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Dynamics of the Interaction between Cotton Bollworm Helicoverpa armigera and Nucleopolyhedrovirus as Revealed by Integrated Transcriptomic and Proteomic Analyses

2017; Elsevier BV; Volume: 16; Issue: 6 Linguagem: Inglês

10.1074/mcp.m116.062547

1535-9484

Longsheng Xing, C. YUAN, Manli Wang, Zhe Lin, Ben-chang Shen, Zhìhóng Hú, Zhen Zou,

Invertebrate Immune Response Mechanisms

Over the past decades, Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been widely used for biocontrol of cotton bollworm, which is one of the most destructive pest insects in agriculture worldwide. However, the molecular mechanism underlying the interaction between HearNPV and host insects remains poorly understood. In this study, high-throughput RNA-sequencing was integrated with label-free quantitative proteomics analysis to examine the dynamics of gene expression in the fat body of H. armigera larvae in response to challenge with HearNPV. RNA sequencing-based transcriptomic analysis indicated that host gene expression was substantially altered, yielding 3,850 differentially expressed genes (DEGs), whereas no global transcriptional shut-off effects were observed in the fat body. Among the DEGs, 60 immunity-related genes were down-regulated after baculovirus infection, a finding that was consistent with the results of quantitative real-time RT-PCR. Gene ontology and functional classification demonstrated that the majority of down-regulated genes were enriched in gene cohorts involved in energy, carbohydrate, and amino acid metabolic pathways. Proteomics analysis identified differentially expressed proteins in the fat body, among which 76 were up-regulated, whereas 373 were significantly down-regulated upon infection. The down-regulated proteins are involved in metabolic pathways such as energy metabolism, carbohydrate metabolism (CM), and amino acid metabolism, in agreement with the RNA-sequence data. Furthermore, correlation analysis suggested a strong association between the mRNA level and protein abundance in the H. armigera fat body. More importantly, the predicted gene interaction network indicated that a large subset of metabolic networks was significantly negatively regulated by viral infection, including CM-related enzymes such as aldolase, enolase, malate dehydrogenase, and triose-phosphate isomerase. Taken together, transcriptomic data combined with proteomic data elucidated that baculovirus established systemic infection of host larvae and manipulated the host mainly by suppressing the host immune response and down-regulating metabolism to allow viral self-replication and proliferation. Therefore, this study provided important insights into the mechanism of host-baculovirus interaction. Over the past decades, Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been widely used for biocontrol of cotton bollworm, which is one of the most destructive pest insects in agriculture worldwide. However, the molecular mechanism underlying the interaction between HearNPV and host insects remains poorly understood. In this study, high-throughput RNA-sequencing was integrated with label-free quantitative proteomics analysis to examine the dynamics of gene expression in the fat body of H. armigera larvae in response to challenge with HearNPV. RNA sequencing-based transcriptomic analysis indicated that host gene expression was substantially altered, yielding 3,850 differentially expressed genes (DEGs), whereas no global transcriptional shut-off effects were observed in the fat body. Among the DEGs, 60 immunity-related genes were down-regulated after baculovirus infection, a finding that was consistent with the results of quantitative real-time RT-PCR. Gene ontology and functional classification demonstrated that the majority of down-regulated genes were enriched in gene cohorts involved in energy, carbohydrate, and amino acid metabolic pathways. Proteomics analysis identified differentially expressed proteins in the fat body, among which 76 were up-regulated, whereas 373 were significantly down-regulated upon infection. The down-regulated proteins are involved in metabolic pathways such as energy metabolism, carbohydrate metabolism (CM), and amino acid metabolism, in agreement with the RNA-sequence data. Furthermore, correlation analysis suggested a strong association between the mRNA level and protein abundance in the H. armigera fat body. More importantly, the predicted gene interaction network indicated that a large subset of metabolic networks was significantly negatively regulated by viral infection, including CM-related enzymes such as aldolase, enolase, malate dehydrogenase, and triose-phosphate isomerase. Taken together, transcriptomic data combined with proteomic data elucidated that baculovirus established systemic infection of host larvae and manipulated the host mainly by suppressing the host immune response and down-regulating metabolism to allow viral self-replication and proliferation. Therefore, this study provided important insights into the mechanism of host-baculovirus interaction. Lepidopteran insect pests cause vast damage to crops worldwide. Baculoviruses, which comprise enveloped viruses with large, circular, and double-stranded DNA genomes, are important biological agents in controlling lepidopteran insects. Several hundred baculovirus species (1.Jehle J.A. Lange M. Wang H. Hu Z. Wang Y. Hauschild R. Molecular identification and phylogenetic analysis of baculoviruses from Lepidoptera.Virology. 2006; 346: 180-193Crossref PubMed Scopus (186) Google Scholar) have been found from Lepidoptera. Their genomes range from 80 to 180 kb in size, encoding ∼90 to 180 genes. Baculoviruses usually involve a narrow host range, infecting one or several closely related insect species. During the replication cycle, two morphological forms exist, the budded virus (BV), 1The abbreviations used are: BV, budded virus; HearNPV, H. armigera nucleopolyhedrovirus; MF, mock fat body sample; IF, infected fat body sample; DEG, differentially expressed gene; qRT-PCR, quantitative real-time RT-PCR; FDR, false discovery rate; AMP, antimicrobial peptide; PRR, pattern recognition receptors; CM, carbohydrate metabolism; GO, Gene Ontology; DEP, differentially expressed proteins; NPA, normalized peak area; AAM, amino acid metabolism; GOEA, GO Enrichment Analysis; RPKM, reads per kilobase of exon model per million; OB, occlusion body; ODV, occlusion-derived virus; RNA-seq, RNA-sequencing; NGS, next-generation sequencing; PCA, principal component analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPP, pentose-phosphate pathway; CP, control protein; IP, infected protein; IM, infected mRNA; AcMNPV, A. californica multiple nucleopolyhedrovirus; 20E, 20-hydroxyecdysone. 1The abbreviations used are: BV, budded virus; HearNPV, H. armigera nucleopolyhedrovirus; MF, mock fat body sample; IF, infected fat body sample; DEG, differentially expressed gene; qRT-PCR, quantitative real-time RT-PCR; FDR, false discovery rate; AMP, antimicrobial peptide; PRR, pattern recognition receptors; CM, carbohydrate metabolism; GO, Gene Ontology; DEP, differentially expressed proteins; NPA, normalized peak area; AAM, amino acid metabolism; GOEA, GO Enrichment Analysis; RPKM, reads per kilobase of exon model per million; OB, occlusion body; ODV, occlusion-derived virus; RNA-seq, RNA-sequencing; NGS, next-generation sequencing; PCA, principal component analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPP, pentose-phosphate pathway; CP, control protein; IP, infected protein; IM, infected mRNA; AcMNPV, A. californica multiple nucleopolyhedrovirus; 20E, 20-hydroxyecdysone. which causes systemic infection in the host, and the occlusion-derived virus (ODV), which infects per os the host insect and transmits infection horizontally (2.Kamita S.G. Nagasaka K. Chua J.W. Shimada T. Mita K. Kobayashi M. Maeda S. Hammock B.D. A baculovirus-encoded protein tyrosine phosphatase gene induces enhanced locomotory activity in a lepidopteran host.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2584-2589Crossref PubMed Scopus (123) Google Scholar). Baculoviruses not only could be used as a biopesticide to regulate insect populations in agriculture with high efficiency and specificity but also can be developed and widely used in various applications, such as baculovirus-insect cell expression systems, transduction vectors for protein expression in mammalian cells, and potential gene therapy vectors. The cotton bollworm (Helicoverpa armigera), belonging to Noctuidae, is one of the most destructive pest insects in agriculture worldwide (3.Wu K.M. Lu Y.H. Feng H.Q. Jiang Y.Y. Zhao J.Z. 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To our knowledge, this is the first study to integrate transcriptomic and proteomic approaches to provide a framework for genome-wide gene expression profiles in H. armigera larvae infected with HearNPV at both the mRNA and protein levels, which might contribute to a better understanding of the antiviral immunity mechanism of H. armigera against baculovirus infection, thus providing insights into the development of novel biological control strategies and facilitating more practical applications for baculovirus-based engineering technology. Mock and infected fat body samples were collected from naive and infected H. armigera larvae at 72 h post-infection (hpi), respectively. The samples include three independent biological replicates for each group (n = 3, five larvae per replicate), and each biological replicate consists of three technical replicates. Protein was extracted from the fat body samples and separated by electrophoresis on SDS-polyacrylamide gel. Proteins in the fat body were identified by searching mass spectrometry data against an in-house sequence database of H. armigera proteins. To confirm the occurrence of proteins in one group, we adopted a stringent criteria that each protein must be identified at least twice in both biological and technical replicates. Protein abundances were quantified by label-free quantification using Peak Area Calculation Quantification method and compared by Student's t test to determine significant differences (p < 0.05 and fold change ≥1.5 or <0.67) between the infected and mock group. Pearson correlation coefficients were calculated to analyze the correlations of protein levels and mRNA levels in the fat body. A similar method was used to examine the possible correlations between mRNA and protein level changes. The H. armigera nucleopolyhedrovirus G4 strain (34.Chen X. IJkel W.F. Tarchini R. Sun X. Sandbrink H. Wang H. Peters S. Zuidema D. Lankhorst R.K. Vlak J.M. Hu Z. The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome.J. Gen. Virol. 2001; 82: 241-257Crossref PubMed Scopus (203) Google Scholar) was used to infect H. armigera larvae, which were reared on an artificial diet at 27 ± 1 °C under relative humidity of 80% and a 16:8-h (light/dark) photoperiod. Helicoverpa zea cells were cultured in Grace's insect medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum. Occlusion bodies (OBs) were amplified from infected larvae. Day 2 third instar cotton bollworm larvae with a similar body size and weight were used for per os infection in this study. Day 2 third instar H. armigera larvae were selected and divided into the infected and control groups, which had been starved for 16 h before infection. Each larva in the infected group was fed for 10 min on 2 μl of 10% sucrose solution containing edible blue dye contaminated with HearNPV at a final concentration of ∼6.7 × 107 OBs/μl (LC99). For the control group, each larva was provided only 2 μl of 10% sucrose solution containing edible blue dye. The time at which the edible blue dye was consumed was defined as 0 hpi. Hereafter, the larvae in the two groups were transferred to the normal artificial diet. The larvae were harvested at four time points after infection, 0, 24, 48, and 72 h, respectively, and then were dissected for collection of the fat body. Fat body samples were frozen immediately in liquid nitrogen and stored at −80 °C for later use. Briefly, fat body samples were homogenized thoroughly in Qiazol lysis buffer (Qiagen, Hilden, Germany) using a tissue grinder. Total RNA was extracted from the fat body of larvae using the Qiagen RNeasy kit according to the manufacturer's instructions. The integrity of total RNA was measured using the Nanodrop 2000 spectrophotometer, and the RNA samples were separated by gel electrophoresis. In a subsequent procedure, mRNA was purified from 100 μg of total RNA using an mRNA purification kit (Ambion, Austin, TX). A total of 10 μg of mRNA was used to construct a cDNA library for RNA-seq following the Illumina library construction protocol. Using a magnetic stand and beads, DNA fragments of the appropriate sizes were isolated from the initial PCR products. Quality assessment of the cDNA libraries was evaluated using the Agilent Bioanalyzer 2100 system. cDNA libraries were paired-end sequenced on the Hiseq 2000 platform. The obtained raw sequencing reads were preprocessed to remove adaptor sequences and reads of low quality and short size, followed by the filtering of bacterial and viral genomes except for HearNPV. Next, the clean reads were de novo assembled into primary transcripts using Trinity (35.Grabherr M.G. Haas B.J. Yassour M. Levin J.Z. Thompson D.A. Amit I. Adiconis X. Fan L. Raychowdhury R. Zeng Q. Chen Z. Mauceli E. Hacohen N. Gnirke A. Rhind N. et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome.Nat. Biotechnol. 2011; 29: 644-652Crossref PubMed Scopus (12590) Google Scholar). For each library, a set of transcripts was produced from the assembly. To remove redundant transcripts, CAP3 (36.Huang X. Madan A. CAP3: A DNA sequence assembly program.Genome Res. 1999; 9: 868-877Crossref PubMed Scopus (4080) Google Scholar) was used to assemble overlapping transcripts into larger contigs. Additionally, the CD-Hit suite (37.Huang Y. Niu B. Gao Y. Fu L. Li W. CD-HIT Suite: a web server for clustering and comparing biological sequences.Bioinformatics. 2010; 26: 680-682Crossref PubMed Scopus (1630) Google Scholar) was employed to remove redundant transcript sequences to obtain non-redundant unigenes. To predict the functions of these transcripts, the Blast2GO (38.Conesa A. Götz S. García-Gómez J.M. Terol J. Talón M. Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.Bioinformatics. 2005; 21: 3674-3676Crossref PubMed Scopus (8558) Google Scholar) pipeline was used to annotate the unigene datasets. Bowtie was employed to map reads to the corresponding transcripts; subsequently, RSEM (39.Li B. Dewey C.N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.BMC Bioinformatics. 2011; 12: 323-338Crossref PubMed Scopus (10800) Google Scholar) was applied to calculate the normalized gene expression value, expressed in reads per kilobase of exon model per million (RPKM) mapped reads. Based on the gene expression values, the DEGseq (40.Wang L. Feng Z. Wang X. Wang X. Zhang X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data.Bioinformatics. 2010; 26: 136-138Crossref PubMed Scopus (2809) Google Scholar) package in the R environment was implemented for differential gene expression analysis. The genes were considered differentially expressed if they had a p value 1.5 (up-regulated genes) or <0.67 (down-regulated genes). To determine the enriched GO terms, the significantly up- and down-regulated genes were subjected to GOEA using DAVID (41.Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25346) Google Scholar). First, the corresponding DEG orthologs in Drosophila melanogaster were identified using InParanoid. Next, the orthologs from D. melanogaster were submitted to the DAVID annotation server for GOEA. The entire gene set of D. melanogaster was used as the reference. The enriched GO terms were depicted as a bar plot, with an adjusted p value (Padj) of <0.05 deemed to indicate statistical significance. The KOBAS 2.0 (42.Xie C. Mao X. Huang J. Ding Y. Wu J. Dong S. Kong L. Gao G. Li C.Y. Wei L. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases.Nucleic Acids Res. 2011; 39: W316-W322Crossref PubMed Scopus (2909) Google Scholar) web server was employed for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis following the procedure from annotation to identification. In the annotation step, KEGG orthology was used to annotate the corresponding input GenInfo Identifier entries, which were obtained by a BLAST search against the non-redundant protein sequence database (NCBI Nr). Subsequently, the enriched metabolic pathways and biological processes were identified from the preprocessed datasets. The entire gene set of D. melanogaster was used as the reference for identification of enriched pathways. For statistical analysis, the binomial test method was employed to determine statistical significance using the Benjamin and Hochberg method for false discovery rate (FDR) correction. PCA of eight RNA-seq libraries was performed using the mdatools package within the R statistical environment. The top three principal components, PC1, PC2, and PC3, were selected to show the differential effects on global gene expression levels in eight libraries. Total RNA was extracted from the fat body as described previously (14.Xiong G.H. Xing L.S. Lin Z. Saha T.T. Wang C. Jiang H. Zou Z. High throughput profiling of the cotton bollworm Helicoverpa armigera immunotranscriptome during the fungal and bacterial infections.BMC Genomics. 2015; 16: 321-341Crossref PubMed Scopus (77) Google Scholar). The RNA samples were treated with DNase I (Invitrogen) to remove the genomic contaminants. In total, 2 μg of total RNA were used for synthesis of first-strand cDNA using reverse transcriptase Moloney murine leukemia virus (Promega, Fitchburg, WI) and oligo-(dT) primers according to the manufacturer's instructions. qRT-PCR was performed to measure the expression levels of the genes of interest. qRT-PCR was carried out using the Stratagene Mx3005p real-time PCR system and SYBR solution (Tiangen, China) based on the manufacturer's instructions. The specificity