Supporting InformationZou et al. 10.1073/pnas.1405032111SI Materials and MethodsConstruction of Transgenic Strains. Mutagenesis of Thr669 of cdc-48.2 to alanine or glutamic acid residue was performed by theChameleon Site Directed Mutagenesis Kit (Stratagene) usingcdc-48.2 cDNA as template. Mutagenized fragments were con-firmed by DNA sequencing. The vectors Pcdc-48.2::cdc-48.2(Thr669), Pcdc-48.2::cdc-48.2(Ala669), and Pcdc-48.2::cdc-48.2(Glu669) was generated by subcloning a 1.9-kb promoter frag-ment of cdc-48.2 (corresponding to nucleotide −1919 to −1 rela-tive to the translational start site) into an expression vector(pPD95.75) in which one of cdc-48.2 cDNA (Thr669, Ala669, andGlu669) was fused at its 3′ end in-frame with GFP and the3′-UTR of unc-54. The three constructs were coinjected with themarker plasmid pRF4 containing rol-6(su1006) into gonads of cdc-48.2(tm659), ormpk-1(n2521)mutants by standard techniques (1),respectively. The transgenic worms were confirmed before assay.

RNA Interference. The clones of genes for RNAi were from theAhringer library (2). RNAi feeding experiments were performedon synchronized L1 larvae at 20 °C for 40 h.

Infection with Bacteria. Synchronized populations of worms werecultivated at 20 °C until the young adult stage (i.e., within 12 hbeyond the L4 stage). For all pathogen assays, 75 μg/mL 5′-fluoro-2′-deoxyuridine was added to the assay plates to abolish the growthof progeny. Pseudomonas aeruginosa strain PA14 (a gift fromK. Zhu, Institute of Microbiology, Chinese Academy of Sciences,Beijing), then seeded on slow-killing plates as described (3, 4).Caenorhabditis elegans killing assays were prepared by spreadinga 100-μL drop of P. aeruginosa on the nematode growth media(NGM) agar in 60-mm diameter Petri plates. Pathogenic bacteriawere incubated first for 24 h at 37 °C and then for 24 h at 25 °C.Fifty to 60 young adult worms were transferred to the pathogenplates at 25 °C. The number of living worms was counted at 12-hintervals. Immobile worms unresponsive to touch were scored asdead. Three plates of approximately 50–60 animals per plate weretested per assay, and all experiments were performed three timesindependently.

Autophagy Analysis. After 12 h of bacterial infection, the trans-genic worms carrying GFP::LGG-1 were immediately mounted inM9 onto microscope slides. The slides were viewed by using aZeiss Axioskop 2 plus fluorescence microscope (Carl Zeiss) witha digital camera. GFP::LGG-1–positive puncta were counted inthe seam cells (5) or the intestinal cells (6). Three plates of eachgenotype were performed per assay, and all experiments wereperformed three times independently.

Detection of Bacterial Accumulation. Colony forming units (CFU)in the intestine of worms were determined according to themethod described (7), with some modifications. Briefly, synchro-nized young adult worms were exposed to plates seeded withP. aeruginosa PA14 expressing GFP (a gift from K. Zhu) for 24 hat 25 °C. Then worms were transferred to an NGM plate seededwith E. coli OP50 for 15 min to eliminate P. aeruginosa stuck tothe body of the worms for three times. Fifty nematodes per con-dition were transferred into 50 μL of PBS plus 0.1% Triton X-100and ground. The lysates were serially diluted by 10-fold in steril-ized water and spread over Luria–Bertani (LB) agar plates. Af-ter 1 d of incubation at 37 °C, colonies of P. aeruginosa/GFPwere counted.

Western Blotting. After worms were homogenized in liquid ni-trogen, the homogenate was lysed on ice for 30 min in lysis buffer(BioTeKe). The lysates (50 μg) of total protein were loaded perwell and separated by SDS-PAGE using 10% (wt/vol) poly-acrylamide gels. Proteins were then transferred to immobilon-PSQ transfer PVDFmembrane (Millipore). Primary antibodiesweremonoclonal MAPKYT antibody for detection of activated MPK-1(1:400 dilution; Sigma), anti-GFP antibodies (1:3000 dilution; Af-finity Biosciences), and anti-actin antibodies (1:400 dilution; Sigma).The secondary antibody was a peroxidase-coupled anti-rabbit ormouse IgG (1:5000 dilution; Abmart). The membrane was exposedto Kodak X-Omat film (Kodak), and the film was developed.

Quantitative Real-Time PCR. Total RNA was isolated from youngadult worms with TRIzol Reagent (Invitrogen). Random-primedcDNAs were generated by reverse transcription of the total RNAsamples with SuperScript II (Invitrogen). A real-time PCR analysiswas conducted by using the Roche LightCycler 480System (RocheApplied Science) using SYBRPremix-Ex TagTM GC (Takara).The primers used for PCR were as follows: lin-3: 5′- TCA ATGACG ACG ACA CGC -3′ (F), 5′- CTC CAG AAC TTC GAGACGG -3′; bec-1: 5′- GAT TGCTCTGACGCTCTTCG-3′ (F),5′- GCT CCT TCT CCT CAT CGG AAA-3′(R); asp-4: 5′- GGAATC ACC TTT GTC GCT GC -3′(F), 5′-AGA GTC TGG GTTACG GTT GAG-3′(R); clp-1: 5′-GCT GTC GCT GGA AATGTG ATT -3′ (F), 5′- GTC TTG TGG TTC GGT AGT TGG -3′(R); act-1: 5′-GGG CGA AGA AGG AAA TGG TC-3′ (F), 5′-CAG GTG GCG TAG GTG GAG AA -3′ (R).

Lifespan Analysis. All lifespan measurements were performed onNGM agar plates of heat-killed E. coli OP50 or P. aeruginosa PA14at 20 °C, starting with day 1 adults (8). Worms were transferred tonew plates during each day of their reproductive period and afterthat were transferred every third day. Lifespan was monitored everyday. Animals that did not move when gently prodded and displayedno pharyngeal pumping were marked as dead.

Microscopy Analysis for Necrosis. After young adults were infectedwith P. aeruginosa PA14 for 12 h, the worms were stained in M9medium containing 1 mM acridine orange (Sangon Biotech) (9),20 mg/mL uranine (Sangon) (10), or 20 μM (Z-Phe-Arg) 2-R110[(Z-FR) 2-R110; Invitrogen] (9) for 2 h. After washing with M9 me-dium for three times, the worms were mounted in M9 onto micro-scope slides. The red fluorescence of acridine orange and the greenfluorescence of uranine and (Z-FR)2-R110 were monitored by usinga Nikon e800 fluorescence microscope (Nikon). For intestinal de-generation assay, worms exposed toP. aeruginosaPA14 for 12hwerestained with DAPI diluted in PBS (final concentration of 10 μg/mL)for 30 min. Then the intestinal nuclei were visualized and counted.

Detection of Apoptosis. After synchronized mid-L4 worms werestarved for 48 h, the worms were stained in M9 medium containing20 μMSYTO12 green (Invitrogen) for 90min (11). Then the wormsweremounted inM9ontomicroscope slides. The green fluorescencewas monitored by using a Nikon e800 fluorescence microscope.

Statistics.The results presented in each figure are the average of threeindependent experiments. Differences in survival rates were analyzedby using the log-rank test. The statistical significance of differencesin gene expression, the numbers of GFP::LGG-1 puncta and DAPI-stained cells, and fluorescence intensity was assessed by performinga one-way ANOVA followed by a Student–Newman–Keuls test.Data were analyzed by using SPSS11.0 software (SPSS Inc.).

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required for the Caenorhabditis elegans innate immune response. Proc Natl Acad SciUSA 106(8):2782–2787.

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Fig. S1. Autophagy induced by P. aeruginosa is not due to nutrient limitation. (A and B) Representative images of autophagosomes (GFP::LGG-1 puncta) inthe seam cells and intestinal cells of worms exposed to OP50, PA14, a mixture of OP50 and PA14, or heat-killed (HK) PA14, for 12 h. The arrow denotesa representative autophagosome. The numbers of GFP::LGG-1 puncta in the seam cells and intestinal cells were counted (Right). These results are mean ± SD ofthree independent experiments performed in triplicate. **P < 0.01 versus OP50 alone. (C) The expression of Pgst-4::gfp was only up-regulated in worms after12 h starvation. Right shows quantification of GFP levels. **P < 0.01 versus OP50 alone. (Scale bars: A, 10 μm; B, 20 μm; C, 100 μm.)

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Fig. S2. Inhibition or activation of the ERK pathway affects autophagy and survival of worms after P. aeruginosa PA14 infection. (A and B) U0126 (25 μM)suppressed autophagy in wild-type (WT) worms after P. aeruginosa PA14 infection. (A) Seam cells. (B) Intestinal cells. **P < 0.01 versus control. (C) A gain-of-function mutation let-60(n1046) resulted in enhanced resistance to the killing by PA14. P < 0.05 versus WT. (D) ksr-1(ku68) mutants exhibited enhancedsusceptibility, whereas lip-1(zh15) mutants exhibited enhanced resistance to P. aeruginosa PA14 infection. P < 0.001 versus WT. (E and F) The numbers of GFP::LGG-1 puncta in the seam cells (E) and intestinal cells (F) were counted in ksr-1(ku68), or lip-1(zh15) mutants. *P < 0.05; **P < 0.01 versus WT.

Fig. S3. The ERK signaling in the intestine is required for pathogen resistance. (A) Intestinal-specific RNAi of let-60, lin-45, mek-2, and mpk-1 significantlyreduced the survival of worms after P. aeruginosa PA14 infection. P < 0.01 versus WT+empty vector (EV). (B and C) Epidermal- (B) or muscular- (C) specificknockdown of these genes did not influence the survival of worms after P. aeruginosa PA14 infection.

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Fig. S4. CDC-48.2-mediated pathogen resistance depends on autophagy. (A) RNAi of cdc-48.1 or cdc-48.2 significantly reduced autophagy in worms exposedto P. aeruginosa PA14. **P < 0.01 versus EV (empty vector). (B) A mutation in cdc-48.2(tm659), but not cdc-48.1(tm544), led to enhanced sensitivity toP. aeruginosa PA14 infection. P < 0.01 versus WT. (C) PA14 infection did not result in the aggregation of CDC48.2-GFP in the intestine. (Scale bars: 20 μm.) (D)Knockdown of bec-1 by RNAi suppressed GFP::LGG-1 puncta in cdc-48.2(tm659) worms expressing Pcdc-48.2::cdc-48.2(Glu669). **P < 0.01 versus EV. (E) bec-1RNAi significantly reduced the survival of cdc-48.2(tm659) worms expressing Pcdc-48.2::cdc-48.2(Glu669) after PA14 infection. P < 0.01 versus EV.

Fig. S5. Mutations in the components of the ERK signaling shorten lifespan. Worms were grown on plates containing heat-killed E. coli OP50 (A) andP. aeruginosa PA14 (B). P < 0.001 versus WT.

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Fig. S6. Necrosis is not detected inmpk-1(n2521)mutants, or worms subjected to bec-1 RNAi grown on E. coli OP50. (A) DIC images of worms and fluorescencemicroscopy of uranine-labeled intestine of worms are shown after P. aeruginosa PA14 infection. (B) DIC images and fluorescence microscopy of acridineorange-labeled intestine in worms grown on E. coli OP50. (C) DAPI-stained nuclei of intestine in worms grown on E. coli OP50. Lower shows quantification ofnuclei. (D and E) Knockdown of asp-4 and clp-1 by RNAi did not influence the survival of WT worms (D) and pmk-1(km25) mutants (E) after P. aeruginosa PA14infection. (F) Double RNAi of bec-1 and asp-4 or double RNAi of bec-1 and clp-1 down-regulated the expression of bec-1 and asp-4 or bec-1 and clp-1. **P <0.01 versus WT+EV (empty vector). (Scale bars: 10 μm.)

Fig. S7. P. aeruginosa PA14 infection does not induce apoptosis in the germ line. (A) Fluorescence microscopy of SYTO 12-labeled germ line of wild-type (WT)and mpk-1(n2521) mutants are shown after P. aeruginosa PA14 infection. However, germ-line apoptosis was not observed. (Scale bars: 10 μm.) (B) Knockdownof ced-4 by RNAi did not affect the immune-deficient phenotype of mpk-1(n2521) mutants after PA14 infection.

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Table S1. The substrates of MPK-1 are involved in innateimmunity to P. aeruginosa

RNAi TD50, h (mean ± SEM, n = 3) P value

Vector 59.8 ± 4.5 —

mtr-4 57.2 ± 3.5 —

ddx-19 57.9 ± 3.3 —

rha-2 58.9 ± 4.3 —

cgh-1 59.9 ± 4.0 —

hel-1 48.1 ± 3.3 <0.01hrd-1 42.0 ± 3.9 <0.01toe-3 59.3 ± 2.0 —

toe-4 59.2 ± 4.5 —

cdc-48.2 40.1 ± 5.8 <0.01rskn-1 42.9 ± 4.4 <0.001cdtl-7 56.2 ± 4.5 —

ttbk-2 58.2 ± 4.1 —

rskd-1 56.2 ± 3.8 —

toe-1 60.2 ± 3.7 —

C30G12.6 53.2 ± 3.5 —

par-5/ftt-1 60.3 ± 4.0 —

pac-1 63.2 ± 5.7 —

toe-2 55.2 ± 5.3 —

toe-5 61.2 ± 4.9 —

cya-1 58.2 ± 5.3 —

pole-2 76.2 ± 8.8 —

mrps-5 60.6 ± 4.0 —

mrs-1 62.3 ± 5.2 —

rps-8 89.2 ± 5.3 >0.001irs-1 56.8 ± 2.6 —

zim-2 57.2 ± 4.8 —

dis-3 57.9 ± 4.5 —

rop-1 58.3 ± 2.3 —

gsk-3 65.3 ± 3.3 —

dcr-1 55.3 ± 4.3 —

drsh-1 38.1 ± 3.3 <0.001rab-5 45.0 ± 5.9 <0.05rba-1 61.3 ± 6.3 —

F56D2.6 58.8 ± 5.4 —

eif-3.D 59.3 ± 4.7 —

C05C9.1 54.4 ± 5.8 —

D1007.5 39.1 ± 2.9 <0.001C05C10.2 47.0 ± 3.7 <0.05F55F10.1 63.2 ± 5.1 —

tbx-9 56.6 ± 4.5 —

unc-101 58.6 ± 4.1 —

mtx-1 56.2 ± 5.8 —

inf-1 80.2 ± 4.3 >0.01csn-5 57.7 ± 5.5 —

pgk-1 46.0 ± 2.0 <0.001T03F6.3 57.9 ± 3.7 —

rga-3 59.9 ± 3.8 —

gpdh-2 60.0 ± 5.8 —

ugt-58 62.2 ± 4.2 —

C05C10.3 36.0 ± 3.0 <0.001F42G9.1a 56.9 ± 4.9 —

kin-19 58.2 ± 5.1 —

C06A8.1a 56.5 ± 3.8 —

F45G2.9 60.2 ± 3.6 —

rfs-1 53.2 ± 4.5 —

rba-2/lin-53 63.2 ± 3.0 —

abl-1 59.2 ± 5.9 —

rps-0 82.2 ± 7.6 >0.01rpn-2 65.2 ± 4.5 —

rpt-3 58.1 ± 6.1 —

ptp-1 62.2 ± 5.7 —

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Table S1. Cont.

RNAi TD50, h (mean ± SEM, n = 3) P value

trs-1 58.5 ± 4.5 —

dhs-21 58.5 ± 2.1 —

F01F1.9 59.8 ± 3.8 —

rfc-5 61.2 ± 5.7 —

rfc-4 57.7 ± 4.5 —

ima-3 56.8 ± 6.5 —

mcm-6 58.9 ± 2.1 —

cdk-1/ncc-1 56.9 ± 4.8 —

F12F6.7 64.4 ± 3.7 —

ubc-21 86.2 ± 3.1 >0.001hda-1 55.2 ± 5.5 —

C36A4.4 58.2 ± 6.8 —

tbg-1 59.6 ± 7.6 —

nrs-1 59.2 ± 4.6 —

T09A5.11 39.0 ± 2.2 <0.001crt-1 63.2 ± 5.5 —

cts-1 60.1 ± 4.0 —

T12A2.2 63.2 ± 4.9 —

gpa-7 62.2 ± 5.3 —

sec-10 85.2 ± 7.6 >0.01T06E8.1 63.2 ± 2.0 —

cct-1 49.0 ± 5.8 0.017T07C4.2 61.2 ± 1.5 —

F45E4.1 55.2 ± 1.9 —

T19A6.2 58.8 ± 5.8 —

F57C9.1a 59.1 ± 1.3 —

sns-8 59.3 ± 2.2 —

plk-3 59.6 ± 5.6 —

eif-3.B 49.8 ± 6.4 0.019Y51H4A.7 59.8 ± 1.3 —

F43G9.1 59.1 ± 4.1 —

swp-1 60.8 ± 6.9 —

F54D5.7 62.8 ± 2.5 —

B0035.5 61.1 ± 2.2 —

F55C5.8 58.6 ± 5.8 —

mog-5 57.8 ± 4.4 —

mog-1 57.9 ± 2.5 —

T27F2.1 84.2 ± 8.4 >0.05K12D12.1 63.6 ± 5.0 —

F59E10.1 49.7 ± 2.4 0.035W08E3.3 60.8 ± 6.5 —

F09E5.5 59.5 ± 7.9 —

drs-1 58.6 ± 6.0 —

F18A1.5 55.1 ± 4.1 —

F01G4.6 59.6 ± 5.2 —

cct-6 59.8 ± 2.3 —

cct-4 40.7 ± 4.0 <0.001R03D7.1 57.8 ± 5.7 —

F59B2.3 62.2 ± 6.5 —

unc-32 79.0 ± 6.0 >0.01F53H4.1 58.1 ± 2.1 —

cdc48.1 43.1 ± 4.7 <0.001C01G6.6a 59.3 ± 4.9 —

Y75B8A.31 59.0 ± 4.2 —

ZC101.2 62.3 ± 6.0 —

pek-1 63.1 ± 6.7 —

C02F5.3 57.0 ± 5.1 —

W02D9.1 59.3 ± 2.0 —

F11A10.1 57.7 ± 5.9 —

F36A4.7 90.2 ± 5.7 >0.001C26E6.4 41.7 ± 2.1 <0.001ZC168.4 63.1 ± 4.1 —

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Table S1. Cont.

RNAi TD50, h (mean ± SEM, n = 3) P value

C37C3.2 79.2 ± 8.8 —

T05H4.6a 57.3 ± 3.3 —

R10E4.4 59.1 ± 3.4 —

F10C2.4 59.6 ± 4.0 —

K10B2.1 63.0 ± 5.2 —

pis-1 61.1 ± 4.0 —

met-2 64.1 ± 5.5 —

C14B9.8 60.8 ± 4.8 —

F52E4.7 59.8 ± 6.9 —

F48E8.5 74.1 ± 7.9 —

F08B1.1 60.1 ± 2.5 —

C30C11.2 59.9 ± 5.9 —

R74.1 58.8 ± 6.1 —

W08D2.5 63.9 ± 5.5 —

egl-45 61.7 ± 4.2 —

T07D4.3 63.3 ± 5.6 —

D1005.1 56.0 ± 2.3 —

F25B5.5 57.8 ± 4.4 —

F28D1.10 59.1 ± 5.4 —

F39C12.1 59.6 ± 5.9 —

B0334.8 55.8 ± 4.3 —

Y46G5A.4 58.8 ± 5.0 —

Table S2. RNAi of necrosis-associated genes does not rescuesusceptibility of mpk-1(n2521) mutants on P. aeruginosa PA14

Strain RNAi TD50, h (mean ± SEM, n = 3) P value

mpk-1 vector 35.6 ± 5.5 —

asp-1 38.2 ± 3.5 NDasp-2 33.4 ± 2.8 NDclp-3 37.5 ± 4.1 NDasp-3 35.8 ± 6.7 NDasp-5 36.6 ± 3.6 NDtra-3/clp-5 32.2 ± 4.3 NDclp-6 33.1 ± 7.9 NDclp-7 38.6 ± 3.1 NDasp-6 34.9 ± 6.7 ND

ND, P > 0.05 versus mpk-1 mutants + empty vector.

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