Functional Analysis Of The Arf-GAP Proteins In Arthrobotrys Ologospora

As a typical nematode-trapping fungus,the Arthrobotrys oligospora can produce special three-dimensional networks through the specialization of nutritional hyphae to catch and infect nematodes.It is also a representative strain to study the interaction between nematode-trapping fungi and nematodes.ADP-ribosyhtion factor(Arfs)is one of the subfamilies of small GTPases.As a molecular switch controlling various important cellular processes,Arf-GAP proteins regulate the transition from activation state to inactivation state of Arf proteins.In this paper,four Arf-GAP proteins-coding genes including Gcs1,Gts1,Age1 and Age2 in A.oligospora were characterized.Several techniques such as gene knockout,phenotypic comparison and transcriptomic analysis were used to explore the functions of four Arf-GAP proteins in mycelial growth,differentiation,sporulation,stress resistance and pathogenicity in A.oligospora.The main experimental results:1.Functional domain,biochemical properties and cluster analysis of Arf-GAP proteinsThe biochemical properties of Arf-GAP proteins were analyzed,the GCS1 shares the smallest molecular weight(MW)of 39.2 KDa,GTS1 shares the largest MW of 72.9 KDa,AGE1 and AGE2 are close in size.Several domains,such as ArfGTPase-activated protein(IPR001164),Arf-GAP domain(IPR038508)and ArfGAP/RecO zinc finger structure(IPR037278),were all found in Arf-GAP proteins.The phylogenetic analysis showed that the four Arf-GAP proteins of A.oligospora were divided into two branches,GCS1,GTS1 and AGE2 were clustered on the same branch,while AGE1 was clustered on the other branch.2.Knock-out of four Arf-GAP proteins-coding genes and phenotypic analysisAccording to the principle of homologous recombination,four knockout mutant strains of Arf-GAP protein-encoding genes were obtained.The phenotypic characteristics of wild-type strain(AoWT)and mutants were compared.Thephenotypes of ?AoGcs1 and ?AoAge1 mutants showed no difference from the AoWT,while the phenotypes of ?AoGts1 and ?AoAge2 mutants showed impotantly change.The ?AoGts1 and ?AoAge2 growth became slower,aerial hyphae were decreased,and the conidal yields were also decreased.The hyphal septa were increased in the?AoGts1 mutant;meanwhile both of the hyphal septa and branch of the ?AoAge2mutant were increased obviously.Moreover,the ?AoGts1 mutant is more sensitive to chemical agents,such as osmotic and cell wall interference agents,while low concentration of oxidants can promote its growth.Similarly,the ?AoAge2 mutant is alos more sensitive to above chenmical stressors.Bioassay analysis showed that the?AoAge2 mutant can not produce traps and kill nematodes.3.Transcriptomic sequencing analysis of AoWT and ?AoAge2 mutantSamples collected from the four time points of AoWT and ?AoAge2 induced by nematodes,and then the data are analyzed using Venn,GO and KEGG enrichment.GO and KEGG enrichment analyses revealed that AoAge2 is involved in the biological processes of nuclear,transmembrane transport,cell cycle and ribosomal protein complex in the regulation of A.oligospora.Innovations of this paper:1.The four Arf-GAP proteins encoding genes was deleted in the A.oligospora,and the functions of AoGts1 and AoAge2 in colony morphology,mycelium branching,septal formation,stress resistance and spore number were reported.2.According to transcriptomic analysis between AoWT and ?AoAge2 mutant,AoAge2 is involved in regulation of multiple biological processes,such as ribonucleoprotein complex,the cell nucleus,membrane transport and cell cycle.