| Pine wood nematode disease(PWD)is a major worldwide quarantine forest disease caused by Bursaphelenchus xylophilus,which has caused serious damage in the economic and ecological value.In order to explore the potential of nematode trapping fungi to effectively control the damage of pine nematode disease,this study explored the molecular mechanisms of nematode trapping fungi in the predation process of pine nematodes using multi-omics techniques.Arthrobotrys conoides(Ac),which has a high predation rate,was analyzed by whole-genome sequencing using Pacbio technology to obtain the coding genes.We sampled the mycelium of Arthrobotrys conoides at different stages of the nematode predatory process and performed transcriptome sequencing to analyze the expression of various genes in different stages,and to explore their significance in biological processes.Using metabolome sequencing technology,we analyzed the differences of metabolic substances secreted by Ac during nematode predation process,and searched for active metabolites.Based on proteome sequencing techniques,we analyzed the differences in protein expression of the fungi in predatory process.Based on the joint multi-omics analysis,the key genes,proteins and metabolites were identified,and key pathways were screened to elucidate the predation mechanism of Ac on pine nematodes.The results of the study are as follows:1.The nematode trapping fungi were isolated from pine nematode infected wood and soil,and morphologically identified as Monacrosporium ellipsosporum,A.cladodes,A.oligospora,A.conoides,and A.dactyloides.Based on the results of the predation rate,A.conoides was selected for the followup study.2.During the interactions between Arthrobotrys conoides and Bursaphelenchus xylophilus,the process was divided into recognition period,adhesion period,penetration period,and decomposition period.The 0-14 h was the recognition period,with no obvious structural features of mycelium appeared.The initial adhesive rings appeared on the mycelium from 15 h to 26 h,and the number of three-dimensional network gradually increased from 26 h to 48 h,when the number of threedimensional network reached the peak and the structure was very clear and obvious.48h-80 h was the decomposition period,when the nematode body wall was degraded and a large number of new conidia were formed.3.Genome sequencing was performed by Pacbio technology.After assembly and optimization,the genome size was 39,588,692 bp,with 10 contigs assembled,the longest fragment was 7,352,175 bp.There were 8,623 coding genes with a total length of 12,786,567 bp and an average length of 1,483 bp,with a total length of 32.3% of the genome.Among the non-codingRNAs(ncRNAs),115 tRNAs,10 snRNAs,and 2 miRNAs were obtained.Gene annotation was performed in 12 databases.Based on bioinformatics analysis,a total of 962 signaling proteins,1,450 transmembrane structural proteins,and 741 secreted proteins were obtained.4.The transcriptome sequencing time points of the predatory process were R(Recognition period)10h,A(Adhesion period)26h,P(Penetration period)48h,and T(Decomposition period)80h.The transcriptome of Ac mycelium during predation on pine nematodes was sequenced.The functional sets of various weighted association analyses were calculated using the R package WGCNA to construct networks.In the Rvs CK group,a total of 579 differential genes were screened,including 194 upregulated genes and 385 down-regulated genes.In the Avs CK group,a total of 3,083 differential genes were screened,of which 1,336 were up-regulated and 1,747 were down-regulated.In the Pvs CK group,a total of 1,100 differential genes were screened,of which 469 were up-regulated and 631 were downregulated.In the Tvs CK group,a total of 2,036 differential genes were screened,of which 837 were up-regulated and 1,199 were down-regulated.15 up-regulated and 15 down-regulated genes were selected respectively to be validated for real-time fluorescence quantitative PCR assays.Their expression trends were analyzed according to the treatment groups at different periods and compared with the RNA-seq transcriptome sequencing results to verify the authenticity and reliability of the transcriptome sequencing results.5.The hub genes were selected by WGCNA analysis,most of the genes with high gene connectivity are located in blue,Turquoise functional module.The subtilase family of serine proteases has 38 genes in Ac.In which the most abundant conserved domain was Peptidases＿S8＿PCSK9＿Proteinase K＿like,which has 18 sequences.These sequences containing Peptidases＿S8＿S53 superfamily,Inhibitor＿I9,and Peptidases＿S8＿5 belonged in different subfamilies.A total of 12 conserved motifs were identified,and motif 5 was found to be present in all 23 sequences,and motif 2 was found to be present in all 28 sequences.6.The metabolome sequencing was used to identified the mycelium at decomposition stages of Ac.Based on the annotation of KEGG,HMDB,and Lipidmaps databases,the total number of metabolites identified in positive ion mode was 790,with 420 has significant differences.The total number of significantly up-regulated metabolites was 267,and down-regulated metabolites was 154.In the negative ion mode,the total number of metabolites identified was 606,with 330 has significant differences.The total number of significantly up-regulated metabolites was 206,and the downregulated metabolites was 124.7.Proteome sequencing was performed to identify Ac mycelium at decomposition stages.A total of 4,112 proteins were functionally annotated based on the combined results of GO,KEGG,COG,and IPR database annotations.A total of 1,465 proteins were annotated in all four functional databases.With the difference fold-change >1.2,a total of 527 up-regulated proteins and 820 down-regulated proteins were identified.The up-regulated pathways included penicillin and cephalosporin biosynthesis pathways,pyruvate metabolism pathway and antibiotic biosynthesis pathway.8.For the association analysis of transcriptome and metabolome,the number of genes whose type of quantifiable and significant difference was down-regulated was 1,199,and the significantly different metabolites were 124 in the negative ion mode and 154 in the positive ion mode.The number of genes with quantifiable and significantly different types as up-regulated was 837,and the significantly different metabolites were 206 in the negative ion mode and 267 in the positive ion mode.The key metabolites involved in the positive ion mode of the metabolome were Linoleoyl ethanolamide,16β-Hydroxystanozolol,5-Hydroxytryptophol,Riboflavin,L-Threonic acid-1,4-lactone.The key metabolites involved in the positive ion mode of the metabolome are LPE15:1,DGlucosamine 6-phosphate,D-Raffinose,N-Oleoyl Glycine,N-Oleoyl Glycine,Galacturonic acid.Based on protein metabolic association analysis,the number of down-regulated significantly different proteins was 47,and the number of significantly different metabolites was 124 in the negative ion mode and 154 in the positive ion mode.The number of up-regulated significantly different proteins was 17,and the number of significantly different metabolites was 206 in the negative ion mode and 267 in the positive ion mode.A total of 15 pathways were found to be co-enriched.Among them,tryptophan metabolism ko00380,biosynthetic pathway of secondary metabolites ko01110,and biosynthesis of antibiotics ko01130 were enriched both in positive and negative ion mode.Based on the transcription and proteomic association analysis,a total of 18 metabolic pathways were found to be co-enriched.Including MAPK signaling pathway-yeast,starch and sucrose metabolism,carbon metabolism,cyanogenic amino acid metabolism,glycerophospholipid metabolism,fatty acid metabolism,longevity regulation pathway,tryptophan metabolism,nitrogen metabolism,and m RNA monitoring pathway.Combined transcriptome and metabolome analysis and the key genes were identified,including A0498,A1075,A1562,A2072,A2341,A2596,A2640,A2731,A4103,A4740,A5689,A6505,A6623,A6948,A7585,A7616,A7655,A8002,A8081,A8483. |
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