Figure 4: Involvement of RNAi genes in the effects of OxyS on C. elegans.

(a) Repelling effect of OxyR E. coli on various mutants of the C. elegans RNAi/microRNA pathways in food comparison assays. N=36 worms for each group. (b) che-2 mRNA levels in N2, alg-1 and rde-4 worms were shown by real-time PCR. (c) Levels of che-2 mRNA in alg-2, rrf-3, sid-1 and sid-2 worms fed with K12 and OxyR food. In a and c, two alleles of sid-1 (qt2 & pk3321) and sid-2 (qt42 & gk505) were examined, and data from only one allele for each gene (pk3321 and qt42) were presented, as both alleles of each gene gave similar results. (d) haf-6 but not haf-2 mutant showed similar distribution as N2 worms in the food comparison assays. N=36 worms. (e) OxyS had no effect on the level of che-2 mRNA in haf-2 worms, whereas the suppressive effect was present in haf-6 worms. (f) Comparison of food searching ability for worms fed on OxyS or che-2 RNAi bacteria. N=40 worms for each group. (g) Worms fed on OxyS or che-2 RNAi bacteria examined with food comparison assay. N=36 worms for each group. (h) Real-time PCR of che-2 mRNA levels to compare the effect of OxyS to che-2 dsRNA-based feeding RNAi. In e–h, 2 out of 10 trials with che-2 feeding RNAi showed significant effect in behavioural tests, and the data shown were from these two trials only. (i) Injection of the 17-nt complementary ssRNA suppressed the expression of Pche-2::che-2::gfp. Scramble small RNA (scRNA NC) was used as a negative control. N=20 worms for each group. P values were determined by two-tailed Student's t-test. *P<0.05; **P<0.01. All data are from three repeat experiments. Error bars represent s.e.m.