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Oppert B.,U.S. Department of Agriculture | Guedes R.N.C.,Federal University of Viçosa | Aikins M.J.,Kansas State University | Perkin L.,U.S. Department of Agriculture | And 18 more authors.
BMC Genomics | Year: 2015

Background: Phosphine is a valuable fumigant to control pest populations in stored grains and grain products. However, recent studies indicate a substantial increase in phosphine resistance in stored product pests worldwide. Results: To understand the molecular bases of phosphine resistance in insects, we used RNA-Seq to compare gene expression in phosphine-resistant and susceptible laboratory populations of the red flour beetle, Tribolium castaneum. Each population was evaluated as either phosphine-exposed or no phosphine (untreated controls) in triplicate biological replicates (12 samples total). Pairwise analysis indicated there were eight genes differentially expressed between susceptible and resistant insects not exposed to phosphine (i.e., basal expression) or those exposed to phopshine (>8-fold expression and 90 % C.I.). However, 214 genes were differentially expressed among all four treatment groups at a statistically significant level (ANOVA, p < 0.05). Increased expression of 44 cytochrome P450 genes was found in resistant vs. susceptible insects, and phosphine exposure resulted in additional increases of 21 of these genes, five of which were significant among all treatment groups (p < 0.05). Expression of two genes encoding anti-diruetic peptide was 2- to 8-fold reduced in phosphine-resistant insects, and when exposed to phosphine, expression was further reduced 36- to 500-fold compared to susceptible. Phosphine-resistant insects also displayed differential expression of cuticle, carbohydrate, protease, transporter, and many mitochondrial genes, among others. Gene ontology terms associated with mitochondrial functions (oxidation biological processes, monooxygenase and catalytic molecular functions, and iron, heme, and tetrapyyrole binding) were enriched in the significantly differentially expressed dataset. Sequence polymorphism was found in transcripts encoding a known phosphine resistance gene, dihydrolipoamide dehydrogenase, in both susceptible and resistant insects. Phosphine-resistant adults also were resistant to knockdown by the pyrethroid deltamethrin, likely due to the increased cytochrome P450 expression. Conclusions: Overall, genes associated with the mitochondria were differentially expressed in resistant insects, and these differences may contribute to a reduction in overall metabolism and energy production and/or compensation in resistant insects. These data provide the first gene expression data on the response of phosphine-resistant and -susceptible insects to phosphine exposure, and demonstrate that RNA-Seq is a valuable tool to examine differences in insects that respond differentially to environmental stimuli. © 2015 Oppert et al.


Oppert B.,U.S. Department of Agriculture | Guedes R.N.C.,Federal University of Viçosa | Aikins M.J.,Kansas State University | Perkin L.,U.S. Department of Agriculture | And 16 more authors.
BMC Genomics | Year: 2015

Background: Phosphine is a valuable fumigant to control pest populations in stored grains and grain products. However, recent studies indicate a substantial increase in phosphine resistance in stored product pests worldwide. Results: To understand the molecular bases of phosphine resistance in insects, we used RNA-Seq to compare gene expression in phosphine-resistant and susceptible laboratory populations of the red flour beetle, Tribolium castaneum. Each population was evaluated as either phosphine-exposed or no phosphine (untreated controls) in triplicate biological replicates (12 samples total). Pairwise analysis indicated there were eight genes differentially expressed between susceptible and resistant insects not exposed to phosphine (i.e., basal expression) or those exposed to phopshine (>8-fold expression and 90 % C.I.). However, 214 genes were differentially expressed among all four treatment groups at a statistically significant level (ANOVA, p < 0.05). Increased expression of 44 cytochrome P450 genes was found in resistant vs. susceptible insects, and phosphine exposure resulted in additional increases of 21 of these genes, five of which were significant among all treatment groups (p < 0.05). Expression of two genes encoding anti-diruetic peptide was 2- to 8-fold reduced in phosphine-resistant insects, and when exposed to phosphine, expression was further reduced 36- to 500-fold compared to susceptible. Phosphine-resistant insects also displayed differential expression of cuticle, carbohydrate, protease, transporter, and many mitochondrial genes, among others. Gene ontology terms associated with mitochondrial functions (oxidation biological processes, monooxygenase and catalytic molecular functions, and iron, heme, and tetrapyyrole binding) were enriched in the significantly differentially expressed dataset. Sequence polymorphism was found in transcripts encoding a known phosphine resistance gene, dihydrolipoamide dehydrogenase, in both susceptible and resistant insects. Phosphine-resistant adults also were resistant to knockdown by the pyrethroid deltamethrin, likely due to the increased cytochrome P450 expression. Conclusions: Overall, genes associated with the mitochondria were differentially expressed in resistant insects, and these differences may contribute to a reduction in overall metabolism and energy production and/or compensation in resistant insects. These data provide the first gene expression data on the response of phosphine-resistant and -susceptible insects to phosphine exposure, and demonstrate that RNA-Seq is a valuable tool to examine differences in insects that respond differentially to environmental stimuli. © 2015 Oppert et al.


PubMed | Kansas State University, Oklahoma State University, LifeTechnologies, North Carolina State University and 2 more.
Type: | Journal: BMC genomics | Year: 2015

Phosphine is a valuable fumigant to control pest populations in stored grains and grain products. However, recent studies indicate a substantial increase in phosphine resistance in stored product pests worldwide.To understand the molecular bases of phosphine resistance in insects, we used RNA-Seq to compare gene expression in phosphine-resistant and susceptible laboratory populations of the red flour beetle, Tribolium castaneum. Each population was evaluated as either phosphine-exposed or no phosphine (untreated controls) in triplicate biological replicates (12 samples total). Pairwise analysis indicated there were eight genes differentially expressed between susceptible and resistant insects not exposed to phosphine (i.e., basal expression) or those exposed to phopshine (>8-fold expression and 90 % C.I.). However, 214 genes were differentially expressed among all four treatment groups at a statistically significant level (ANOVA, p<0.05). Increased expression of 44 cytochrome P450 genes was found in resistant vs. susceptible insects, and phosphine exposure resulted in additional increases of 21 of these genes, five of which were significant among all treatment groups (p<0.05). Expression of two genes encoding anti-diruetic peptide was 2- to 8-fold reduced in phosphine-resistant insects, and when exposed to phosphine, expression was further reduced 36- to 500-fold compared to susceptible. Phosphine-resistant insects also displayed differential expression of cuticle, carbohydrate, protease, transporter, and many mitochondrial genes, among others. Gene ontology terms associated with mitochondrial functions (oxidation biological processes, monooxygenase and catalytic molecular functions, and iron, heme, and tetrapyyrole binding) were enriched in the significantly differentially expressed dataset. Sequence polymorphism was found in transcripts encoding a known phosphine resistance gene, dihydrolipoamide dehydrogenase, in both susceptible and resistant insects. Phosphine-resistant adults also were resistant to knockdown by the pyrethroid deltamethrin, likely due to the increased cytochrome P450 expression.Overall, genes associated with the mitochondria were differentially expressed in resistant insects, and these differences may contribute to a reduction in overall metabolism and energy production and/or compensation in resistant insects. These data provide the first gene expression data on the response of phosphine-resistant and -susceptible insects to phosphine exposure, and demonstrate that RNA-Seq is a valuable tool to examine differences in insects that respond differentially to environmental stimuli.


News Article | December 23, 2015
Site: www.nature.com

No statistical methods were used to predetermine sample size. The experiments were not randomized. In vivo transfer colitis and EAE mouse experiments were blinded, but cell culture and in vitro studies were not. EEF1A1-LSL.EGFPL10 (lox-stop-lox-EGFP–L10 knockin at the Ef1a1 locus) transgenic mice, RORγ/γt-deficient animals and Ddx5fl/fl mice have been previously described elsewhere31, 47, 48. Conditional mutant mice were bred to CD4-Cre transgenic animals (Taconic) and maintained on the C57BL/6 background. We bred heterozygous mice to yield 6–8-week-old Ddx5+/+CD4-Cre+ (subsequently referred to as wild type) and Ddx5fl/flCD4-Cre+ (referred to as DDX5-T) littermates for experiments examining DDX5 in peripheral T-cell function. DDX5 conditional mutant mice were also bred to IL7R-Cre transgenic animals (Jackson Laboratory) (with Ddx5 deleted in common early lymphoid progenitors; referred to as DDX5-clpKO) for experiments examining DDX5 functions during T-cell development in the thymus. RmrpG270T knock-in mice were generated using CRISPR-Cas9 technology by the Rodent Genetic Engineering Core (RGEC) at New York University Langone Medical Center. Guide RNA and homology directed repair donor template sequences are provided in Supplementary Table 1. Heterozygous crosses provided Rmrp+/+ (wild-type) and RmrpG270T/G270T littermates for in vivo studies. All animal procedures were in accordance with protocols approved by the Institutional Animal Care and Use Committee of the New York University School of Medicine (Animal Welfare Assurance number: A3435-01). Steady-state small intestines were collected for isolation of lamina propria mononuclear cells as previously described45. For detecting SFB colonization, SFB-specific 16S primers were used49. Universal 16S and/or host genomic DNA was quantified simultaneously to normalize SFB colonization of each sample. All primer sequences are listed in Supplementary Table 1. For the adoptive transfer model of colitis, 5 ×105 CD4+CD25−CD44low CD45RBhiCD62Lhi T cells were isolated from mouse splenocytes by FACS sorting and administered i.p. into Rag2−/− mice as previously described50. Animal weights were measured approximately weekly. Between weeks seven and eight, large intestines were collected for H&E staining and isolation of lamina propria mononuclear cells as previously described45. The H&E slides from each sample were examined in a double-blind fashion. The histology scoring (scale 0–24) was based on the evaluation of criteria described previously51. For induction of active EAE, each mouse was immunized subcutaneously on day 0 with 100 μg of MOG35–55 peptide, emulsified in CFA (Complete Freund’s Adjuvant supplemented with additional 2 mg ml−1 Mycobacterium tuberculosis), and injected i.p. on days 0 and 2 with 100 ng per mouse of pertussis toxin (Calbiochem). The EAE scoring system was as follows: 0, no disease; 1, limp tail; 2, weak/partially paralysed hind legs; 3, completely paralysed hind legs; 4, complete hind and partial front leg paralysis; 5, complete paralysis/death. In transfer colitis and EAE experiments, animals of different genotypes were co-housed and weighed and scored blindly. For statistical power level of 0.8, probability level of 0.05, anticipated effect size of 2, minimum sample size per group for two-tailed hypothesis is 6. Two-tailed unpaired Student’s t-test was performed using Prism (GraphPad Software). We treated a P value of less than 0.05 as a significant difference. All experiments were performed at least twice. Mouse T cells were purified from lymph nodes and spleens of 6–8-week-old mice, by sorting live (DAPI−), CD8−CD19−CD4+CD25−CD44low/intCD62L+ naive T cells using a FACSAria (BD). Detailed antibody information is provided in Supplementary Table 1. Cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Sigma) supplemented with 10% heat-inactivated FBS (Hyclone), 50 U penicillin-streptomycin (Invitrogen), 4 mM glutamine and 50 μM β-mercaptoethanol. For T-cell polarization, 200 μl of cells was seeded at 0.3 × 105 cells per ml in 96-well plates pre-coated with goat anti-hamster IgG at a 1:20 dilution of stock (1 mg ml−1, MP Biomedicals). Naive T cells were activated with anti-CD3 ε (2.5 μg ml−1) and anti-CD28 (10 μg ml−1). Cells were cultured for 4–5 days under T 17-polarizing conditions (0.1–0.3 ng ml−1 TGF-β, 20 ng ml−1 IL-6), T 1- (10 ng ml−1 IL-12, 10 U ml−1 IL-2), T 2- (10 ng ml−1 IL-4, 10 U ml−1 IL-2), or T - (5 ng ml−1 TGF-β, 10 U ml−1 IL-2) conditions. Human T cells were isolated from peripheral blood of healthy donors using anti-human CD4 MACS beads (Miltenyi). Human CD4+ T cells were cultured in 96-well U bottom plates in 10 U ml−1 of IL-2, 10 ng ml−1 of IL-1β, 10 ng ml−1 of IL-23, 1 μg ml−1 of anti-IL-4, 1 μg ml−1 of anti-IFNγ and anti-CD3/CD28 activation beads (LifeTechnologies) at a ratio of 1 bead per cell, as previously described52. For cytokine analysis, cells were incubated for 5 h with phorbol 12-myristate 13-acetate (PMA) (50 ng ml−1; Sigma), ionomycin (500 ng ml−1; Sigma) and GolgiStop (BD). Intracellular cytokine staining was performed according to the manufacturer’s protocol (Cytofix/Cytoperm buffer set from BD Biosciences and FoxP3 staining buffer set from eBioscience). A LSR II flow cytometer (BD Biosciences) and FlowJo (Tree Star) software were used for flow cytometry and analysis. Dead cells were excluded using the Live/Dead fixable aqua dead cell stain kit (Invitrogen). Custom Rmrp and predesigned Malat1 Stellaris RNA fluorescence in situ hybridization (FISH) probes were purchased from BiosearchTech and used to label mouse Rmrp and Malat1 RNA in cultured T 17 cells according to the manufacturer’s protocol. Control and human DDX5-specific short interfering RNAs (siRNAs) were obtained from Cell Signaling. Synthesis of ASOs was performed as previously described53. All ASOs were 20 nucleotides in length and had a phosphorothioate backbone. The ASOs had five nucleotides at the 5′ and 3′ ends modified with 2’-O-methoxyethyl (MOE) for increased stability. ASOs and siRNA sequences are provided in Supplementary Table 1. siRNA and ASOs were introduced into mouse T 17 cells by Amaxa nucleofection as previously described8. Wild-type and helicase-dead mutant DDX5 were described previously54. DDX5 and Rmrp were subcloned into the mouse stem-cell virus (MSCV) Thy1.1 vectors for retroviral overexpression and rescue assays in T cells. Retrovirus production was carried out in Plat-E cells (Cell Biolabs, Inc., not tested for mycoplasma) as previously described55. Spin transduction was performed 24 h after in vitro T-cell activation by centrifugation in a Sorvall Legend RT at 700 g for 90 min at 32 °C. Aqua−Thy1.1+ live and transduced cells were analysed by flow cytometry after 5 days of culture in T 17-polarizing conditions. A ROR luciferase reporter was constructed with four RORE sites replacing the Gal4 (UAS) sites from the pGL4.31 vector (luc2P/GAL4 UAS/Hygro) from Promega (C935A) as described in ref. 56. Naive CD4+ T cells were cultured in T 17-polarizing conditions for 72 h. Nucleofection (Amaxa Nucleofector 4D, Lonza) was then used to introduce 1 μg RORE–firefly luciferase reporter construct and 1 μg control Renilla luciferase construct according to the manufacturer’s instructions. Luciferase activity was measured using the dual luciferase reporter kit (Promega) at 24 h after transfection. Relative luciferase units (RLU) were calculated as a function of firefly luciferase reads over those of Renilla luciferase. DMSO or 2 μM RORγ inhibitor (ML209) were used in luciferase experiments as described in ref. 57. Cultured T 17 cells (100 × 106) were lysed in 25 mM Tris (pH 8.0), 100 mM NaCl, 0.5% NP-40, 10 mM MgCl , 10% glycerol, 1× protease inhibitor and PhosphoSTOP (Roche) on ice for 30 min, followed by homogenization with a 25-gauge needle. The RORγ/γt-specific antibody used for pull-down assays was previously described8. Co-immunoprecipitated complexes were collected with protein G dynabeads (Dynal, Invitrogen). Detailed antibody information is provided in Supplementary Table 1. Mass spectrometry and the Mascot database search to identify protein complex composition were both performed by the Central Proteomics Facility at the Dunn School of Pathology, Oxford, UK. Twenty million cells cultured in T 17-polarizing conditions for 48 h were lysed in 10 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl , 0.5 mM dithiothreitol (DTT), 100 μg ml−1 cycloheximide, 1% NP-40, 30 mM DHPC, 1× protease inhibitor and PhosphoSTOP (Roche). Ribosome-TRAP immunoprecipitation was first performed using 2 μg of anti-GFP antibody (Invitrogen) and collected in 20 μl of protein G magnetic dynabeads. The supernatant was removed for subsequent RIP pull-down using anti-DDX5 (Abcam) or anti-RORγt antibodies and collected with protein G dynabeads. TRAP-seq samples were washed with high-salt wash buffer (10 mM HEPES (pH 7.4), 350 mM KCl, 5 mM MgCl , 1% NP-40, 0.5 mM DTT and 100 μg ml−1 cycloheximide). RIP-seq samples were washed three times with 25 mM Tris (pH 8.0), 100 mM NaCl, 0.5% NP-40, 10 mM MgCl , 10% glycerol, 1× protease inhibitor and PhosphoSTOP (Roche). Enrichment of target proteins was confirmed by immunoblot analysis. Complementary DNAs (cDNAs) were synthesized from TRIzol (Invitrogen)-isolated RNA, using Superscript III kits (Invitrogen). RNA-seq libraries were prepared and sequenced at Genome Services Laboratory, HudsonAlpha. Sequencing reads were mapped by Tophat and transcripts called by Cufflinks. Pull-down enrichment was calculated for each transcript as a ratio of FPKM recovered from TRAP-seq and RIP-seq samples compared to those from 5% input. For RNA-seq analysis, volcano scores for wild-type, DDX5-T and RORγt-knockout T 17 cells were calculated for each transcript as a function of its P value and fold change between mutant and wild-type controls. BAM files were converted to .tdf format for viewing with the IGV Browser Tool. Ingenuity Pathway Analysis (IPA, Qiagen) was used to identify enriched Gene Ontology terms in the DDX5–RORγt coregulated gene set. The ChIRP-seq assay was performed largely as described previously58. Mouse T 17 cells were cultured as above and in vivo RNA–chromatin interactions were fixed with 1% glutaraldehyde for 10 min at 25 °C. Antisense DNA probes (designated ‘odd’ or ‘even’) against Rmrp were designed by Biosearch Probe Designer (1, 5′-TAGGAAACAGGCCTTCAGAG-3′; 2, 5′-AACATGTCCCTCGTATGTAG-3′; 3, 5′-CCCCTAGGCGAAAGGATAAG-3′; 4, 5′-AACAGTGACTTGCGGGGGAA-3′; 5, 5′-CTATGTGAGCTGACGG ATGA-3′). Probes modified with BiotinTEG at the 3′ end were synthesized by Integrated DNA Technologies (IDT). Isolated RNA was used in RT–qPCR analysis (Stratagene) to quantify enrichment of Rmrp and depletion of other cellular RNAs. Isolated DNA was used for qPCR analysis or to make deep sequencing libraries with the NEBNext DNA library prep master mix set for Illumina (NEB). Library DNA was quantified on the high sensitivity bioanalyzer (Agilent) and sequenced from a single end for 75 cycles on an Illumina NetSeq 500. Sequencing reads were first trimmed of adaptors (FASTX Toolkit) and then mapped using Bowtie to a custom bowtie index containing single-copy loci of repetitive RNA elements (ribosomal RNAs, small nuclear RNAs, and noncoding Y RNAs59). Reads that did not map to the custom index were then mapped to mm9. Mapped reads were separately shifted towards the 3′ end using MACS and normalized to a total of 10 million reads. Even and odd replicates were merged as described previously58 by taking the lower of the two read density values at each nucleotide across the entire genome. These processing steps take raw FASTQ files and yield processed files that contain genome-wide Rmrp-occupied chromatin association maps, where each nucleotide in the genome has a value that represents the relative binding level of the Rmrp RNA. MACS parameters were as follows: band width = 300; model fold = 10, 30; P-value cutoff = 1 × 105. The full pipeline is available at https://github.com/bdo311/chirpseq-analysis. ChIRP-qPCR was performed on DNA purified after treatment with RNase (60 min, 37 °C) and proteinase K (45 min, 65 °C). The primers used for qPCR are listed in Supplementary Table 1. For enrichment analysis, we tested for the enrichment of Rmrp ChIRP peaks among ChIP peak sets for key T 17 transcription factors, CTCF, RNA Pol II and several histone marks8. Assay for transposase-accessible chromatin sequencing (ATAC)-seq was performed, according to published protocol60, on cultured T 17-polarized cells in vitro for 48 h (unpublished data). Because of differences in ChIP antibody affinities and the bias in the selection of ChIP and ChIRP factors, we used peaks generated from ATAC-seq data as a background setting for the enrichment analysis. In our analysis, we considered all ChIRP and ChIP peaks that fell within ±500 base pairs of ATAC-seq peaks, and then calculated the overlap among the ChIRP and ChIP sets, using the hypergeometric distribution to estimate significance. For in vitro binding assays, pcDNA3.1-Rmrp vectors were used for T7 polymerase-driven in vitro transcription (IVT) reactions (Promega). Haemagglutinin (HA)–DDX5 and Flag–RORγt were in vitro transcribed and translated using an in vitro transcription and translation (TNT) system according to the manufacturer’s protocol (Promega). Alternatively, pGEX4.1-DDX5 (wild-type and helicase-dead mutant) constructs were transformed into BL21 to synthesize recombinant full-length GST–hDDX5 proteins. Full-length His-tagged human RORγt was purified in three steps through Ni-resin, S column and gel-filtration (AKTA). Then, 0.5 μg of each recombinant protein was incubated in the presence or absence of 200 μM ATP, 300 ng in vitro transcribed Rmrp in co-immunoprecipitation buffer containing 25 mM Tris (pH 8.0), 100 mM NaCl, 0.5% NP-40, 10 mM MgCl , 10% glycerol, 1× protease inhibitor, RNaseInhibitor (Invitrogen) and PhosphoSTOP (Roche). GST–DDX5 was enriched on glutathione beads (GE); HA–DDX5, Flag–RORγt and His–RORγt were enriched using anti-HA (Covance), anti-Flag (Sigma) and anti-His antibodies (Santa Cruz Bio) coupled to anti-mouse immunoglobulin dynabeads (Dynal, Invitrogen). T 17 cells were cultured on glass coverslips for 48 h and fixed in 4% paraformaldehyde in PBS for 5 min at room temperature. Fixed cells were permeabilized with 0.1% bovine serum albumin (BSA), 0.1% Triton and 10% normal serum in PBS for 1 h. Cells were then incubated with primary antibodies (DDX5 (Abcam) or RORγt (eBiosciences)) in 0.1% BSA and 0.2% Triton PBS overnight at 4 °C. Secondary antibodies (anti-goat Alexa 488 or anti-rat Alexa 647 (Molecular Probe)) were incubated at 4 °C for 1 h. Stained cells were washed three times with 0.5% Tween and 0.1% BSA in PBS. DAPI was used to stain DNA inside the nucleus. Immunofluorescence images were captured on a Zeiss 510 microscope at 40×. T 17-polarized cells were crosslinked with 1% paraformaldehyde (EMS) and incubated with rotation at room temperature. Crosslinking was stopped after 10 min with glycine to a final concentration of 0.125 M and incubated for a further 5 min with rotation. Cells were washed with 3× ice-cold PBS and pellets were either flash-frozen in liquid N or immediately resuspended in Farnham lysis buffer (5 mM PIPES, 85 mM KCl, 0.5% NP-40). Hypotonic lysis continued for 10 min on ice before cells were spun down and resuspended in RIPA buffer (1× PBS, 1% NP-40, 0.5% SDS, 0.5% Na-deoxycholate), transferred into TPX microtubes and lysed on ice for 30 min. Nuclear lysates were sonicated for 40 cycles of 30 s ‘ON’ and 30 s ‘OFF’ in 10-cycle increments using a Biorupter (Diadenode) set on high. After pelleting debris, chromatin was precleared with protein G dynabeads (dynabeads, TFS) for 2 h with rotation at 4 °C. For immunoprecipitation, precleared chromatin was incubated with anti-RORγt antibodies (1 μg per 2 × 106 cells) overnight with rotation at 4 °C and protein G was added for the final 2 h of incubation. Beads were washed and bound chromatin was eluted. ChIP-qPCR was performed on DNA purified after treatment with RNase (30 min, 37 °C) and proteinase K (2 h, 55 °C) followed by reversal of crosslinks (8–12 h, 65 °C). The primers used for qPCR have been described previously5. For analysis of mRNA transcripts, gene specific values were normalized to the Gapdh housekeeping gene for each sample. All primer sequences are listed in Supplementary Table 1.

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