The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, wild-type C. elegans strains vary dramatically in their response to exogenous RNAi, challenging our characterization of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains, but dampens it further in others. Across strains, we observe variability in expression of known RNAi genes and strain-specific instances of pseudogenization and allelic divergence. Our results support the conclusions that Argonautes share overlapping functions, that germline RNAi incompetence is strain-specific but likely caused by genetic variants at common genes, and that RNAi pathways are evolving rapidly and dynamically. This work expands our understanding of RNAi by identifying conserved and variable pathway components, and it offers new access into characterizing gene function, identifying pathway interactions, and elucidating the biological significance of RNAi.

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Though natural systems harbor genetic and phenotypic variation, research in model organisms is often restricted to a reference strain. Focusing on a reference strain yields a great depth of knowledge but potentially at the cost of breadth of understanding. Furthermore, tools developed in the reference context may introduce bias when applied to other strains, posing challenges to defining the scope of variation within model systems. Here, we evaluate how genetic differences among 5 wild Caenorhabditis elegans strains affect gene expression and its quantification, in general and after induction of the RNA interference (RNAi) response. Across strains, 34% of genes were differentially expressed in the control condition, including 411 genes that were not expressed at all in at least 1 strain; 49 of these were unexpressed in reference strain N2. Reference genome mapping bias caused limited concern: despite hyperdiverse hotspots throughout the genome, 92% of variably expressed genes were robust to mapping issues. The transcriptional response to RNAi was highly strain- and target-gene-specific and did not correlate with RNAi efficiency, as the 2 RNAi-insensitive strains showed more differentially expressed genes following RNAi treatment than the RNAi-sensitive reference strain. We conclude that gene expression, generally and in response to RNAi, differs across C. elegans strains such that the choice of strain may meaningfully influence scientific inferences. Finally, we introduce a resource for querying gene expression variation in this dataset at https://wildworm.biosci.gatech.edu/rnai/.

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