A lifespan assay was used to compare the wwp-1 mutants to slow killing by P. aeruginosa PA14. This graph represents combined data from three experiments. The lifespan of wwp-1 mutants, including wwp-1(gk372) and wwp-1(ok1102), feeding on P. aeruginosa PA14 bacteria are statistically shorter than wild-type N2 animals.

In the canonical DAF-2 insulin/IGF-1 signaling pathway in C. elegans, DAF-2 regulates DAF-16 through the activation of PI3K, composed of AGE-1 and AAP-1 for the catalytic subunit and the regulatory subunit respectively. PI3K activates the activity of PDK-1, which eventually inhibits the nuclear translocation and transcriptional activity of DAF-16. Here we demonstrate that the attenuated DAF-2 signal can also regulate an additional DAF-16-independent signal arm that diverges form PDK-1 to WWP-1 to defense against Cry5B PFT.

daf-2(e1370), daf-16(mu86), daf-2(e1370);daf-16(mu86) and wwp-1(ok1102) mutant worms were fed dsRNA to wwp-1 (clone 8A15) to knock down wwp-1 gene in all these strains (plaid boxes). The worms fed on E. coli HT115 transformed with the RNAi empty vector (pL4440 vector) were used as controls (solid boxes). After developing to L4 stage, all RNAi knock down worms are exposed to purified Cry5B at 25°C. Lethality was determined after 6 days. Knockdown of wwp-1 gene in animals, including N2, daf-2(e1370), daf-16(mu86), and daf-2(e1370);daf-16(mu86), increased their sensitivity to Cry5B statistically significant. However, the sensitivity of wwp-1(ok1102) animals to Cry5B cannot be further enhanced by wwp-1 RNAi.

(A, B) Comparisons of the daf-2(e1370), daf-16(mu86), and daf-2(e1370);daf-16(mu86) mutant animals to wild-type N2 animals in 40 µg/ml purified Cry5B or 8 µg/ml Cry21A indicate daf-2(e1370) mutant and daf-2(e1370);daf-16(mu86) double mutant are all statistically resistant to Cry5B and Cry21A compared with N2. * indicates P<0.001 (P<0.05 in B) relative to wild-type N2. ^# indicates P<0.001 (P<0.05 in B) relative to daf-2(e1370);daf-16(mu86) mutant. (C) Comparisons of the mutants in the canonical DAF-2 insulin-like signaling pathway, including daf-2(e1370), age-1(hx546), aap-1(m889), daf-18(e1375), pdk-1(sa680), pdk-1(sa709), akt-1(sa573), akt-1(ok525), akt-1(mg144), akt-2(ok393), sgk-1(ok538), daf-16(mu86), daf-2(e1370);daf-16(mu86), to wild-type N2 animals in E. coli-expressed Cry5B liquid toxicity assay. * indicates P<0.01 relative to wild-type N2. (D, E) Dose-dependent mortality were performed using purified Cry5B toxin to quantitatively compare sensitivities of wild-type N2 to the daf-2(e1370), pdk-1(sa680), pdk-1(sa709), pdk-1(mg142), akt-1(ok525), akt-1(sa573), akt-2(ok393), and sgk-1(ok538) mutants. This semi-log graph represents three independent experiments, and each data point shows the mean and standard deviations from three experiments.

(A) A lifespan assay was used to compare the normal lifespan of wwp-1 mutant worms and the wild-type N2 worms. This graph represents combined data from three independent experiments. The lifespan of wwp-1 mutants, including wwp-1(gk372) and wwp-1(ok1102), are statistically shorter than wild-type N2 animals. (B) A dose-dependent mortality assay comparing sensitivity to CuSO₄ revealed the wwp-1 mutants, wwp-1(gk372), wwp-1(gk397), and wwp-1(ok1102) are not hypersensitive compared to wild-type N2. Lethality was determined after 6 days of CuSO₄ exposure, the same time frame as the Cry5B lethality assay. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (C) A dose- dependent mortality assay comparing sensitivity to H₂O₂ revealed wwp-1 mutants are not hypersensitive to this toxic insult compared to wild-type N2. Lethality was determined after 4 hours of H₂O₂ exposure. Data, plotted semi-log, are the mean and standard deviation of three independent experiments.

(A) Two novel PDK-1 interacting protein identified by the Worm Interactome Database. Two bait-prey interactions, including H42K12.1(pdk-1)-Y65B4BR.4(wwp-1) and H42K12.1(pdk-1)-C10F3.5(pcm-1), were identified by using H42K12.1(pdk-1) as the bait in the high-throughput yeast two hybrid screens [28]. (B) Predicted genomic structure of wwp1. Boxes and lines denote exons and introns respectively. The predicted functional domains of WWP1, including a C2 domain, four WW domains and a HECT domain, are indicated. The regions corresponding to the interfering dsRNA clones, 8A5 and 8A15 are underlined. The mutation regions of the three wwp-1 mutant alleles used in this study are indicated as gray boxes. (C) A Cry5B dose-dependent mortality assay was performed to quantitatively compare sensitivities of wild-type N2 to the wwp-1(ok1102) and pcm-1(qa201) mutants. Only wwp-1(ok1102) mutant animals showed statistically (p<0.01) hypersensitivity to Cry5B compared to N2. (D) A Cry5B dose-dependent mortality assay was performed to quantitatively compare sensitivities of wild-type N2 to three wwp-1 mutants, including wwp-1(gk372), wwp-1(gk397), wwp-1(ok102). All wwp-1 mutants showed statistically hypersensitive to Cry5B. (E) RNAi sensitive rrf-3(pk1426);glp-4(bn2) mutant worms after feeding on pL4440 (empty vector control), daf-2, or two wwp-1(8A5 and 8A15) dsRNA expressing E. coli were exposed to purified Cry5B at 25°C. Lethality was determined after 6 days. Animals knocked down for the daf-2 gene were statistically resistant to Cry5B whereas animals knocked down for the wwp-1 gene by two independent wwp-1 RNAi clones were statistically hypersensitive to Cry5B.

(A) Comparison of the reduction-of-function daf-2(e1370) mutant animals to wild-type N2 animals and the known Cry5B resistant bre-3(ye28) animals on Cry5B-expressing E. coli plates indicates daf-2(e1370) animals are resistant to Cry5B. The experiment was performed three times. Two representative worms are shown for each strain 48 hours after feeding either on E. coli with (Cry5B) or without Cry5B (No Toxin). (B) A dose-dependent mortality assay was performed using purified Cry5B toxin to quantitatively compare sensitivities of wild-type N2 to the bre-3(ye28) and daf-2(e1370) mutants. Lethality was determined after 6 days. This semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. Note, that although daf-2 mutant animals are highly resistant based on their ability to stay alive, they clearly are not as resistant as bre-3 animals since the latter are alive and robustly active and healthy whereas the former are alive but sickly at the end of this 6 day assay. (C) The dose-dependent mortality assay for Cry21A spore-crystal lysates. The fact that spores are present in these assays is due to the fact we currently do not have purified Cry21A. The presence of spores increases the toxicity of the Cry protein. Hence, the curves in (B) and (C) cannot be directly compared. (D) RNA interference E. coli-expressed Cry5B toxin plate assay. RNAi sensitive rrf-3(pk1426) worms after feeding on pL4440 (empty vector control), bre-3, or daf-2 genes dsRNA expressing E. coli were transferred to the E. coli-expressing either Cry5B (Cry5B), Cry21A (Cry21A) or without toxin (No Toxin) bacterial lawns for 48 hours. Knock down of the daf-2 gene leads to resistant to both Cry5B and Cry21A toxins. The experiment was performed three times and two representative worms are shown for each RNAi knock down strain.