gpmk1 signaling pathway.And Gia1 regulates meiosis and the development of asci via the Gpmk1 signaling pathway.Domain swapping analysis revealed that the C-terminal region of Gip1 was responsible for the difference in downstream signaling pathways.Overexpression of GIP1,but not GIA1,affected colony growth rates and conidia germination,suggesting that Gip1 and Gia1 may recognize different ligands.In the protein structure predicted with Alpha Fold2,the NT and EL3 regions of Gip1/Gia1 were adjacent to each other.Domain swapping analysis showed that the N-terminal region and extracellular loop 3 of Gia1/Gip1 were responsible for the difference in ligand recognition,and that the extracellular loop 1 and extracellular loop 2 stabilized the protein structure by forming intramolecular disulfide bonds.In this study,we have discovered for the first time of spontaneous complementation mutations during the sexual reproduction in fungi.The gip1 mutant was unstable during sexual reproduction stage and a total of 10 spontaneous suppressors were collected from7-dpf carrot agar plates.The original gip1 mutant and three suppressor strains（S1,S5,and S7）were selected for genome sequencing.To our surprise,all selected suppressor mutations were identified in the VEA gene.We then amplified and sequenced the VEA gene in the remaining 7 suppressor strains,and found that all of the remaining 7 suppressor strains also had mutations in the VEA gene,suggesting that Ve A was associated with the function of Gip1 during sexual reproduction.Based on RNA-seq analysis,a total of 16 genes that were previously known to be important for the formation of protoperithecia and perithecia showed reduced transcription level in the gip1 mutant.Based on qRT-PCR analysis,some of down-regulated genes in the gip1 mutant were significantly up-regulated in the S2suppressor strain.Interestingly,the suppressor strain S1 only produced protoperithecia,but the ve A^E433fs strain produced normal perithecia with cirrhi.These results further suggest that the GIP1 gene not only influences the formation of protoperithecia,but also the maturation of perithecia in F.graminearum.It was found that Gip1 recognizes two ligands and Gia1 recognizes one,indicating that the ancestor of Gip1/Gia1 should have recognized three or more ligands.In this study,we preliminarily exclude the possibility that pheromones and oxylipins are Gip1/Gia1 ligands by gene knockout and phenotypic observation,which further indicates that Gip1/Gia1belongs to non-pheromone receptors.By engineering yeast and introducing the P_FUS1-GFP vector,we established an efficient fluorescence reporter system for ligand screening.Under the treatment of mycelium and perithecia samples,only the perithecia sample was able to trigger fluorescence,indicating that the ligand of Gia1 was specifically produced during the sexual reproduction in F.graminearum.After high temperature and proteinase K treatment,the perithecia sample failed to trigger fluorescence,indicating that the ligand of Gia1 might be proteins or peptides.Taken together,our data suggest that signal recognition for sexual structure differentiation and meiosis is widespread in F.graminearum and other Sordariomycetes fungi.And non-pheromone receptors,Gip1/Gia1 and their orthologs,play specific roles in the differentiation of sexual structures and meiosis in Sordariomycetes fungi.