| O.oligospora is a typical strain in the family of nematode-capturing fungi,in which vegetative hyphal can be switched to the adhesive predacious status in response to nitrogen limitation and nematode presence.The species is commonly seen as the model for the mechanisms underlying the fungi-nematodes interaction,and the vegetative-predacious cells transformation.Many researches confer Arrestin protein family as major scaffold proteins in fungi and animals with the action of signal transduction,regulating cell growth and development,pathogenicity,receptors endocytosis,and apoptosis.Our previous finding documented that ammonia can be used as an inducer for trapping nematode-trapping fungi.Tanscriptome data showed that Arrestin-related genes were significantly different during the process of ammonia induced O.oligospora trap formation.Therefore,in this study,we focused on the function of Arrestin encoding genes of O.oligospora via the approaches of homologous recombination,FM4-64 staining,TEM observation,DAPI staining,CFW staining,RT-PCR assays,trap production and other biological comparison of phenotypes.The main experimental results are as follows:1.Functional domain,physicochemical properties and cluster analysis of Arrestin proteinThe functional domain analysis of Arrestin protein showed that Arrestin protein had C-terminal domain（IPR014752）,Arrestin like N-terminal,（IPR011021）and VPS26（IPR028934）.The physicochemical properties of Arrestin protein showed that the molecular weight of 4g345 was the largest,the molecular weight of 76g674 was the smallest,the molecular weight of 117g61 and 193g74 were close,and their isoelectric points were also close.According to the phylogenetic analysis of Arrestin proteins,seven Arrestin proteins clustered and evolved into two branches.Among them,54g332 and 4g345 clustered in one branch,193g25,117g61,76g674,193g74 and 43g250 clustered in another branch.Seven Arrestin proteins had the closest genetic relationship with A.flagrans.2.Deletion of Arrestin-related genes and phenotypic comparisonSeven Arrestin-related gene mutants were obtained by homologous recombination technique,and the phenotypic characteristics of the mutants and wild-type strain were compared.The growth rate of Δ193g25,Δ54g332,Δ117g61,Δ193g74 and Δ43g250 in TYGA medium was slightly lower than that of wild-type strain,Δ193g25,Δ54g332,Δ117g61,Δ193g74 and Δ43g250 produce significantly less traps induced by ammonia;Under the induction of C.elegans,the number of traps produced by Δ193g25,Δ54g332 and Δ43g250 were lower than those produced by wild-type strain;FM4-64 staining showed that the fluorescence intensity ofΔ54g332,Δ117g61 and Δ43g250 decreased,indicating that the endocytosis ability decreased;Under TEM observation,the cell membranes of Δ54g332,Δ117g61 andΔ43g250 became curved and uneven,and some organelles were damaged;CFW staining showed that Δ54g332 and Δ43g250 hyphae had more septum,and the conidiaspore would be deformed;DAPI staining showed that the nuclear number of hyphae and conidial nuclei of seven Arrestin-related gene mutants were significantly reduced;The sporulation number and spore germination rate of Δ54g332,Δ117g61,Δ193g74 and Δ43g250 were significantly lower than those of wild-type strain;Δ54g332,Δ117g61,Δ193g74 and Δ43g250 were more sensitive to the synthesis of cell wall interference agents and hyperosmotic chemical reagents,but less sensitive to oxidants,and appear high concentration to promote growth,Δ4g345 and Δ76g674were involved in the anti-oxidation process.3.Transcription level analysis of phenotype related genes of Arrestin-related genesThe results of RT-PCR analysis of spore production,stress resistance and G protein subunit related genes of the mutant strain and wild-type strain showed that the mutant strain of Arrestin-related genes could affect the spore production,stress resistance and G protein related genes transcription level in O.oligospora,these results indicate that Arrestin-related genes play an important role in the biological processes of O.oligospora,such as growth and development,morphogenesis,sporulation,pathogenicity and stress resistance.4.RNA-Seq data analysis of wild-type and mutant strains of Δ54g332 andΔ43g250High-throughput transcriptome sequencing of wild-type strain and mutant strains by RNA-seq technique was performed.GO enrichment analysis showed thatΔ54g332 and Δ43g250 were involved in membrane formation;KEGG enrichment analysis showed that Δ54g332 and Δ43g250 were involved in the Glycosphingolipid metabolism pathway.The innovation of this paper:1.For the first time,Arrestin-related gene was knocked out in O.oligospora,Based on phenotypic and transcriptomic analysis,it was found that Δ193g25,Δ54g332,Δ117g61,Δ193g74 and Δ43g250 were involved in the regulation of trap formation,membrane endocytosis and morphogenesis.2.RT-PCR analysis showed that Arrestin-related genes were involved in sporulation,stress resistance and G protein subunit expression. |
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