Extended Data Fig 2: sRNA and bacterial lawn training of C. elegans RNAi pathway mutants.

a, Wild-type and sid-2(qt42) worms were trained on OP50 or PA14 bacterial lawns for 24 h and tested for learned PA14 avoidance. b, c, Wild-type and dcr-1(mg375) worms were trained on OP50 or PA14 lawns (b) or OP50 or PA14 sRNA (c). d, Wild-type, dcr-1(mg375) and dcr-1(mg375);vha-6p::dcr-1 worms were trained on OP50 or PA14 bacterial lawns for 24 h and tested for learned PA14 avoidance. Training and choice assays for dcr-1(mg375) and dcr-1(mg375);vha-6p::dcr-1 worms were performed on the same plates. For choice assays, transgenic worms expressing the rescue construct and pharyngeal mCherry were counted using a fluorescence dissecting microscope. Non-transgenic, non-fluorescent dcr-1(mg375) sibling worms from the same plates were also counted and the results are shown. e, Wild-type and sid-1(pk3321) worms were trained on OP50 or PA14 bacterial lawns for 24 h and tested for learned PA14 avoidance. sid-1 mutants have a constitutively high naive avoidance, but are able to learn avoidance after training on a PA14 lawn, but not after training on sRNAs. f–j, Wild-type, rde-1(ne219) (f, g), rde-2(pk1657) (h, i) or rde-4(ne305) (j) worms were trained on OP50 or PA14 bacterial lawns (f, h, j) or sRNA (g, i) for 24 h and tested for learned PA14 avoidance. rde-1, rde-2 and rde-4 mutants are able to learn avoidance following bacteria lawn training, but do not additionally avoid PA14 following sRNA training. k, l, Wild-type and mut-7(pk720) worms were trained on OP50 or PA14 lawns (k) or sRNA (l) for 24 h and tested for learned PA14 avoidance. These mutants (in e–l) have high naive preference after training on sRNAs only. m–p, Wild-type and alg-1(gk214) (m, n) or drh-1(ok3495) (o, p) worms were trained on OP50 or PA14 bacterial lawns (m, o) or sRNA (n, p) for 24 h and tested for learned PA14 avoidance. Biological replicates: 3 (d, f, h–p), 4 (a–c, g), 6 (e). For choice assays, each dot represents an individual choice assay plate (average of 115 worms per plate) with data shown from all replicates. For box plots, centre line is the median, box extends from the 25th to the 75th percentile; whiskers denote minimum–maximum values. Two-way ANOVA (a–p), Tukey’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, n.s., not significant. Mean differences are shown using Cumming estimation plots⁴⁷, with each graphed as a bootstrap sampling distribution. Mean differences are depicted as dots; 95% confidence intervals are indicated by the ends of the vertical bars. See Supplementary Table 4 for exact sample sizes (n) and P values.