The xbp-1 pathway functions in the intestine to protect against Cry5B PFT Mosaic and expression analyses have shown that the targeting of intestinal cells by the PFT Cry5B is both necessary and sufficient to intoxicate worms [15, 16] . If the ire-1-xbp-1 pathway is functioning directly to protect against the effects of the PFT, then we would predict that the ire-1-xbp-1 pathway should function in the target cells of the toxin, the intestinal epithelial cells. Alternatively, the pathway might be functioning indirectly to protect against the effects of the PFT (e.g., it might hypothetically function in neurons that then sends protective signals to the intestine). Consistent with the first hypothesis, that the pathway is functioning directly in the target cells to protect against the PFT, we previously noted that a marker for downstream activation of the pathway, hsp-4, is turned on exclusively in intestinal cells ( Figure 1B , middle panel), although the pathway is capable of being activated throughout the worm by a more general stress, such as heat shock ( Figure 1B, right panel) . elegans fed E. coli expressing Cry5B compared to control E. coli not expressing Cry5B. The time the worms were allowed to feed on the E. coli before total RNA was prepared for RT-PCR is indicated at the top, and the positions of the nucleotide size markers are indicated at the left. (B) Compared to worms fed control non-Cry5B expressing E. coli, in vivo activation of hsp-4::GFP occurs specifically in the intestines of worms fed Cry5B expressing E. coli at 20uC for 8 hours. As a comparison for GFP induction, separate worms on control bacteria were heat shocked at 30uC for 8 hours to induce the ER stress response by causing unfolded proteins. The heat shock worms have a strong increase in GFP throughout the body including the head, intestine and hypodermis. Thus, although the entire worm is capable of activating the ire-1-xbp-1 pathway as judged by hsp-4 induction, activation in Cry5B-fed animals is occurring only in those cells targeted by the PFT. Images taken by light microscopy are compared to images with fluorescence microscopy. Scale bar is 0.2 mm. The experiment was performed three times, and representative worms are shown. (C) Aerolysin induces activation of IRE1 in mammalians cells. Exposure of HeLa cells to proaerolysin (2 ng/mL) leads to increased production of spliced XBP1 protein as shown on this immunoblot (upper) and quantitated relative to no toxin control (lower). DTT (10 mg/mL for 2 h) was used as a positive control. Positions of molecular weight markers (kDa) are indicated on right side of the figure. A nonspecific antibody-reacting band was used as a loading control and normalization of the XBP1 signal in each lane. doi:10.1371/journal.ppat.1000176.g001 Figure 2 . Loss of specific UPR pathways cause hypersensitivity to PFT but not other toxins or a pathogenic bacteria. (A) Comparison of ER stress response mutants to wild-type N2 on 25% Cry5B-expressing E. coli plates indicate ire-1(v33) and xbp-1(zc12) are hypersensitive to Cry5B intoxication. Two representative worms are shown for each strain 48 hours after feeding either on E. coli without Cry5B or on E. coli of which 25% expressed Cry5B. Scale bar is 0.2 mm. (B) A lethal concentration assay was performed using purified Cry5B toxin to quantitatively compare sensitivities of wild-type N2 and the ER stress mutants. Lethality was determined after 8 days. This semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. (C) A Cry5B developmental inhibition assay was performed beginning with synchronized worms at the first larval stage. Worms were grown on plates containing different percentages of Cry5B-expressing E. coli (% Cry5B as indicated under the figure), and the percent of worms reaching the L4 stage or adulthood 72 hours later is indicated. ire-1(v33) was included only on the plates with 0% Cry5B. Data are presented as mean and standard deviation. (D) A lethal concentration assay comparing sensitivity to CuSO 4 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 8 days of CuSO 4 exposure, the same time frame as the Cry5B lethality assay. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (E) A lethal concentration assay comparing sensitivity to H 2 O 2 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 4 hours of H 2 O 2 exposure. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (F) A lifespan assay was used to compare the ER stress mutants to slow killing by P. aeruginosa PA14. This graph represents combined data from three experiments. doi:10.1371/journal.ppat.1000176.g002