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Upon sensing viral RNA, mammalian RIG-I-like receptors (RLRs) activate downstream signals using caspase activation and recruitment domains (CARDs), which ultimately promote transcriptional immune responses that have been well studied. In contrast, the downstream signaling mechanisms for invertebrate RLRs are much less clear. For example, theCaenorhabditis elegansRLR DRH-1 lacks annotated CARDs and up-regulates the distinct output of RNA interference. Here, we found that similar to mammal RLRs, DRH-1 signals through two tandem CARDs (2CARD) to induce a transcriptional immune response. Expression of DRH-1(2CARD) alone in the intestine was sufficient to induce immune gene expression, increase viral resistance, and promote thermotolerance, a phenotype previously associated with immune activation inC. elegans. We also found that DRH-1 is required in the intestine to induce immune gene expression, and we demonstrate subcellular colocalization of DRH-1 puncta with double-stranded RNA inside the cytoplasm of intestinal cells upon viral infection. Altogether, our results reveal mechanistic and spatial insights into antiviral signaling inC. elegans, highlighting unexpected parallels in RLR signaling betweenC. elegansand mammals. 

Abstract Orsay virus infection in the nematodeCaenorhabditis eleganspresents an opportunity to study host‐virus interactions in an easily culturable, whole‐animal host. Previously, a major limitation ofC. elegansas a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen,C. eleganshas emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability ofC. elegansenables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection ofC. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of Orsay virus filtrate Support Protocol: SynchronizeC. elegansdevelopment by bleaching Basic Protocol 2: Orsay virus infection Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT‐PCR Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus‐infected intestinal tissue 

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematodeCaenorhabditis elegans. SKN-1/NRF has 3 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 inskn-1aand its activating enzymesddi-1andpng-1show constitutive expression of immune response programs against natural eukaryotic pathogens ofC.elegans. These programs 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-1amutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs 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 theC.elegansepidermis and intestine. 

The immune system continually battles against pathogen-induced pressures, which often leads to the evolutionary expansion of immune gene families in a species-specific manner. For example, thepalsgene family expanded to 39 members in theCaenorhabditis elegansgenome, in comparison to a single mammalianpalsortholog. Our previous studies have revealed that two members of this family,pals-22andpals-25, act as antagonistic paralogs to control the Intracellular Pathogen Response (IPR). The IPR is a protective transcriptional response, which is activated upon infection by two molecularly distinct natural intracellular pathogens ofC.elegans–the Orsay virus and the fungusNematocida parisiifrom the microsporidia phylum. In this study, we identify a previously uncharacterized member of thepalsfamily,pals-17, as a newly described negative regulator of the IPR.pals-17mutants show constitutive upregulation of IPR gene expression, increased immunity against intracellular pathogens, as well as impaired development and reproduction. We also find that two other previously uncharacterizedpalsgenes,pals-20andpals-16, are positive regulators of the IPR, acting downstream ofpals-17. These positive regulators reverse the effects caused by the loss ofpals-17on IPR gene expression, immunity, and development. We show that the negative IPR regulator protein PALS-17 and the positive IPR regulator protein PALS-20 colocalize inside and at the apical side of intestinal epithelial cells, which are the sites of infection for IPR-inducing pathogens. In summary, our study demonstrates that severalpalsgenes from the expandedpalsgene family act as ON/OFF switch modules to regulate a balance between organismal development and immunity against natural intracellular pathogens inC.elegans. 

Abstract Although the type‐I interferon (IFN‐I) response is considered vertebrate‐specific, recent findings about the Intracellular Pathogen Response (IPR) in nematodeCaenorhabditis elegansindicate that there are similarities between these two transcriptional immunological programs. The IPR is induced during infection with natural intracellular fungal and viral pathogens of the intestine and promotes resistance against these pathogens. Similarly, the IFN‐I response is induced by viruses and other intracellular pathogens and promotes resistance against infection. Whether the IPR and the IFN‐I response evolved in a divergent or convergent manner is an unanswered and exciting question, which could be addressed by further studies of immunity against intracellular pathogens inC. elegansand other simple host organisms. Here we highlight similar roles played by RIG‐I‐like receptors, purine metabolism enzymes, proteotoxic stressors, and transcription factors to induce the IPR and IFN‐I response, as well as the similar consequences of these defense programs on organismal development.