Phenotypic And Transcriptomic Analyses Of The Gene AoRic8 Deleted Mutant In Arthrobotrys Oligospora

Arthrobotrys oligospora can capture and infect nematodes via forming special three-dimensional networks to capture and infect nematodes.Cholinesterase(Ric8)is a conserved guanine nucleotide exchange factor(GEF),which is involved in the regulation of G protein signals and regulates many different biological processes in filamentous fungi.In this paper,the wild-type(AoWT)and ΔAoRic8 mutant strains of Arthrospora oligospora were selected as the research objects,phenotypic differences between the mutant and AoWT strain were compared.At the same time,transcriptomic sequencing was used to compare the differentially expressed genes(DEGs)betweenΔAoRic8 and AoWT strains after induced by nematodes at four time points.The GO and KEGG analyses were performed on DEGs,phenotypic verification and metabolomics experiments were performed on partial key DEGs.Moreover,the interaction between AoRic8 and proteins related to protein kinase A(PKA)signaling pathway was predicted and verified by yeast two-hybrid.The main experimental results:1.Comparison of phenotypic characteristics of ΔAoRic8 mutant and AoWT.Compared with AoWT,the ΔAoRic8 mutants showed a slower growth rate on TG,TYGA,PDA,WA,CMY media,significantly reduced in spore production,decreased in spore germination rate,and significantly reduced in the mycelial differentiation after germination,and no traps was produced and no nematodes was killed,and the hydrolytic activity of extracellular proteases was also reduced.The growth of ΔAoRic8mutant was inhibited by chemical stressors,and they become more sensitive to different chemicals(such as NaCl and Sorbitol).Moreover,compared with AoWT,the transcriptional levels of some genes related to sporulation,cell wall biosynthesis,oxidative stress,and extracellular proteases were significantly down-regulated in theΔAoRic8 mutant strain.2.Transcriptome analysis of ΔAoRic8 mutant and AoWT strainThe mycelia of ΔAoRic8 mutant and AoWT strain were induced by nematodes,andcollected at four time points,then the samples were analyzed by RNA-sequencing.Transcriptomic analysis showed that some genes related to cell wall,cell membrane biosynthesis,lipid metabolism and other related processes were down-regulated in theΔAoRic8 mutant.At the same time,some genes related to gene replication and repair,membrane synthesis,nitrogen metabolism,and genes related to the synthesis of secondary metabolites were also down-regulated.3.Phenotypic verification based on the transcriptomic analysisBased on the transcriptomic analysis,several experiments such as CFW staining,FM4-64 staining,and transmission electron microscopy were performed for verification of DEGs,the results showed that the mycelium of the ΔAoRic8 mutant became swollen,the endocytosis of the ΔAoRic8 mutant was weakened,and the intracellular lipid was reduced.In summary,these phenotypic results were agreement with transcriptomic analysis.Moreover,the yeast two-hybrid experiments showed that there was an interaction between AoRic8 and AoGα1,AoGβ,AoPKA and AoSom1,AoStuA,and AoSom1 and AoStuA.4.Metabonomics analysis of ΔAoRic8 mutant and AoWT strainThe AoWT and ΔAoRic8 mutant strains were cultured in a PDA liquid medium and shaked with flask,and then ultrasonically crushed and extracted with ethylacetate,and the extract was dissolved in an analytical grade methanol to a volume of 1 ml,then the samples were analyzed using LC-MS.Mass spectrometry and chromatogram analyses showed that many compounds were significantly down-regulated in ΔAoRic8 mutant compared with the AoWT strain,among which arthrobotrisin A,B,and C were significantly down-regulated.Innovations of this paper:1.In this paper,the DEGs between the ΔAoRic8 mutant and AoWT strain was compared by transcriptomic analysis,and verification of related phenotypes further revealed the function of AoRic8 in the growth and development,sporulation and trap formation of A.oligospora.2.The differences of metabolites between the ΔAoRic8 mutant and AoWT strainwas compared using LC-MS,which laid a foundation for further elucidating the regulatory mechanism of AoRic8 on secondary metabolism in A.oligospora,such as oligosporon.