Molecular Mechanism Of Chlamydospore Formation Of Trichoderma Virens GV29-8

Trichoderma spp.are widely applied biocontrol agents which are antagonistic to a variety of plant pathogens.Chlamydospores are a type of propagules produced by many fungi that have thick walls and are highly resistant to adverse environmental conditions.The chlamydospore preparations of Trichoderma spp.have a longer shelf life and highly control efficy to diseases than conidial preparations,which have a better application potential.However,mass production of chlamydospores has been proven difficult.Previously,we have developed a new medium and fermentation technology to induce mass production of chlamydospores in Trichoderma spp..In the study,we performed a comprehensive analysis of transcriptome dynamics during chlamydospore formation(CF).Combined with the results of transcriptome analysis,the key genes and pathways affecting the CF of Trichoderma virens GV29-8were obtained,which provided theoretical guidance for clarifying the molecular mechanism of CF,obtaining genetic improvement strains and controlling of fermentation technology in Trichoderma.The main results of the study were as follows:1.Through several systematic observations of the CF process of T.virens GV29-8 strain,eight morphologically significant time point samples that represented significant events during CF were selected for transcriptome sequencing,3 biological replicates were selected at each time point.Including the mycelium growth stage(20h/TVS1,24h/TVS2 and 26h/TVS3);early stage of CF(28h/TVS4 and32h/TVS5);middle stage of CF(38h//TVS6);flourishing stage of CF(45h/TVS7);late stage of CF(56h/TVS8).A total of 206.07 GB of clean bases were obtained from 24 samples,with Q30 greater than90.85%,and the overall mapping ratios ranged from 79.34% to 85.96%.A total of 19,737 genes were obtained from 24 samples.Among them,7332 were new genes predicted by stringtie software.According to PCA analysis,8 sampling time points were divided into 4 stages: the mycelium growth stage(S1),early and middle stage of CF(S2),flourishing stage of CF(S3),and late stage of CF and mycelia initial autolysis(S4).2.A total of 6462 DEGs were obtained by comparing transcriptome data of samples at adjacent stages.GO enrichment analysis showed that organonitrogen compound metabolic(GO:1901564)was the most significantly enriched GO term in S2 vs S1.There were 126 DEGs,144 of which were down-regulated and related to the synthesis of organic nitrogen compounds.In S3 vs S2,tetrapyrrole binding(GO:0046906)and heme binding(GO:0020037)were the most significantly enriched GO terms.A total of 52 DEGs were recognized in these two GO terms,mainly were P450 family genes,and most of which were involved in the synthesis of toxic secondary metabolites.In S4 vs S3,lipid metabolic process(GO:0006629)was the most significantly enriched GO term,in which 54 DEGs were enriched.These DEGs were mainly involved in phospholipid,glycerolipid and fatty acid metabolic.KEGG enrichment analysis showed that the ribosome(tre03010)pathway category was the most significantly enriched pathway and the DEGs in the ribosomal pathway were all ribosomal protein genes and down-regulated in S2 vs S1.N-glycan biosynthesis(tre00510)categories were also significantly enriched in S3 vs S2.There were 14 DEGs included in the N-glycan biosynthesis pathway.Among them,10 were up-regulated and involved in mannan synthesis,3 down-regulated DEGs were involved in mannose degradation.A total of 5699 DEGs were obtained by comparing transcriptome data at 8adjacent time points.KEGG enrichment analysis showed that the most abundant pathway for the TVS5 vs TVS4 and TVS6 vs TVS5 were cell cycle-yeast.WGCNA analysis showed that the pink module exhibited high positive correlations with S1 were mainly contained ribosomal protein genes.Genes in this module were significant enrichment in the ribosome pathway(tre03010).Orange module genes(59)were positively associated with S2.Genes related to oxidation-reduction and transmembrane transport processes were included in this module.Phenylalanine metabolism(tre00360),glutathione metabolism(tre00480),tryptophan metabolism(tre00380)and biosynthesis of secondary metabolites(tre01110)pathways were all enriched.The genes in the black and purple modules that highly positively correlated with S4 were mainly involved in metabolic,oxidation-reduction,and transport processes.fatty acid metabolism(tre01212),biosynthesis of unsaturated fatty acids(tre01040),amino sugar and nucleotide sugar metabolism(tre00520),and starch and sucrose metabolism(tre00500)were all enriched.Among them,a total of 16 genes were differentially expressed in amino sugar and nucleotide sugar metabolism pathway(tre00520),which are mainly related to chitin metabolism.Metabolic and oxidation-reduction genes were dominant in dark red and green modules that negatively correlated with S4,and fatty acid degradation(tre00071)pathway was enriched in this module.3.According to the analysis of transcriptome data,it was found that the amino sugar and nucleotide sugar metabolism pathway enriched more DEGs.Among them,the gene expression level of chitinase gene(Ech2)and chitin synthetase gene(Chs1＿7926/Chs1＿8917)have a large variation.Adding Glc NAc,an intermediate metabolite of chitin metabolism,at 25g/L and 45g/L significantly inhibited CF in T.virens Gv29-8.Based on this,to analysis the effects of these 3 genes on the CF of T.virens GV29-8,the knockout,overexpression and complementary strains of chitinase(Ech2)and chitin synthase(Chs1＿7926/Chs1＿8917)genes were constructed.The results showed that the yield of the chlamydospores were significantly increased in the Ech2 mutant.At the same time,the growth rate and the yield of the conidia were accelerated,the biomass was reduced.Deletion of the chitin synthase gene(Chs1＿7926/Chs1＿8917)blocked chitin biosynthesis of the cell wall,resulting in mycelium dysplasia and an inability to form normal chlamydospores.At the same time,the growth rate,biomass,conidial yield,and conidial germination rate were significantly reduced.Adding 25 g/L Glc NAc to the chlamydospore inducing medium,the abnormality of the mycelia recovered slightly,but still can’t formed the complete chlamydospores.The growth rate and conidia production were increased in the mutant strain.In summary,through GO,KEGG,STC and WGCNA analysis of the transcriptome data of T.virens GV29-8 at different stages of CF,the metabolic pathways and genes that may play an important role in each stage of CF have been preliminarily identified.It was speculated that the differential expression of DEGs in the organonitrogen compound metabolism pathway may be involved in the assimilation and absorption of exogenous nitrogen in the early growth stage(S1),resulted in subsequent nitrogen deficiency(S2).At the same time,The differential expression of DEGs related to secondary metabolism during mycelium growth was accompanied by the accumulation of secondary metabolites and reactive oxygen free radicals;The resulting nitrogen-deficient and toxin enriched medium may stimulate cell differentiation by initiating cell cycle DEGs regulation to induce morphological transformation of mycelia into chlamydospores.High expression of DEGs relating to glycogen,lipid,mannan,and chitin synthetic metabolic pathways during the flourishing(S3)and late stages(S4)of CF may be conducive to energy storage and cell wall construction in chlamydospores.By construction of knockout,overexpression and complementary strains of chitinase and chitin synthase genes,preliminarily clarified the effect of key genes in amino sugar and nucleotide sugar metabolism pathway on CF of T.virens GV29-8 strain.Our results provide a new perspective for understanding the genes of pathways involved in CF of Trichoderma spp.