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C. elegans interprets bacterial non-coding RNAs to learn pathogenic avoidance

Abstract

Caenorhabditis elegans must distinguish pathogens from nutritious food sources among the many bacteria to which it is exposed in its environment1. Here we show that a single exposure to purified small RNAs isolated from pathogenic Pseudomonas aeruginosa (PA14) is sufficient to induce pathogen avoidance in the treated worms and in four subsequent generations of progeny. The RNA interference (RNAi) and PIWI-interacting RNA (piRNA) pathways, the germline and the ASI neuron are all required for avoidance behaviour induced by bacterial small RNAs, and for the transgenerational inheritance of this behaviour. A single P. aeruginosa non-coding RNA, P11, is both necessary and sufficient to convey learned avoidance of PA14, and its C. elegans target, maco-1, is required for avoidance. Our results suggest that this non-coding-RNA-dependent mechanism evolved to survey the microbial environment of the worm, use this information to make appropriate behavioural decisions and pass this information on to its progeny.

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Fig. 1: sRNA of PA14 is sufficient to induce C. elegans pathogen avoidance.
Fig. 2: dsRNA transport, processing machinery, the PRG-1 piRNA pathway, and the germline are required for avoidance induced by bacterial sRNA.
Fig. 3: P11 is required and sufficient for learned avoidance behaviour.
Fig. 4: P11 induces avoidance through its target maco-1.

Data availability

Sequencing data are available from: BioProject under accession number PRJNA553700. Any data related to the study that are not provided in the Article and its Supplementary Information can be obtained upon reasonable request from the corresponding author.

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Acknowledgements

We thank the C. elegans Genetics Center for strains; the Genomics Core Facility at Princeton University; BioRender for model figure design software; W. Wang for helping to develop methods to sequence bacterial small RNAs; the laboratory of C.T.M. for discussion; and R. Clausen and J. Ashraf for assistance. C.T.M. is the Director of the Glenn Center for Aging Research at Princeton and an HHMI-Simons Faculty Scholar. This work was supported by a Pioneer Award to C.T.M. (NIGMS DP1GM119167), the Glenn Foundation for Medical Research (GMFR CNV1001899), the HHMI-Simons Faculty Scholar Program (AWD1005048), T32GM007388 (NIGMS) support of R.S.M. and G.D.V., and a Pioneer Award to Z.G. (DP1A1124669).

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Authors

Contributions

R.K., R.S.M., G.D.V., Z.G. and C.T.M. designed experiments. R.K. and R.S.M. performed experiments and analysed data. G.D.V. constructed P11 E. coli and PA14 mutant strains. L.R.P. and R.K. analysed small-RNA-seq data. R.K., R.S.M. and C.T.M. wrote the manuscript.

Corresponding author

Correspondence to Coleen T. Murphy.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 PA14 sRNAs induce maternal PA14 avoidance and increased daf-7 expression in the ASI neurons.

a, Worms exposed to a PA14 bacterial lawn for 24 h learn to avoid PA14 in a choice assay, but PA14 DNA exposure alone does not induce maternal avoidance of PA14. b, Training with large RNA (>200 nt) isolated from bacterial lawns of PA14 is not sufficient for maternal PA14 avoidance. c, Bioanalyzer results of isolated PA14 total RNA and fractionated small and large RNAs. RNA levels were normalized for worm training. d, ΔlasR sRNA exposure does not induce PA14 learned avoidance. e, Development of progeny of PA14 sRNA-trained mothers was not delayed compared to progeny of OP50-trained mothers. n = 6 plates per condition with 23–142 progeny per plate, mean ± s.e.m. f, pmk-1(km25) worms learn to avoid PA14 when exposed to PA14 bacteria lawns. g, pmk-1(km25) is not required for PA14 sRNA-induced pathogenic learning. h, irg-1p::gfp expression is induced by PA14 bacterial lawn exposure, but not by PA14 sRNAs alone. i, GFP intensity from h was quantified, n = 33, 24, 43 and 35 worms, left to right. j, Model of PA14 bacteria lawn and sRNA learning. k, Location of ASI and ASJ neurons. l, The 24-h exposure of worms to a PA14 lawn or PA14 sRNA increases daf-7p::gfp expression in the ASI neurons. Two-way ANOVA, Tukey’s multiple comparison test. n = 35, 41, 41 and 49 neurons, left to right. m, ΔlasR sRNA exposure does not induce changes in daf-7p::gfp ASI expression. One-way ANOVA, Tukey’s multiple comparison test. n = 38, 27 and 29 neurons, left to right. Biological replicates: 2 (a), 3(bd, fi, m), 6 (l). 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. One-way (d, m) and Two-way ANOVA (a, b, eg, i, l), 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.

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. fj, 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 el) have high naive preference after training on sRNAs only. mp, 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, hp), 4 (ac, 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 (ap), 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.

Extended Data Fig. 3 sRNA and bacterial lawn training of C. elegans sRNA pathway and germline mutants.

ae, Wild-type and prg-1(n4357) (a), prg-1 germline rescue (b), rrf-1(pk1417) (c), rrf-3(pk1426) (d), or hpl-2(ok916) worms (e) were trained on OP50 or PA14 bacterial lawns or sRNA (a, right) for 24 h and tested for learned PA14 avoidance. f, g, Representative images of OP50- or PA14-trained prg-1;daf-7::gfp (f) or glp-1;daf-7::gfp (g) mothers. ASI neuron = white arrow, ASJ neuron = grey arrow. h, Germline-less glp-1 mutants induce daf-7p::gfp expression in the ASJ neuron after PA14 lawn exposure. n = 42 (OP50), 46 (PA14) neurons, two-tailed Student’s t-test. i, Wild-type and meg-3(tm4259) meg-4(ax2026) worms were trained on OP50 or PA14 bacterial lawns for 24 h and tested for learned PA14 avoidance. Biological replicates: 3 (ai). 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 (ae, i), Tukey’s multiple comparison test. ****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.

Extended Data Fig. 4 sid-1, sid-2 and dcr-1 F1 progeny of plate and sRNA trained parent worms.

ac, Progeny of sid-1 (a), sid-2 (b) and dcr-1 (c) mutants are defective in both transgenerational pathogen avoidance following maternal bacterial lawn (left) and PA14 sRNA training (right). Biological replicates: 3 (ac). 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 (ac), Tukey’s multiple comparison test. **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.

Extended Data Fig. 5 RNA sequencing of sRNAs from PA14.

a, sRNA training protocol using RNA isolated from PA14 cultures grown at 25 °C or 15 °C on plates, or in liquid culture. b, Principal component analysis of PA14 sRNA sequencing. c, Upregulated sRNAs in 25-°C grown PA14 compared to 15 °C-grown and liquid-grown PA14 sRNAs. The outermost grey circle represents the PA14 genome, with previously identified sRNAs that were upregulated (DESeq2, adjusted P ≤ 0.05) in the 25-°C plate condition relative to 15-°C plate grown PA14 (pink) or liquid-grown PA14 (green) are indicated. Overlapping sRNAs upregulated in the 25-°C plate condition relative to both other conditions are noted (blue dots). sRNAs annotated with white lines. d, Venn diagram showing the number of PA14 sRNAs upregulated at 25-°C growth conditions compared to liquid growth (blue circle) or 15-°C growth (red circle). The six shared upregulated sRNAs are shown.

Extended Data Fig. 6 Identification and testing of the differentially regulated PA14 sRNA.

a, P11 expressed in E. coli induces PA14 avoidance in a choice assay between PA14 and E. coli strain MG1655. Worms were trained on OP50, PA14, E. coli strain MG1655 containing empty vector (control), or E. coli strain MG1655 expressing PA14 small RNAs (red). Following training, a choice assay was performed between E. coli strain MG1655 and PA14. Similar to Fig. 3c, worms trained on PA14 or E. coli expressing P11 exhibited PA14 avoidance behaviour. One-way ANOVA with Tukey’s multiple comparison test. b, PA14 plate-trained worms do not avoid E. coli expressing P11 compared to OP50 in a choice assay. Two-tailed Student’s t-test. c, PA14 ΔP11 bacteria grown on NGM plates produce pyocyanin (blue pigment on plates) similar to PA14. d, e, Model showing induced learning pathways for wild-type worms on isolated PA14 ΔP11 sRNA (d) or on PA14 ΔP11 bacteria lawns (e). f, Exposure to E. coli expressing P11 does not affect total brood size (left) (two-tailed Student’s t-test), or the number of progeny hatched per day (right). Two-way ANOVA with Tukey’s multiple comparison test. n = 15 worms were analysed per condition. g, Worms exposed to control (top) or E. coli expressing P11 appear healthy after 24 h of training. Biological replicates: 2 (a), 3 (b, c, g). 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. **P ≤ 0.01, ****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.

Extended Data Fig. 7 P11 region of homology with maco-1 and behaviour of vhp-1 mutants.

a, Wormbase depiction of the maco-1 genomic locus and the region of P11 homology (red). b, Model depicting induced learning pathways in wild-type or maco-1 mutant worms as a result of OP50 or PA14 lawn exposure. c, F1 progeny of OP50 or PA14 sRNA-exposed maco-1 mutant mothers were tested for PA14 avoidance behaviour. d, vhp-1expression is increased in PA14-exposed mothers, but not in F1 progeny3. Mean; DESeq2 adjusted P values are shown. P0, n = 6 replicates per condition; F1, n = 4 replicates per condition. e, The vhp-1(sa336) mutation is a null allele (early stop codon)40. Wild-type and vhp-1(sa366) worms were trained on OP50 or PA14 bacterial lawns for 24 h and tested for learned PA14 avoidance (top). f, Wild-type and vhp-1(sa366) worms were trained on OP50 or PA14 sRNA for 24 h and tested for learned PA14 avoidance. g, Progeny of vhp-1(sa366) PA14 lawn-trained worms inherit PA14 avoidance behaviour. h, F1 progeny of OP50 or PA14 lawn-exposed maco-1 mutant mothers were tested for PA14 avoidance behaviour. Biological replicates: 3 (c, eh). 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 (c, eh), 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.

Supplementary information

Estimation statistics for pooled and individual choice assay data

Supplementary Information . Mean differences are shown using Cumming estimation plots, 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. Each panel shows the effect sizes for either pooled data or individual biological replicates from data shown in the main figures. The numbering of figure panels corresponds to the main figures.

Reporting Summary

DESeq2 results comparing annotated small RNAs isolated from PA14 bacteria grown at 25 

Supplementary Table 1 °C or 15 °C on plates. Complete DESeq2 results are reported. All differentially expressed genes (DESeq2, adjusted p-value < 0.05) are shown separately. In addition, genes upregulated at 25 °C compared to 15 °C and a summary of overlapping targets are described. N = 6 biological replicates for 25 °C, and 4 biological replicates for 15°C growth conditions.

DESeq2 results comparing annotated small RNAs isolated from PA14 bacteria grown at 25 

Supplementary Table 2 °C on plated or in liquid. Complete DESeq2 results are reported. All differentially expressed genes (DESeq2, adjusted p-value < 0.05) are shown separately. In addition, genes upregulated at 25°C compared to liquid growth conditions are provided. N = 6 biological replicates for 25°C, and 4 biological replicates for liquid growth conditions.

P11 homology to C. elegans coding and noncoding RNAs

Supplementary Table 3 . PA14 P11 homology with worm sequences was determined by BLAST. Results are shown for homology with worm mRNAs, piRNAs, and additional ncRNAs annotated on Wormbase.

Statistics reporting

Supplementary Table 4 . Statistics were generated using Prism 8. Detailed results from all figures are provided.

Primers used for bacterial cloning

Supplementary Table 5 .

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Kaletsky, R., Moore, R.S., Vrla, G.D. et al. C. elegans interprets bacterial non-coding RNAs to learn pathogenic avoidance. Nature 586, 445–451 (2020). https://doi.org/10.1038/s41586-020-2699-5

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