Impairment of the SKN-1A/NRF1 proteasome surveillance pathway triggers tissue-specific protective immune responses against distinct natural pathogens in C. elegans

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF upregulates proteostasis capacity after blockade of the proteasome, and also promotes resistance against bacterial infection in the nematode C. elegans. SKN-1/NRF has three isoforms, and the SKN-1A/NRF1 isoform in particular regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1, show constitutive expression of immune response programmes against natural eukaryotic pathogens of C. elegans. These programmes are the Oomycete Recognition Response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the Intracellular Pathogen Response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programmes are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner, against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

We performed a chemical mutagenesis screen on animals carrying the chil-27p::GFP 137 reporter, which is not expressed in wild-type animals under standard growth 138 conditions, but is strongly induced upon recognition of oomycete pathogens [2,28]. 139 We obtained several independent mutants with constitutive epidermal expression of skn-1c isoforms as the entire skn-1c sequence is shared by skn-1a ( Figure S2A).

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Because PNG-1 is required specifically for activation of the SKN-1A isoform and SKN-160 1C does not undergo sequence editing [17], the involvement of SKN-1C in activating 161 chil-27p::GFP seemed unlikely. However, to directly test this possibility, we performed 162 wdr-23 RNAi on skn-1a(mg570) mutant animals. WDR-23 is a WD40 protein known 163 to specifically suppress SKN-1C function, inhibition which is released for example 164 under oxidative stress to allow SKN-1C activation [31,32]. We found that wdr-23 RNAi 165 did not suppress chil-27p::GFP induction in skn-1a(mg570) mutants ( Figure S2B). This 166 result suggests that activation of SKN-1C cannot rescue the constitutive expression of 167 chil-27p::GFP in skn-1a(mg570) mutants, thus the constitutive response is likely due 168 to loss of the skn-1a isoform. proteasomal activity by BTZ drug treatment [35], oomycete recognition response [3] 180 and skn-1a loss-of-function [35]. A significant overlap was identified between all these 181 datasets ( Figure 1D, S1B, and supplementary table S1). Wild-type animals treated 182 with BTZ show induction of chil-27 as previously described [15], and this induction is   genes takes place to combat the infection [3]. Having discovered that proteasome 199 inhibition can activate the ORR, we wanted to determine whether this inhibition is 200 required in a tissue-specific manner or not. To address this question, we performed 201 tissue-specific rescue of skn-1a(mg570) mutants by expressing skn-1a under a rab-202 3p (pan-neuronal), dpy-7p (epidermal) or vha-6p (intestinal) promoter. We found that 203 only the epidermal rescue of skn-1a repressed expression of chil-27p::GFP ( Figure   204 2A). We further investigated this question by performing tissue-specific RNAi of the 205 proteasomal subunit rpt-5 [10,36,37], where we found that only epidermal RNAi of 206 rpt-5 was able to induce chil-27p::GFP expression ( Figure 2B). We also tested the 207 survival of tissue-specific rescued lines of skn-1a in the presence of M. humicola and 208 found only epidermal overexpression of skn-1a to rescue the enhanced oomycete 209 resistance phenotype ( Figure 2C). These findings demonstrate that proteasome 210 impairment, or rescue of proteasome surveillance function specifically in the 211 epidermis, regulates the ORR and oomycete resistance.

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Previous work has led to the identification of other epidermal regulators of the ORR, 214 namely the receptor tyrosine kinase OLD- 1 [38] that is specific to oomycete 215 recognition, and the PALS-22/PALS-25 antagonistic paralogs [15,28], which regulate 216 the immune response against both oomycetes and microsporidia. We thus asked 217 whether activation of ORR upon proteasome dysfunction requires old-1 or pals-25. 218 Here, we performed skn-1 RNAi on animals carrying a deletion in old- 1(ok1273) or in 219 pals-25(jy81), along with wild-type animals as control, and found no difference in chil-220 27p::GFP induction ( Figure S4). These results suggest that epidermal proteasome 221 dysfunction acts either downstream of OLD-1 and PALS-25-mediated signalling, or as 222 a parallel trigger leading to the activation of ORR.

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Oomycete extract exposure does not cause broad proteasome dysfunction.

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Since epidermal proteasome dysfunction triggers the ORR, we tested whether 227 impairment of proteasome function occurs upon exposure to oomycete extract.

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Proteasomal subunit expression is observed as a bounce-back response upon 229 proteasome dysfunction [18]. Even though significant overlap was obtained between 230 ORR, and genes upregulated upon proteasome dysfunction ( Figure 1D), none of the 231 proteasomal components were found to be induced as a part of the ORR ( Figure 3A).   Table S1). To investigate 260 whether loss of skn-1a also leads to increased resistance against intestinal pathogens, 261 we assayed the skn-1a(mg570) mutant for resistance against the intestinal pathogen 262 N. parisii. Here, we found that skn-1a(mg570) mutants had increased resistance 263 towards N. parisii ( Figure 4C), which was rescued in this case specifically by intestinal 264 (vha-6p) expression of skn-1a ( Figure 4D). These results demonstrate that impairment 265 of the SKN-1A proteasome surveillance pathway also induces resistance to intracellular pathogens of the intestine, and this impairment can be rescued specifically 267 by SKN-1A function in the intestine.

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We have previously reported that approximately half of IPR genes are also present in 270 the ORR list [3], which was rather surprising given the distinct infection strategies and 271 tissue tropism between oomycetes and microsporidia [41]. We hypothesised that the 272 IPR and ORR programmes might share significant overlap because they both relate 273 to regulation by the proteasome. Remarkably, we observed that almost all common 274 ORR and IPR genes (40 out of 41 genes) were also regulated by BTZ treatment 275 ( Figure 4B and Table S1). We reasoned that these shared immune response genes 276 may be induced in different tissues following an ORR or IPR trigger. To test this   In this assay, pals-22(icb89) mutants and skn-1 RNAi treated animals were used as 295 positive controls, as both have been shown to result in enhanced susceptibility towards 296 PA14 infection [15,22]. The susceptibility of skn-1a(mg570) animals to PA14 was 297 found to be comparable to wild-type animals, while both skn-1 RNAi and pals-22 298 mutants showed increased susceptibility as expected ( Figure S6). The fact that skn-1 299 RNAi targets both a and c isoforms, but skn-1a(mg570) animals are not 300 hypersusceptible to PA14 suggests that requirement of SKN-1 to combat PA14 301 infection is more likely to be associated with the function of the SKN-1C isoform. Taken 302 together, these results suggest that different skn-1 isoform perturbations can lead to 303 different host immunity outcomes in a pathogen-specific way.  skn-1a(mg570) controls were also picked in the same manner and pulse infection was 448 performed as described above. To remove any potential bias, the order in which 449 strains were picked was randomized from assay to assay, and the 30 hpi endpoints   in TRIzol (Invitrogen) followed by RNA extraction using isopropanol/ethanol 477 precipitation. RNA was quantified using NanoDrop (Thermo Scientific) and its quality 478 was analyzed by gel electrophoresis. cDNA was synthesized using 2 µg RNA with 479 Superscript IV (Invitrogen) and Oligo(dT) primers as per manufacturer's instructions.

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Real-time PCR was performed using qPCR primer pairs listed in Table S2 and  To generate constructs for tissue-specific rescue of skn-1a(mg570) mutant, skn-1a 497 fragment was amplified from N2 cDNA using primers skn-1a_fullF and skn-1a_fullR.