Phenotype And Transcriptome Analysis Of StuA Gene Mutant Strain Of Arthrobotrys Oligospora

Nematophagous fungi are an important group of soil microoganisms,which serve as natural enemies of nematodes and play important roles in maintaining nematode population dynamics in natural environments.Arthrobotiys oligospora is the model fungus to study the interaction between nematode-trapping fungi and nematodes,it can live in the soil by saprophytic life,and also form adhesive three-dimentional nets to capture nematodes.The formation of nematode-trapping device is an indication of its transition from saprophytic life to parasitic life.In 2011,our group reported the genome sequence of A.oligospora,which provided a basis for furtherly studying the molecular mechanism of trap formation.In this study,the?AoStuA mutants were chosen,and the phenotypic characteristics of the wild type A.oligospora(WT)and ?AoStuA were compared.Moreover,the gene expressional level of the WT and the mutans were analyzed when they were induced by nematode extract at different time points by transcriptome sequencing,and GO/KEGG enrichment analysis of significant DEGs(differentially expressed genes)were carried out The main experimental results were described as follows.1.Phenotypic analysis of AAoStuA mutants.The growth and conidia production of ?AoStuA mutants were significantly reduced,and the morphology of majority of conidia was abnormal,and the germination rate of spore was lower than the WT.The mycelial differentiation of AAoStuA mutants was weakened.Importantly,?AoStuA strains could not form traps and kill nematodes.Its resistance to NaCl,H2O2 and SDS became weak than the WT,and the extracellular proteolytic activity was decreased.2.Transcriptional levels of phenotype-related genes were analyzed during the trap formation.The total RNA was extracted from the mycelia of the WT and mutants,which were induced by nematode extract at different time points,and the cDNA was obtained by reverse transcription.Some genes were selected for Real time PCR,including genes involved in G protein regulatory pathway,peroxisome biogenesis,conidiation and serine proteases.When the AoStuA gene was deleted,the transcript of most of above genes was down-regulated,and genes located the up-and downstream of AoStuA,such as the Ras2(AOL_s00215g7)was significantly up-regulated;two genes AOL_s00004g650 and AOL_s00080g270 of the MCM family and the gene AOL_s00076g653 of the ORC family were significantly down-regulated;genes involved in peroxisome biogenesis,such as Pexl(AOL_s00054g771),Pex3(AOL_s00215g610),Pexll(AOL_s00083g431),and Pexl4(AOL_s00004g492),were significantly down-regulated,and two genes AOL_s00083g 15 8 and AOL_s00215g205 of PMP34 protein family,as well as genes AOL_s0054g405 and AOL_s00006g18 of MPV17 protein family were also down-regulated.3.Transcriptome sequencing analysis of A.oilospora and ?AoStuA mutants.The total RNA was extracted from A.oilospora and ?AoStuA mutants under induced by nematode extract.Eight sets of transcriptome data by RNA-seq sequencing.Through the data analysis,some genes were differentially expressed in AAoStuA mutants,and some differentially expressed genes were predicted to involved in trap formation,such as the MAPK pathway genes(AOL_s00076g411,AOL_s00083g305,AOL_s00076g496,AOL_s00054g653 and AOL_s00007g300),peroxisome pathway genes(AOL_s00054g476 and AOL_s00173g103),glutathione metabolism pathway gene(AOL_s00043g230),and dicarboxylate metabolic pathway genes(AOL_s00006g210 and AOL_s00006g411).In addition,AOL_s0018 8g243 is a kind of catalase,it is also involved in peroxisomal pathway and other channels.The potential functions of some genes was predicted by comparison with the reference species,which laid a foundation for further study on the molecular mechanism of trap formation.The main innovations of this study:1.The phenotypic characteristics of the WT and the AAoStuA mutants were compared,and the function of the AoStuA gene in the growth and development of the A.oligospora was speculated.2.The transcriptional levels of some genes were analyzed by Real time PCR at different time points.It was confirmed that the AoStuA gene affected the expression of several genes involved in peroxisome biogenesis,and it also affected the transcript of genes located in its upstream and downstream.Moreover,several genes involved in cell wall synthesis,hydrogen peroxide synthesis,spore production and protease synthesis were significantly down-regulated,which resulted in the phenotypic difference between the WT and mutants.3.Using RNA-seq technique,the transcriptional levels of the WT and the?AoStuA mutants were compared at different time points,and some genes related to the regulation of trap formation were predicted,which laid a foundation for further explanation of the mechanism of trap formation in A.oligospora.