Pathogen infection and cholesterol deficiency activate the C. elegans p38 immune pathway through a TIR-1/SARM1 phase transition
- Program in Innate Immunity, Division of Infectious Diseases and Immunology, University of Massachusetts Chan Medical School, United States
- Program in Chemical Biology, University of Massachusetts Chan Medical School, United States
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, United States
Figures
Figure 1 with 1 supplement
Multimerization of TIR-1/SARM1 and its intrinsic NAD+ glycohydrolase activity are required for activation of the p38 PMK-1 innate immune pathway during pathogen infection.
(A) Images of animals expressing TIR-1::wrmScarlet in the indicated conditions. All tir-1::wrmScarlet animals were treated with glo-3(RNAi) to deplete autofluorescent gut granules. Representative …
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Figure 1—source data 1
Figure 1B Quantification of the number of TIR-1::wrmScarlet puncta present in the last posterior pair of intestinal epithelial cells.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig1-data1-v3.xlsx
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Figure 1—source data 2
Figure 1F Quantification of p38 immunoblot analysis of lysates from the indicated genotypes.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig1-data2-v3.xlsx
Figure 1—figure supplement 1
Multimerization of TIR-1/SARM1 and its intrinsic NAD + glycohydrolase activity are required for activation of the p38 PMK-1 innate immune pathway during pathogen infection.
(A) Immunoblot analysis of lysates from the indicated genotypes probed with an antibody that recognizes the FLAG epitope. (B) Images of T24B8.5p::gfp immune reporter expression in the indicated …
Figure 2 with 2 supplements
TIR multimerization and phase transition superactivates its intrinsic NAD + glycohydrolase activity.
(A) NADase activity of purified TIR at increasing TIR protein concentrations is shown. Activity was assessed by incubating TIR protein with 1 mM ε-NAD and monitoring the rate at which the …
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Figure 2—source data 1
Figure 2A NADase activity of purified TIR at increasing TIR protein concentrations.
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Figure 2—source data 2
Figure 2B NADase activity of 2.5 μM TIR incubated in the presence of 25% (w/v) of macro- (PEG 8000, PEG 3350, and dextran) and micro- (sucrose and glycerol) viscogens.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig2-data2-v3.xlsx
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Figure 2—source data 3
Figure 2C Dose dependency of macroviscogens on the NADase activity of TIR.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig2-data3-v3.xlsx
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Figure 2—source data 4
Figure 2D Steady-state kinetic analysis of 2.5 μM TIR incubated in 0%–30% (w/v) of PEG 3350.
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Figure 2—source data 5
Figure 2H NADase activity of TIR protein in each fraction and at each concentration of PEG 3350.
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Figure 2—source data 6
Figure 2I Steady-state kinetic analysis of TIR wild-type, oligomerization mutant (TIRG747P, TIRH833A), and catalytic mutants (TIRE788Q and TIRE788A) in 25% PEG 3350.
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Figure 2—source data 7
Figure 2J Quantification of SDS-PAGE analysis of TIR: wild-type, oligomerization mutant (TIRG747P) and catalytic mutant (TIRE788Q), precipitation in the presence of 25% PEG 3350.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig2-data7-v3.xlsx
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Figure 2—source data 8
Figure 2K Quantification of SDS-PAGE analysis of TIR: wild-type and catalytic mutant (TIRE788A), precipitation in the presence of 25% PEG 3350.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig2-data8-v3.xlsx
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Figure 2—source data 9
Figure 2L Effect of 1,6-hexanediol on TIR NADase activity.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig2-data9-v3.xlsx
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Figure 2—source data 10
Figure 2M NADase activity of TIR protein incubated with either 25% PEG 3350 or 500 mM citrate before (precentrifugation) and after centrifugation, the supernatant and precipitant fractions.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig2-data10-v3.xlsx
Figure 2—figure supplement 1
TIR multimerization and phase transition superactivates its intrinsic NAD + glycohydrolase activity.
(A) Enzyme concentration dependence in the presence and absence of either 25 % PEG 3350 or 500 mM sodium citrate (n = 2). (B) Dose-dependence of TIR NADase activity on citrate concentration as …
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Figure 2—figure supplement 1—source data 1
1A enzyme concentration dependence in presence and absence of either 25% PEG 3350 or 500 mM sodium citrate.
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Figure 2—figure supplement 1—source data 2
1B Dose dependency of citrate on the NADase activity of TIR.
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Figure 2—figure supplement 1—source data 3
1C Steady-state kinetic analysis of 2.5 μM TIR incubated in 0–1000 mM sodium citrate.
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Figure 2—figure supplement 1—source data 4
1G NADase activity of TIR protein in each fraction and at each concentration of citrate.
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Figure 2—figure supplement 1—source data 5
1H Steady-state kinetic analysis of TIR wild-type, oligomerization mutant (TIRG747P, TIRH833A), and catalytic mutants (TIRE788Q and TIRE788A) in 500 mM sodium citrate.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig2-figsupp1-data5-v3.xlsx
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Figure 2—figure supplement 1—source data 6
1I Quantification of SDS-PAGE analysis of TIR: wild-type, oligomerization mutant (TIRG747P) and catalytic mutant (TIRE788Q), precipitation in the presence of 500 mM sodium citrate.
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Figure 2—figure supplement 1—source data 7
1J Quantification of SDS-PAGE analysis of TIR: wild-type and catalytic mutant (TIRE788A), precipitation in the presence of 500 mM sodium citrate.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig2-figsupp1-data7-v3.xlsx
Figure 2—figure supplement 2
TIR multimerization and phase transition superactivates its intrinsic NAD + glycohydrolase activity.
(A) SDS-PAGE analysis of TIR protein incubated first with 25% PEG 3350 or 500 mM citrate and subsequently with 2% 1,6-hexanediol; analysis was performed on TIR samples taken precentrifugation (C) or …
Figure 3 with 1 supplement
Cholesterol scarcity activates intestinal innate immune defenses.
Images of T24B8.5p::gfp (A) and irg-5p::GFP (B) transcriptional immune reporters in wild-type animals grown on standard nematode growth media ( + 5 μg/mL cholesterol) and in the absence of …
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Figure 3—source data 1
Figure 3C qRT-PCR data of the indicated innate immune effector genes in wild-type C. elegans growing in the presence (+5 μg/mL) and absence (+0 μg/mL) of supplemented cholesterol.
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Figure 3—source data 2
Figure 3I qRT-PCR data of T24B8.5 in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media (+5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig3-data2-v3.xlsx
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Figure 3—source data 3
Figure 3J qRT-PCR data of irg-4 in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media (+5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated.
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Figure 3—source data 4
Figure 3K qRT-PCR data of irg-5 in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media (+5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated.
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Figure 3—source data 5
Figure 3L qRT-PCR data of K08D8.4 in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media (+5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated.
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Figure 3—source data 6
Figure 3O Colony-forming units (CFUs) of P. aeruginosa, isolated from the intestines of animals with the indicated genotypes, quantified after 24 hr of bacterial infection.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig3-data6-v3.xlsx
Figure 3—figure supplement 1
Cholesterol scarcity activates intestinal innate immune defenses.
Figure 4 with 1 supplement
Cholesterol scarcity activates the p38 PMK-1 innate immune pathway.
(A) Gene set enrichment analysis (GSEA) of p38 PMK-1 targets in the 0 μg/mL cholesterol mRNA-seq experiment. Fold change in expression of significantly differentially expressed genes (fold-change > …
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Figure 4—source data 1
Figure 4C Quantification of p38 immunoblot analysis of lysates from wild-type C. elegans grown on standard nematode growth media in the presence ( + 5 μg/mL cholesterol) and in the absence ( + 0 μg/mL cholesterol) of supplemented cholesterol.
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Figure 4—source data 2
Figure 4F Quantification of p38 immunoblot analysis of lysates from the indicated C. elegans strains.
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Figure 4—source data 3
Figure 4H qRT-PCR data of T24B8.5 in the indicated mutant animals grown on standard nematode growth media ( + 5 μg/mL cholesterol).
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Figure 4—source data 4
Figure 4I qRT-PCR data of irg-4 in the indicated mutant animals grown on standard nematode growth media ( + 5 μg/mL cholesterol).
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig4-data4-v3.xlsx
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Figure 4—source data 5
Figure 4J qRT-PCR data of irg-5 in the indicated mutant animals grown on standard nematode growth media ( + 5 μg/mL cholesterol).
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig4-data5-v3.xlsx
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Figure 4—source data 6
Figure 4K qRT-PCR data of K08D8.4 in the indicated mutant animals grown on standard nematode growth media ( + 5 μg/mL cholesterol).
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig4-data6-v3.xlsx
Figure 4—figure supplement 1
Cholesterol scarcity activates the p38 PMK-1 innate immune pathway.
(A) Gene set enrichment analysis (GSEA) of p38 PMK-1 targets in the mRNA-seq experiment as described in Figure 4D, except using a different nhr-8 mutant allele: nhr-8(ok186). (B) Images of …
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Figure 4—figure supplement 1—source data 1
1C Quantification of T24B8.5p::gfp expression in the indicated conditions.
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Figure 5
Sterol stress activates the C. elegans p38 immune pathway through the multimerization and NAD + glycohydrolase activity of TIR-1/SARM1.
(A) Images of tir-1::wrmScarlet and nhr-8(hd117);tir-1::wrmScarlet animals as described in Figure 1A exposed to either 0.1% Tergitol or tir-1(RNAi). Scale bar equals 20 μm (2 µm for the inset …
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Figure 5—source data 1
Figure 5B Quantification of the number of TIR-1::wrmScarlet puncta present in the last posterior pair of intestinal epithelial cells in the indicated strains and conditions.
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Figure 5—source data 2
Figure 5E qRT-PCR data of T24B8.5 in wild-type and mutant animals of the indicated genotypes grown on standard nematode growth media ( + 5 μg/mL cholesterol).
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Figure 5—source data 3
Figure 5F qRT-PCR data of irg-4 in wild-type and mutant animals of the indicated genotypes grown on standard nematode growth media ( + 5 μg/mL cholesterol).
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Figure 6
Sterol scarcity primes p38 PMK-1 immune defenses.
(A, B, C, D, E) Images of the indicated transcriptional immune reporters under the indicated conditions. Scale bars in all images equal 200 μm. (F) A heat map compares the expression levels of the …
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Figure 6—source data 1
Figure 6H mRNA-seq data for T24B8.5 from the conditions indicated showing scaled reads per base.
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Figure 6—source data 2
Figure 6I mRNA-seq data for irg-4 from the conditions indicated showing scaled reads per base.
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Figure 6—source data 3
Figure 6J mRNA-seq data for irg-5 from the conditions indicated showing scaled reads per base.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig6-data3-v3.xlsx
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Figure 6—source data 4
Figure 6K mRNA-seq data for K08D8.4 from the conditions indicated showing scaled reads per base.
- https://cdn.elifesciences.org/articles/74206/elife-74206-fig6-data4-v3.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | OP50 | CaenorhabditisGenetics Center | ||
| Strain, strain background (Escherichia coli) | HT115 | CaenorhabditisGenetics Center | ||
| Strain, strain background (Escherichia coli) | BL21 (DE3) | ThermoFisher Scientific | EC0114 | Chemically competent |
| Strain, strain background (Escherichia coli) | XL1-Blue | Agilent | 200,249 | Chemically competent |
| Strain, strain background (Pseudomonas aeruginosa) | UCBPP-PA14 | PMID:7604262 | ||
| Strain, strain background (Caenorhabditis elegans) | N2; wild-type | CGC, PMID:4366476 | WormBase ID:WBStrain00000001 | Laboratory reference strain/wild type |
| Strain, strain background (Caenorhabditis elegans) | KU25 | PMID:15116070/ | WormBase ID: WBStrain00024040 | Genotype: pmk-1(km25) IV |
| Strain, strain background (Caenorhabditis elegans) | AU3 | PMID:12142542 | WormBase ID: WBStrain00000259 | Genotype: nsy-1(ag3) II |
| Strain, strain background (Caenorhabditis elegans) | ZD101 | PMID:19837372 | WormBase ID:WBStrain00040806 | Genotype: tir-1(qd4) III |
| Strain, strain background (Caenorhabditis elegans) | AA968 | PMID:23931753 | Genotype: nhr-8(hd117) IV | |
| Strain, strain background (Caenorhabditis elegans) | AE501 | PMID:11516648 | WormBase ID:WBStrain00000059 | Genotype: nhr-8(ok186) IV |
| Strain, strain background (Caenorhadbditis elegans) | AU78 | PMID:19837372 | WormBase ID:WBStrain00000262 | Genotype: agIs219 [T24B8.5p::gfp::unc-54–3’UTR; ttx-3p::gfp::unc-54–3’UTR] III |
| Strain, strain background (Caenorhabditis elegans) | AU307 | PMID:24875643 | Genotype: agIs44 [irg-4p::gfp::unc-54–3’UTR; myo-2p::mCherry] | |
| Strain, strain background (Caenorhabditis elegans) | AY101 | PMID:20133945 | WormBase ID:WBStrain00000322 | Genotype: acIs101 [pDB09.1(irg-5p::gfp); pRF4(rol-6(su1006))] |
| Strain, strain background (Caenorhabditis elegans) | TJ356 | PMID:11747825 | WormBase ID:WBStrain00034892 | Genotype: zIs356 [daf-16p::daf-16a/b::gfp + pRF4(rol-6(su1006))] |
| Strain, strain background (Caenorhabditis elegans) | RPW278 | This study | Genotype: nhr-8(hd117);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW317 | This study | Genotype: tir-1(qd4);nhr-8(hd117) | |
| Strain, strain background (Caenorhabditis elegans) | RPW339 | This study | Genotype: tir-1(ums47[E788A]);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW369 | This study | Genotype: tir-1(ums54[ΔSAM]);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW374 | This study | Genotype: tir-1(ums55[G747P]);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW381 | This study | Genotype: tir-1(ums56[H833A]);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW386 | This study | Genotype: tir-1(ums57[tir-1::3xFLAG]);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW387 | This study | Genotype: tir-1(ums58[tir-1[E788A]::3xFLAG]);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW388 | This study | Genotype: tir-1 (ums59[tir-1[ΔSAM]::3xFLAG]);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW 389 | This study | Genotype: tir-1(ums60[tir-1[G747P]::3xFLAG]);agIs219 | |
| Strain, strain background (Caenorhabditis elegans) | RPW403 | This study | Genotype: tir-1(ums63[tir-1::wrmScarlet]) | |
| Strain, strain background (Caenorhabditis elegans) | RPW404 | This study | Genotype: nhr-8(hd117);tir-1(ums63[tir-1::wrmScarlet]) | |
| Antibody | anti-total PMK-1(rabbit polyclonal) | PMID:30668573 | WB(1:1000) | |
| Antibody | anti-Phospho-p38 MAPK (Thr180/Tyr182)(rabbit polyclonal) | Cell Signaling Technology | 9211 | WB(1:1,000) |
| Antibody | anti-FLAG(mouse monoclonal) | Sigma-Aldrich | F1804 | WB(1:1,000) |
| Antibody | anti-alpha-tubulin (mouse monoclonal) | Sigma-Aldrich | T5168 | WB(1:2,000) |
| Antibody | anti-mouse IgG-HRP (goat polyclonal) | Abcam | ab6789 | WB(1:10,000) |
| Antibody | anti-rabbit IgG-HRP(goat polyclonal) | Cell Signaling Technology | 7074 | WB(1:10,000) |
| Chemical compound, drug | Peptone | Gibco | 211820 | |
| Chemical compound, drug | Agar | Fisher | BP9744 | |
| Chemical compound, drug | Cholesterol | Sigma Aldrich | C3045 | |
| Chemical compound, drug | Tri-Reagent | Sigma Aldrich | T9424 | |
| Chemical compound, drug | TERGITOL solution (Type NP-40) | Sigma Aldrich | NP40S | |
| Chemical compound, drug | RIPA Buffer | Cell Signaling Technology, Inc | 89900 | |
| Chemical compound, drug | Halt Protease and Phosphatase inhibitor | ThermoFisher Scientific | 78445 | |
| Chemical compound, drug | NuPAGELDS sample buffer | ThermoFisher Scientific | NP0007 | |
| Chemical compound, drug | 1,6-hexanediol | Sigma Aldrich | 240117 | |
| Chemical compound, drug | Nicotinamide 1,N6-ethenoadenine dinucleotide; ε-NAD | Sigma Aldrich | N2630 | |
| Chemical compound, drug | PEG 400 | Sigma Aldrich | 91893 | |
| Chemical compound, drug | PEG 1500 | Sigma Aldrich | 86101 | |
| Chemical compound, drug | PEG 3350 | Sigma Aldrich | 88276 | |
| Chemical compound, drug | PEG 8000 | Sigma Aldrich | 89510 | |
| Chemical compound, drug | Dextran | Fisher Scientific | ICN16011010 | |
| Chemical compound, drug | Sucrose | Sigma Aldrich | S0389 | |
| Chemical compound, drug | Glycerol | Sigma Aldrich | G5516 | |
| Chemical compound, drug | Sodium citrate | Sigma Aldrich | S4641 | |
| Chemical compound, drug | Kanamycin | Research Products International | K22000 | |
| Chemical compound, drug | IPTG | ThermoFisher Scientific | R0392 | |
| Chemical compound, drug | Pierce EDTA-free protease inhibitor mini tablets | ThermoFisher Scientific | A32955 | |
| Commercial assay or kit | iProof High-Fidelity DNA Polymerase | Bio-Rad Laboratories, Inc | 172–5301 | |
| Commercial assay or kit | iScript gDNA Clear cDNA Synthesis Kit | Bio-Rad Laboratories, Inc | 172–5034 | |
| Commercial assay or kit | iTaq Universal SYBR Green Supermix | Bio-Rad Laboratories, Inc | 1725120 | |
| Commercial assay or kit | DreamTaq Green PCR | ThermoFisher Scientific | K1081 | |
| Commercial assay or kit | DC protein assay | Bio-Rad Laboratories, Inc | 5000116 | |
| Commercial assay or kit | NuPAGE 4%–12% BisTris gels | ThermoFisher Scientific | NP0321BOX | |
| Commercial assay or kit | NuPAGE 3%–8% TrisAcetate gels | ThermoFisher Scientific | EA0375BOX | |
| Commercial assay or kit | SuperSignal West Pico PLUS Chemiluminescent Substrate | Thermo Fisher Scientific | 34577 | |
| Commercial assay or kit | SuperSignal West Femto PLUS Chemiluminescent Substrate | Thermo Fisher Scientific | 34095 | |
| Commercial assay or kit | Strep-Tactin XT Superflow high-capacity resin | IBA Lifesciences | 2-4030-025 | Product discontinued; suitable replacement is Strep-Tactin XT 4Flow high capacity resin (2-5030-025) |
| Commercial assay or kit | Wizard Plus SV Minipreps DNA Purification System | Promega | A1460 | |
| Commercial assay or kit | TALON Metal Affinity Resin | Takara | 635,502 | |
| Peptide, recombinant protein | Lysozyme | Sigma Aldrich | L6876 | |
| Peptide, recombinant protein | DpnI | NEB | R0176S | |
| Peptide, recombinant protein | ADP-ribosyl cyclase | Sigma Aldrich | A9106 | |
| Peptide, recombinant protein | SpCas9 Nuclease | IDT | 1081058 | |
| Recombinant DNA reagent | pET-30a(+) TIR (plasmid) | Loring et al., 2021 | Referred to as pET30a+ Strep-ceTIR-HIS in reference | |
| Software, algorithm | Fiji/imageJ | PMID:22743772 | ||
| Software, algorithm | OASIS 2 | PMID:27528229 | ||
| Software, algorithm | R Console (Version 3.5) | The R Foundation | https://www.r-project.org/ | |
| Software, algorithm | FastQC (Version 0.11.5) | https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ | ||
| Software, algorithm | Kallisto (version 0.45.0) | PMID:27043002 | ||
| Software, algorithm | Sleuth (version 0.30.0) | PMID:28581496 | ||
| Software, algorithm | GSEA (version 4.1.0) | PMID:16199517 | ||
| Software, algorithm | pheatmap (version 1.0.12) | https://cran.r-project.org/web/packages/pheatmap/index.html | ||
| Software, algorithm | DAVID Bioinformatics database | PMID:19131956 | ||
| Software, algorithm | GraphPad Prism 9 | Graphpad | https://www.graphpad.com/scientific-software/prism/ |
Additional files
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Supplementary file 1
Genes significantly differentially expressed in uninfected nhr-8(hd117) mutants compared to wild-type animals in the RNA-seq experiments presented in Figure 3D and Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/74206/elife-74206-supp1-v3.xlsx
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Supplementary file 2
Genes significantly differentially expressed in uninfected nhr-8(ok186) mutants compared to wild-type animals in the RNA-seq experiments presented in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/74206/elife-74206-supp2-v3.xlsx
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Supplementary file 3
Genes significantly differentially expressed in uninfected wild-type animals in the absence (0 μg/mL) versus presence (5 μg/mL) of cholesterol supplementation in the RNA-seq experiments presented in Figure 3E and Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/74206/elife-74206-supp3-v3.xlsx
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Supplementary file 4
Sample sizes, mean lifespan, and p values for the C. elegans pathogenesis assays.
- https://cdn.elifesciences.org/articles/74206/elife-74206-supp4-v3.xlsx
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Supplementary file 5
Genes in Cluster one and Cluster 2 of the heat map shown in Figure 6F.
- https://cdn.elifesciences.org/articles/74206/elife-74206-supp5-v3.xlsx
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Supplementary file 6
Primer, crRNA guide and ssODN sequences designed for this study.
- https://cdn.elifesciences.org/articles/74206/elife-74206-supp6-v3.xlsx
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Supplementary file 7
pmk-1 dependent genes used for GSEA analysis.
- https://cdn.elifesciences.org/articles/74206/elife-74206-supp7-v3.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/74206/elife-74206-transrepform1-v3.pdf
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Source data 1
Raw and annotated gel and blot images 2 of 2.
- https://cdn.elifesciences.org/articles/74206/elife-74206-data1-v3.zip
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Source data 2
Raw and annotated gel and blot images 1 of 2.
- https://cdn.elifesciences.org/articles/74206/elife-74206-data2-v3.zip
