The detection of IRE-1 activity provides a sensitive and responsive measure of ER stress. Most methods used to measure IRE-1 activity require functional IRE-1-XBP-1 output, relying on either detection of the activated spliced form of the xbp-1 mRNA or the transcriptional activity of the resulting XBP-1 protein. In order to follow IRE-1 activity in the absence of a functional XBP-1 protein, we utilized the C. elegans xbp-1(zc12) mutant, which has a C→T mutation that results in an early premature stop codon [4]. We reasoned that we could detect IRE-1-mediated splicing of xbp-1(zc12) mRNA by quantitative RT-PCR (qPCR), as we have done previously for wild type xbp-1 mRNA, as a measure of IRE-1 activity [21].

We anticipated, however, that the xbp-1(zc12) mRNA might be degraded by nonsense-mediated decay (NMD) [22], which would reduce the abundance of xbp-1(zc12) mRNA (Figure 1A). Thus, we constructed a strain carrying xbp-1(zc12) and a null allele of smg-2, the C. elegans homolog of the NMD component Upf1 [23]. Indeed, we observed that the level of xbp-1 mRNA in the xbp-1(zc12) mutant was markedly diminished compared with the level of xbp-1(zc12) mRNA in the smg-2(qd101); xbp-1(zc12) mutant (Figure 1B). These data confirmed that xbp-1(zc12) mRNA is a substrate for the NMD pathway, but that inhibition of NMD permits detection of xbp-1(zc12) mRNA. As expected from the predicted truncated protein product made from translation of the xbp-1(zc12) mRNA (Figure 1A), loss of NMD had no effect on the null phenotype of the xbp-1(zc12) allele, as assessed by the effect of the smg-2(qd101) mutation on expression of an xbp-1-regulated gene, the C. elegans BiP homolog hsp-4 (Figure 1B). We next examined the level of WT xbp-1 mRNA in the smg-2(qd101) mutant, and we observed that NMD inhibition increased the level of xbp-1 mRNA 2-fold relative to WT C. elegans (Figure 1C), which suggests that the NMD complex may function to decrease the level of WT xbp-1 mRNA. This observation is consistent with a prior report suggesting that stress-induced genes may be NMD targets [24], although we hypothesize that the relatively early termination codon present in the xbp-1 mRNA prior to IRE-1-mediated splicing may also contribute to recognition and degradation by the NMD pathway. Consistent with this explanation, after exposing both the WT and smg-2(qd101) strains to tunicamycin for 4 h, the level of IRE-1-spliced xbp-1 mRNA was similar between the two strains (Figure 1C). Furthermore, the loss of NMD did not increase the lethality of either the WT strain or xbp-1(zc12) mutant when grown in the presence of tunicamycin (Figure 1D).

Comparing levels of IRE-1 activity between smg-2(qd101) and smg-2(qd101); xbp-1(zc12) animals, we observed a dramatic elevation in the level of spliced xbp-1 mRNA in the smg-2(qd101); xbp-1(zc12) strain (Figure 1E). To provide a measure for comparison, the basal elevation of spliced xbp-1 mRNA in the smg-2(qd101); xbp-1(zc12) mutant far exceeded the level of spliced xbp-1 mRNA in the smg-2(qd101) mutant even after administration of tunicamycin. Treating the smg-2(qd101); xbp-1(zc12) strain with tunicamycin resulted in only a minor additional increase in spliced xbp-1 mRNA compared with the magnitude of elevation in spliced xbp-1 in that strain under basal conditions (Figure 1E). These data show that XBP-1 deficiency results in a dramatic increase in IRE-1 activity, even in the absence of exogenously administered agents such as tunicamycin.

If the elevated level of IRE-1 activity observed in the smg-2(qd101); xbp-1(zc12) mutant were indicative of increased ER stress due to loss of xbp-1, we might anticipate compensatory activation of the PEK-1 and/or ATF-6 pathways in the absence of XBP-1. We therefore sought to determine levels of PEK-1 activity in an xbp-1 mutant through the detection of eIF-2α phosphorylation. In particular, these measurements would provide an additional measure of ER stress in the xbp-1 mutant that is not dependent on the inactivation of the NMD pathway and its aforementioned experimental caveats. Antibodies raised against mammalian eIF-2α and specifically phosphorylated eIF-2α (P-eIF-2α) cross-react with the highly homologous C. elegans protein [25], [26]. We detected a single band in immunoblots using these antibodies with lysates from WT C. elegans (Figure 2A). We observed that eIF-2α phosphorylation was induced by a 4 h exposure to a high dose of tunicamycin in a PEK-1-dependent manner (Figure 2A and Figure S1A). Of note, eIF-2α phosphorylation appears to be a less sensitive measure of ER stress than IRE-1-mediated xbp-1 mRNA splicing, as we did not observe a significant increase in eIF-2α phosphorylation in response to standard doses of tunicamycin sufficient to induce xbp-1 mRNA splicing.

We next determined PEK-1 activity under basal physiological conditions, specifically in the xbp-1 mutant. We saw induction of PEK-1-mediated eIF-2α phosphorylation relative to WT in the absence of exogenously administered agents to induce ER stress at 16°C (Figure 2B and Figure S1B). The magnitude of the effect of XBP-1 deficiency on PEK-1 activity was comparable to the induction of PEK-1 in WT by treatment with high-dose tunicamycin. We observe no increase in eIF-2α phosphorylation in the xbp-1; pek-1 mutant relative to the pek-1 mutant, confirming that the increase in eIF-2α phosphorylation in the xbp-1 mutant relative to WT is due to activation of PEK-1. In fact, we noticed a slight decrease in eIF-2α phosphorylation in the xbp-1; pek-1 mutant relative to the pek-1 mutant, but the mechanisms underlying this difference are unclear. These data corroborate our observations of increased xbp-1 mRNA splicing in the xbp-1 mutant. Taken together, the increase in levels of IRE-1 and PEK-1 activity in the xbp-1 mutant suggests that XBP-1 deficiency is accompanied by a marked increase in constitutive ER stress under basal physiological conditions.

Previously, we reported that the activation of innate immunity by infection with pathogenic P. aeruginosa induces ER stress, and that XBP-1 serves an essential role in protecting the host against the detrimental effects of immune activation [21]. Our prior ultrastructural analysis of the ER in xbp-1 mutants suggested that disruption of ER homeostasis contributes to this phenotype. One explanation for these observations is that ER homeostasis in the xbp-1 mutant might be minimally perturbed under basal physiological conditions but have a pronounced sensitivity to ER stress from endogenous (e.g. immune activation) or exogenous (e.g. tunicamycin) sources. However, the data in Figure 1 and Figure 2 suggest that even during physiological growth and development, XBP-1 deficiency results in a marked elevation in levels of basal ER stress. We hypothesized, therefore, that under these circumstances, the activation of innate immunity might further increase ER stress levels.

The smg-2(qd101); xbp-1(zc12) strain provided the opportunity to assess levels of ER stress caused by immune activation in the setting of XBP-1 deficiency. Whereas a 4 h exposure of the WT strain to P. aeruginosa PA14 causes a two-fold increase in spliced xbp-1 mRNA relative to exposure to the relatively non-pathogenic bacterial food Escherichia coli OP50 (Figure 3A and [21]), we observe a blunted response to P. aeruginosa infection in the smg-2(qd101) mutant (Figure 3A). This observation is likely due to the 5-fold elevation in spliced xbp-1 mRNA levels in the smg-2(qd101) mutant (Figure 1C), which may buffer the ER from the stress caused by pathogen-induced immune activation. Nevertheless, we observed that the level of spliced xbp-1 mRNA in the smg-2(qd101); xbp-1(zc12) mutant was increased by a 4 h exposure to P. aeruginosa relative to the smg-2(qd101); xbp-1(zc12) mutant treated in parallel with E. coli (Figure 3A). Specifically, under these treatment conditions, the level of spliced xbp-1 mRNA in the smg-2(qd101); xbp-1(zc12) mutant was 20-fold greater than that of the smg-2(qd101) mutant in the absence of additional stress, whereas exposure to P. aeruginosa increased the level of spliced xbp-1 mRNA to over 25-fold that of the smg-2(qd101) mutant (Figure 3A). The total amount of xbp-1 mRNA was unchanged between smg-2(qd101) and smg-2(qd101); xbp-1(zc12) strains, indicating that the increase in spliced xbp-1 mRNA is due to increased IRE-1 activation.

We observed persistent elevation of spliced xbp-1 mRNA after an 11 h exposure to P. aeruginosa, above the elevated basal levels of spliced xbp-1 mRNA in the xbp-1 mutant, suggesting that IRE-1 activity is not attenuated under conditions of physiological ER stress (Figure 3B).

Previously, we established that the ER stress induced by exposure to P. aeruginosa, as well as the lethality of the xbp-1 mutant during infection by P. aeruginosa, are suppressed by a loss-of-function mutation in pmk-1, which encodes a conserved p38 mitogen-activated protein kinase (MAPK) that regulates innate immunity in C. elegans [27]. Our interpretation of these data was that loss of PMK-1 activity diminished the secretory load on the ER by attenuating the innate immune response. In support of this interpretation, we found that the pathogen-induced increase in spliced xbp-1 mRNA in smg-2(qd101); xbp-1(zc12) was suppressed in the smg-2(qd101); xbp-1(zc12); pmk-1(km25) mutant, although the basal levels on E. coli OP50 nevertheless remained markedly elevated (Figure 3A). These data provide quantitative support for a model in which the activation of PMK-1-mediated innate immunity is a physiologically relevant source of ER stress, which in XBP-1-deficient animals exacerbates an already elevated level of ER stress to cause larval lethality.

What are the functional consequences of the elevated ER stress present in the xbp-1 mutant under standard growth conditions, in the absence of infection? The xbp-1 mutant, while viable, exhibits increased sensitivity to exogenously administered ER stress as well as physiological ER stress from immune activation [21], [28]. Inactivation of both xbp-1 and pek-1 was previously reported to result in larval arrest when propagated at 20°C [16]. Our observations of constitutive ER stress in the xbp-1 mutant and increased PEK-1 activity suggest a compensatory functional role for pek-1, and thus we sought to further characterize the larval arrest phenotype of the xbp-1; pek-1 mutant.

Surprisingly, we observed that the xbp-1(tm2482); pek-1(ok275) double mutant exhibited temperature-dependent viability over the physiological temperature range of C. elegans (Figure 4A). The larval development of the xbp-1(tm2482); pek-1(ok275) mutant was similar to that of WT at 16°C. At 20°C, however, approximately half of xbp-1(tm2482); pek-1(ok275) eggs developed to become gravid adults, while the remainder arrested during larval development in the L2 and L3 stages. These arrested larvae died over the course of several days with intestinal degeneration as previously described (Shen et al., 2005). At temperatures greater than 23°C, larval lethality was 100%. At 25°C, 100% of the population died in the L1 and L2 stages after just 2 days. The physiological temperature range for propagation of C. elegans in the laboratory is generally 15°C to 25°C, with optimal reproduction at 20°C. Thus, the observed temperature dependence is observed not at “heat shock” temperatures, but rather, well within the range of physiological temperatures for C. elegans.

The temperature dependence of xbp-1(tm2482); pek-1(ok275) lethality permitted the investigation of whether the larval lethality of the xbp-1; pek-1 mutant is due to a requirement for XBP-1 and PEK-1 at a specific stage of development, or whether the activities of XBP-1 and PEK-1 are required constitutively for viability at other life stages. Specifically, we propagated two xbp-1; pek-1 mutants comprised of different mutant alleles at 16°C until the animals reached the L4 larval stage, then either maintained the mutants at 16°C or shifted them to 25°C to monitor survival. When shifted to 25°C, the xbp-1; pek-1 double mutants exhibited a sharp decrease in survival as compared with the strains maintained at 16°C (Figure 4B). These observations suggest that the activity of either XBP-1 or PEK-1 is not specifically required at a particular developmental stage; instead, the constitutive activities of XBP-1 and PEK-1 are required for survival at physiological temperatures.

Although we previously observed that pek-1 and atf-6 single mutants did not exhibit larval lethality in the presence of pathogenic bacteria [21], our data presented in this paper suggest that PEK-1 functions in parallel to XBP-1 under physiological conditions in C. elegans to maintain ER homeostasis. Because the xbp-1; pek-1 mutant is viable through larval development at 16°C, we were able to ask whether PEK-1 contributes to protection against immune activation in the absence of XBP-1. Populations of synchronized eggs were grown at 16°C with P. aeruginosa as the only food source and development was monitored over time. P. aeruginosa has been shown to exhibit markedly diminished pathogenicity to C. elegans adults at 16°C relative to 25°C [29], and we found this to also be the case during larval development. Specifically, the pmk-1 mutant was able to complete larval development on P. aeruginosa at 16°C (Figure 5A), whereas only half of the pmk-1 eggs grown on P. aeruginosa develop to the L4 stage at 25°C [21], indicating that immune activation is less important for development in the presence of P. aeruginosa grown at 16°C than it is at 25°C. Likewise, the larval development of the xbp-1 mutant, which is severely compromised on P. aeruginosa at 25°C [21], was equivalent to that of WT at 16°C (Figure 5A). Both the diminished pathogenicity of P. aeruginosa at 16°C and the aforementioned temperature-sensitive requirement for UPR function may contribute the survival of the xbp-1 mutant at 16°C. Nevertheless, even under these conditions, the xbp-1(tm2482); pek-1(ok275) mutant exhibited complete larval lethality on P. aeruginosa at 16°C, reminiscent of the larval lethality of xbp-1 on P. aeruginosa grown at 25°C. Eliminating PMK-1-mediated immunity completely rescued this larval lethality (Figure 5A), demonstrating that PEK-1 functions with XBP-1 to protect against PMK-1-mediated immune activation during larval development.

We next asked whether the UPR is required for survival in the presence of pathogen during adulthood. In parallel with our observation that the xbp-1; pek-1 mutant exhibits temperature-sensitive lethality both during larval development and when shifted to a higher temperature from the L4 larval stage, we found that the xbp-1; pek-1 mutant exhibits enhanced lethality relative to the WT strain or either of the single mutants when shifted at the L4 stage to P. aeruginosa at 16°C (Figure S2). These data suggest that the UPR is required for survival during immune activation both in larval development and in adulthood.

Larval arrest of xbp-1; pek-1 mutants has been reported to be accompanied by evidence of intestinal degeneration, including the appearance of vacuoles and light-reflective aggregates in intestinal cells, degradation of intestinal tissues, and distention of the intestinal lumen [16]. We observe similar morphology not only in xbp-1; pek-1 larvae at 23°C on E. coli OP50, but also at 16°C on P. aeruginosa. The similar appearance between xbp-1; pek-1 larvae dying either at 16°C on pathogenic bacteria or at 23°C on E. coli OP50 led us to consider whether ER stress arising from intestinal innate immune activation might contribute in a similar manner to both conditions. We have previously characterized PMK-1-mediated innate immunity and observed both basal and induced components to immunity regulated by PMK-1 [30]. We therefore hypothesized that basal immune activity under standard, non-pathogenic growth conditions could present a low level of ER stress that is severely exacerbated in the absence of intact physiological UPR function, leading to larval lethality of the xbp-1; pek-1 mutants. Consistent with this hypothesis, we observed that pmk-1 loss-of-function was able to partially suppress the larval lethality of the xbp-1; pek-1 double mutant at 23°C and 25°C (Figure 5B).

One explanation for the temperature-sensitive lethality of the xbp-1; pek-1 mutant is that increased temperature leads to increased PMK-1 pathway activation, perhaps as the “non-pathogenic” E. coli OP50 becomes slightly pathogenic. However, the temperature-sensitive lethality is not abrogated by loss of PMK-1; instead, the xbp-1; pmk-1; pek-1 mutant exhibits larval lethality at a temperature several degrees higher than the xbp-1; pek-1 mutant (Figure 5B). Furthermore, the temperature-sensitive larval lethality of the xbp-1; pek-1 mutant on E. coli OP50 was not suppressed by the presence of the bacteriostatic drug ampicillin (Figure S3A). These data indicate that basal immune activation and temperature are distinct contributors to ER stress that function in parallel during growth on E. coli OP50.

The temperature-dependent larval lethality of the xbp-1; pek-1 mutant over a physiological temperature range suggested that UPR signaling might be required for survival in response to thermal stress. Indeed, we observed that the xbp-1 mutant exhibited larval lethality when grown at 27°C, an elevated temperature at which WT N2 C. elegans exhibits a reduced brood size and increased dauer formation (Figure 5C). Similar to our observation that depletion of basal immunity rescued the development of the xbp-1; pek-1 mutant when propagated on E. coli OP50, the temperature-sensitive lethality in the xbp-1 mutant was suppressed in the xbp-1; pmk-1 double mutant (Figure 5C), but not by the presence of ampicillin (Figure S3B).

Unlike the xbp-1 mutant, the development of the pek-1 mutant at 27°C was similar to WT. This is reminiscent of our previous observation that the pek-1 mutant did not exhibit the larval lethality found in xbp-1 when grown on P. aeruginosa at 25°C [21]. However, we next grew the pek-1 mutant on P. aeruginosa at 27°C, reasoning that the elevated temperature would not only increase the ER stress caused by basal growth, but also enhance the pathogenicity of the P. aeruginosa and thereby increase the immune response relative to that at 25°C. Indeed, the pmk-1 mutant exhibited 100% larval lethality on P. aeruginosa at 27°C (Figure 5D), as compared with the 50% lethality we have previously reported for the pmk-1 mutant on P. aeruginosa at 25°C (Richardson et al., 2010). The increased susceptibility of this immune-deficient mutant to P. aeruginosa at 27°C relative to 25°C indicates that the increased temperature causes an increase in P. aeruginosa pathogenicity. On P. aeruginosa at 27°C, the pek-1 mutant exhibited larval lethality relative to the WT strain grown 27°C (Figure 5D). These data further suggest that PEK-1 functions in parallel with XBP-1 to protect C. elegans against the ER stress caused by immune activation.

We showed in Figure 5B and 5C that loss of PMK-1 improves larval development of the xbp-1; pek-1 mutant and the xbp-1 mutant, respectively, in the absence of infection. We suggested that the mechanism behind this phenomenon is that the previously described basal immune activity through the PMK-1 pathway [31] contributes to ER stress. However, we also considered the possibility that the PMK-1 pathway might play an immunity-independent role in exacerbating ER stress in the setting of UPR deficiency. To test this possibility, we examined the ability of WT and UPR mutants to develop in the presence of tunicamycin with or without functional pmk-1. We found that the pmk-1 mutant actually exhibited increased sensitivity to tunicamycin during development. In fact, the pmk-1 mutant exhibited greater lethality at a lower dose of tunicamycin than either the xbp-1 or pek-1 single mutants (Figure 6). These data suggest that the PMK-1 pathway influences ER stress in two ways. First, during infection or under standard growth conditions in the setting of UPR depletion, activation of the PMK-1 pathway generates an increased secretory load that contributes to ER stress. However, when ER stress is induced exogenously with tunicamycin, the PMK-1 pathway activity serves a protective function.

C. elegans strains were constructed and propagated according to standard methods on E. coli OP50 at 16°C [35]. The smg-2(qd101) allele was isolated by K. Reddy and contains a C→T nonsense mutation at nucleotide 1189 of the spliced transcript. The following strains were used in the study: N2 Bristol, ZD627 smg-2(qd101), ZD607 smg-2(qd101);xbp-1(zc12), ZD605 smg-2(qd101);xbp-1(zc12);pmk-1(km25), KU25 pmk-1(km25), RB545 pek-1(ok275), ZD510 xbp-1(tm2482);pek-1(ok275), ZD524 xbp-1(zc12);pek-1(tm629), ZD496 xbp-1(tm2482);pmk-1(km25);pek-1(ok275). All of the alleles used are predicted to be null alleles. Specifically, xbp-1(tm2482) is a 202 bp deletion from nt 231 that causes a frame-shift. The xbp-1(zc12) allele is a nonsense mutation that changes Q34 to an ochre stop. The two alleles exhibit an equivalent phenotype in every assay tested ([21]; this work, and our unpublished data). The pek-1(ok275) allele is a 2013 bp deletion and the pek-1(tm629) allele is a 1473 bp deletion, both of which remove the PEK-1 transmembrane domain and are therefore likely null alleles [6]. Double mutants were made between xbp-1 and pek-1 by crossing strains marked with GFP: xbp-1(III);pT24B8.5::GFP(agIs220)(X) and pT24B8.5::GFP(agIs219)(III);pek-1(X). GFP-negative F2s were singled, propagated, and genotyped by PCR.

For the experiment in Figure 1B, 1C, and 1E, L1 larvae were synchronized by hypochlorite treatment, washed onto E. coli OP50 plates, and grown for 40 h at 16°C to the L3 stage, when they were washed in M9 to plates containing E. coli OP50 or E. coli OP50 with 5 µg/ml tunicamycin. For the experiments in Figure 3, strains were grown and treated as previously described [21]. Specifically, L1 larvae were synchronized by hypochlorite treatment, washed onto E. coli OP50 plates and grown at 20°C for 23 h, then washed in M9 onto treatment plates. After incubation at 25°C for indicated times, worms were washed off plates and frozen in liquid nitrogen. For P. aeruginosa treatment, P. aeruginosa strain PA14 was grown in Luria Broth (LB), and 25 µl overnight culture was seeded onto 10 cm NGM plates. Plates were incubated first at 37°C for 1 d, then at room temperature for 1 d. All RNA extraction, cDNA preparation, qRT-PCR methods and specific primers to detect xbp-1 mRNA were as described previously [21].

For all immunoblots, strains were synchronized by hypochlorite treatment and washed onto E.coli OP50 plates for growth until the L4 stage. For the experiment in Figure 2A, strains were grown at 20°C and L4 worms were then washed in M9 onto treatment plates for incubation at 25°C for 4 hours. For the experiment in Figure 2B, strains were grown at 16°C until the L4 stage and harvested without treatment. All strains were collected and rinsed 2 times in M9. Worm pellets were resuspended in an equal volume of 2× lysis buffer containing 4% SDS, 1oomM Tris Cl, pH 6.8, and 20% Glycerol. After boiling for 15 minutes with occasional vortexing to aid in dissolution, lysates were clarified by centrifugation. Protein samples (50 µg of total lysate loaded per lane) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-rad). Western blots were blocked in 5% milk in PBST and probed with (1∶10,000) anti-eIF2α [26], (1∶1,000) anti-phospho-eIFα (Cell Signaling Technology), or (1∶10,000) anti-tubulin (E7 Developmental Hybridoma Bank, Iowa City). All primary antibodies were diluted in 5% milk in PBST. Following incubation with anti-rabbit or anti-mouse IgG antibodies conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology), signals were visualized with chemiluminescent HRP substrate (Amersham). Quantification of immunoblots was preformed with ImageJ [36].

For all development assays, strains were egg laid on 4–5 prepared plates for no more than 3 h (at least 110 eggs for each strain and treatment). Development was monitored daily for 4 d for experiments conducted at 16°C and 3 d for experiments conducted at all other temperatures. Experiments monitoring development on E. coli OP50 were performed on 6 cm NGM plates. P. aeruginosa PA14 plates were prepared as described [27]. For the data presented in Figure S2, plates were prepared as described [37], except that ampicillin was used instead of carbenicillin.

To monitor L4 survival on E. coli OP50 or P. aeruginosa PA14, strains were incubated at 16°C to the L4 stage, when they were transferred to plates containing FUDR and incubated at either 16°C or 25°C. For each strain, 30 worms were transferred to each of 3–4 plates. Alive vs. dead worms were counted, and worms that died by exploding through the vulva or desiccating on the side of plates were censored.