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Pathogenic bacteria modulate pheromone response to promote mating

Nature volume 613, pages 324–331 (2023)Cite this article

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Abstract

Pathogens generate ubiquitous selective pressures and host–pathogen interactions alter social behaviours in many animals1,2,3,4. However, very little is known about the neuronal mechanisms underlying pathogen-induced changes in social behaviour. Here we show that in adult Caenorhabditis elegans hermaphrodites, exposure to a bacterial pathogen (Pseudomonas aeruginosa) modulates sensory responses to pheromones by inducing the expression of the chemoreceptor STR-44 to promote mating. Under standard conditions, C. elegans hermaphrodites avoid a mixture of ascaroside pheromones to facilitate dispersal5,6,7,8,9,10,11,12,13. We find that exposure to the pathogenic Pseudomonas bacteria enables pheromone responses in AWA sensory neurons, which mediate attractive chemotaxis, to suppress the avoidance. Pathogen exposure induces str-44 expression in AWA neurons, a process regulated by a transcription factor zip-5 that also displays a pathogen-induced increase in expression in AWA. STR-44 acts as a pheromone receptor and its function in AWA neurons is required for pathogen-induced AWA pheromone response and suppression of pheromone avoidance. Furthermore, we show that C. elegans hermaphrodites, which reproduce mainly through self-fertilization, increase the rate of mating with males after pathogen exposure and that this increase requires str-44 in AWA neurons. Thus, our results uncover a causal mechanism for pathogen-induced social behaviour plasticity, which can promote genetic diversity and facilitate adaptation of the host animals.

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Fig. 1: Exposure to pathogenic Pseudomonas strain PA14 suppresses pheromone avoidance in hermaphrodites and induces the pheromone response in AWA.
Fig. 2: Pathogenic Pseudomonas PA14 induces expression of str-44 in AWA.
Fig. 3: STR-44 acts in AWA as a pheromone receptor to regulate pheromone response in pathogen-exposed worms.
Fig. 4: Pathogen exposure regulates str-44 expression and pheromone response via zip-5 to promote mating.

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Data availability

All data generated or analysed during this study are included in the manuscript (and its supplementary information data files and the source data files). The sequencing results have been deposited at NCBI BioProject under accession no. PRJNA789902. Source data are provided with this paper.

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Acknowledgements

We thank the Caenorhabditis Genetics Center, funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for strains; C. I. Bargmann and P. Sengupta for plasmids and strains; N. Pierce, M.-K. Choi and Zhang laboratory members for discussions on the manuscript. A.L.S. is supported by NSERC (RGPIN-2019-06843), C.L. is supported by an NSERC PGS-D, R.A.B. is supported by NIH (GM118775, R35GM144076) and Y.Z. is supported by NIH (NS115484).

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Author notes

  1. These authors contributed equally: Taihong Wu, Minghai Ge

Authors and Affiliations

  1. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA

    Taihong Wu, Minghai Ge, Min Wu, Fengyun Duan, Jingting Liang, Maoting Chen, Xicotencatl Gracida, He Liu, Wenxing Yang & Yun Zhang

  2. Center for Brain Science, Harvard University, Cambridge, MA, USA

    Taihong Wu, Minghai Ge, Min Wu, Fengyun Duan, Jingting Liang, Maoting Chen, Xicotencatl Gracida, He Liu, Wenxing Yang & Yun Zhang

  3. Department of Chemistry, University of Florida, Gainesville, FL, USA

    Abdul Rouf Dar & Rebecca A. Butcher

  4. Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada

    Chengyin Li & Arneet L. Saltzman

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Contributions

T.W., M.G. and Y.Z. conceived the study and designed the experiments. T.W., M.G., F.D., J.L. and W.Y. performed behavioural assays, calcium imaging and gene expression analysis and analysed data. T.W., F.D., X.G. and H.L. generated TRAP–RNA-seq samples. T.W., M.W. and M.C. analysed the TRAP–RNA-seq data. A.R.D. and R.A.B. generated pheromones for the study. T.W., M.G., C.L., A.L.S. and Y.Z. designed ChIP–qPCR experiments. T.W. and M.G. collected samples for ChIP–qPCR assays, and C.L. and A.L.S. performed ChIP–qPCR and analysed data. T.W., M.G., M.W., M.C., C.L., A.L.S. and Y.Z. wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Yun Zhang.

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Extended data figures and tables

Extended Data Fig. 1 Pathogen exposure suppresses avoidance of ascaroside pheromones.

a, b, k, Schematics for bacteria exposure (a), quadrant assay on ascaroside pheromones (b), and histamine treatment during chemotaxis (k). c–j, Avoidance of the mixture of ascaroside pheromones ascr#2, ascr#3 and ascr#5 over a range of concentrations indicated for each ascaroside at equilibrium (c–g) or avoidance of individual ascarosides (h–j, 10 nM each at equilibrium) in wild-type adult hermaphrodites when exposed to E. coli OP50 or P. aeruginosa PA14. Positive avoidance index, avoidance. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different assays. Dots, avoidance indexes of individual assays. P values are derived from two-tailed unpaired t test, asterisks indicate significant difference, **** P < 0.0001, *** P < 0.001, * P < 0.05; ns, not significant. P values are shown in Source data.

Source data

Extended Data Fig. 2 Pathogen exposure induces pheromone response of AWA neurons.

a–d, Traces of GCaMP6 signals in AWA neurons of wild-type adult hermaphrodites in response to pheromones at different concentrations when exposed to OP50 or PA14 (a,c) and quantification of average GCaMP6 signals in AWA (b,d) during pheromone stimulation in a,c, respectively. Phe, pheromone mixture of ascr#2, ascr#3 and ascr#5 (at indicated concentrations for each). Lines in traces, mean; shades, s.e.m. (a,c). Fb is baseline, defined as average GCaMP6 signal in the first 30 s. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different worms (b,d). Dots, average GCaMP6 signals of individual worms during pheromone stimulation (b,d). P values are derived from two-tailed Mann-Whitney test, asterisks indicate significant difference, **** P < 0.0001, ** P < 0.01. e, Paradigm of a long-term exposure to bacterial pathogen PA14. f, Avoidance of ascaroside pheromones in wild-type adult hermaphrodites exposed to OP50 or PA14 in a long-term exposure paradigm as shown in (e). Pheromones, mixture of ascr#2, ascr#3 and ascr#5 (10 nM each at equilibrium). Positive avoidance index, avoidance. Numbers in parentheses, number of different assays. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Dots, avoidance indexes of individual assays. P values are derived from two-tailed unpaired t test, asterisks indicate significant difference, ** P < 0.01. g, h, Traces of GCaMP6 signals in AWA neurons of wild-type adult hermaphrodites in response to ascaroside pheromones when exposed to OP50 or PA14 in the long-term exposure paradigm (g) and quantification of average GCaMP6 signals in AWA (h) during pheromone stimulation in g. Phe, pheromones, individual or mixture of ascr#2, ascr#3 and ascr#5 (1 μM each). Lines in traces, mean;.shades, s.e.m. (g). Fb is baseline, defined as average GCaMP6 signal in the first 30 s. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different worms (h). Dots, average GCaMP6 signals of individual worms during pheromone stimulation (h). P values are derived from One-way ANOVA with Dunnett’s multiple comparisons test, asterisks indicate significant difference, **** P < 0.0001, ** P < 0.01; ns, not significant. i–l, Traces of GCaMP6 signal in AWA neurons of unc-13(e51) (i) or unc-31(e928) (k) mutant adult hermaphrodites in response to pheromones when exposed to OP50 or PA14 and quantification of average GCaMP6 signals (j,l) during pheromone stimulation in i,k, respectively. Phe, Pheromone mixture of ascr#2, ascr#3 and ascr#5 (1 μM each). Lines in traces, mean; shades, s.e.m. (i,k). Fb is baseline, defined as average GCaMP6 signal in the first 30 s. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different worms (j,l). Dots, average GCaMP6 signals of individual worms during pheromone stimulation (j,l). P values are derived from two-tailed unpaired t test, asterisks indicate significant difference, **** P < 0.0001, ** P < 0.01. P values are shown in Source data.

Source data

Extended Data Fig. 3 Different roles of sensory neurons ASK, ASI and ADL in pheromone response and PA14-induced modulation.

a–c, Avoidance of ascaroside pheromones when exposed to E. coli OP50 or P. aeruginosa PA14 (avoidance index) and modulation of pheromone response by PA14 (modulation index) in wild-type adult hermaphrodites and transgenic adult hermaphrodites in which ASK neurons are genetically ablated (a), or ASI neurons are genetically ablated (b), or neurotransmission of ADL is blocked (c). Pheromones, mixture of ascr#2, ascr#3 and ascr#5 (10 nM each at equilibrium). Positive avoidance index, avoidance. Positive modulation index, suppression of avoidance by PA14 exposure. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different assays. Dots, avoidance indexes or modulation indexes of individual assays. P values are derived from Two-way ANOVA with Tukey’s multiple comparisons test (avoidance index in a-c) or two-tailed unpaired t test (modulation index in a and c) or two-tailed Mann-Whitney test (modulation index in b), asterisks indicate significant difference, **** P < 0.0001, *** P < 0.001, * P < 0.05; ns, not significant. P values are shown in Source data.

Source data

Extended Data Fig. 4 Exposure to pathogenic Serratia bacteria induces pheromone response of AWA.

a, Schematic of exposure to different bacteria strains for calcium imaging analysis. b,c, Traces of GCaMP6 signals in AWA neurons of wild-type adult hermaphrodites in response to ascaroside pheromones after 4–6 h exposure to different bacteria strains (b) and quantification of average GCaMP6 signals in AWA (c) during pheromone stimulation in b. Phe, pheromone mixture of ascr#2, ascr#3 and ascr#5 (1 μM each). Lines in traces, mean; shades, s.e.m. (b). Fb is baseline, defined as average GCaMP6 signal in the first 30 s. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values.Numbers in parentheses, number of different worms (c). Dots, average GCaMP6 signals of individual worms during pheromone stimulation (c). P values are derived from Kruskal-Wallis test with Dunn’s multiple comparisons test, asterisks indicate significant difference, *** P < 0.001; ns, not significant. P values are shown in Source data. d, e, Schematics for TRAP-RNAseq (d) and for exposure to different bacteria strains for TRAP-RNAseq analysis (e).

Source data

Extended Data Fig. 5 Exposure to PA14 does not induce AWA expression of several previously identified receptors for pheromone sensing.

a–g, Sample images (a–f) and quantification (g) for srg-36p::gfp, daf-37p::gfp, srd-1p::gfp, srx-44p::gfp, srbc-64p::gfp, and srbc-66p::gfp expression in AWA neurons in adult hermaphrodites exposed to OP50 or PA14 for 4–6 h (reporters are indicated by the name of the promoters in g). Arrows point to cell bodies of several sensory neurons. Lines outline worm bodies. Scale bar (applicable to images in the same row), 20 μm. A, anterior. D, dorsal (arrows point out of the page for OP50 condition in c and for PA14 condition in d). Intensity of fluorescence signals is normalized using average intensity of AWA expression of str-44p::gfp in OP50-exposed worms measured in parallel. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different neurons (g). Dots, signals of individual neurons (g). P values are derived from Two-way ANOVA with Bonferroni’s multiple comparisons test, asterisks indicate significant difference, **** P < 0.0001; ns, not significant. P values are shown in Source data.

Source data

Extended Data Fig. 6 TRAP-RNAseq analysis on AWA neurons in worms exposed to OP50, or PA14, or PA14-gacA(–), or P. fluorescens.

a, Avoidance of ascaroside pheromones in adult transgenic hermaphrodites expressing eGFP::RPL-1 selectively in AWA using gpa-4delta6p promoter exposed to OP50 or PA14 for 4–6 h. Pheromones, mixture of ascr#2, ascr#3 and ascr#5 (10 nM each at equilibrium). Positive avoidance index, avoidance. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, numbers of different assays. Dots, avoidance indexes of individual assays. P values are derived from Two-way ANOVA with Tukey’s multiple comparisons test, asterisks indicate significant difference, ** P < 0.01; ns, not significant. P values are shown in Source data. b, Expression of a transcriptional reporter using an AWA-specific promoter (gpa-4delta6p) in adult transgenic hermaphrodites exposed to OP50 or PA14 for 4–6 h. Intensity of fluorescence signals is normalized using average intensity of AWA expression of gpa-4delta6p::gfp in OP50-exposed worms measured in parallel. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different neurons. Dots, signals of individual neurons. P values are derived from two-tailed Mann-Whitney test; ns, not significant. P values are shown in Source data. c, d, Principal component analysis (c) and hierarchical clustering (d) of samples based on the expression of 806 genes that were differentially expressed in AWA neurons under 4 conditions (exposure to OP50, or PA14, or PA14-gacA(-), or P. fluorescens). log2CPM (counts per million) values calculated using cpm function in edgeR package represent expression levels. OP1–3, samples 1–3 for OP50-exposed hermaphrodites; PA1–3, samples 1–3 for PA14-exposed hermaphrodites; gacA1–3, samples 1–3 for PA14-gacA(-)-exposed hermaphrodites; Pf1–3, samples 1–3 for P. fluorescens-exposed hermaphrodites. e, f, 57 genes showing differential expression in AWA between PA14-exposed worms and OP50-exposed worms (e, FDR < 0.1) and differential expression in AWA between PA14-gacA(-)-exposed worms and OP50-exposed worms (f, FDR < 0.1), but showing no difference between P. fluorescens-exposed worms and OP50-exposed worms. Dashed lines indicate 1 or -1. FC, fold change (in comparison with expression in OP50-exposed worms). P values and FDR are shown in Supplementary Data 5.

Source data

Extended Data Fig. 7 Exposure to PA14 or PA14-gacA(-) induces expression of str-44 in AWA and analysis of str-44 function in pheromone response.

a, Sample images of adult transgenic hermaphrodites expressing str-44p::gfp exposed to OP50, PA14, PA14-gacA(-) or P. fluorescens for 4–6 h. Arrows indicate neuronal cell bodies. Lines outline worm bodies. Scale bar (applicable to images in the same row), 20 μm. A, anterior. D, dorsal. b, Schematic showing the allele str-44(syb5943) that contains the str-44 genomic locus tagged with sequences of T2A peptide and GFP. Boxes indicate protein coding exons. c, Sample images of AWA expression of str-44::T2A-gfp in adult str-44(syb5943) hermaphrodites containing the str-44 genomic locus tagged with a gfp sequence when exposed to OP50 or PA14 for 4–6 h. Dashed lines indicate enlarged views. Arrows indicate neuronal cell bodies. Lines outline worm bodies. Scale bar (applicable to images in the same row), 20 μm. A, anterior. D, dorsal (arrows point slightly out of the page). d, Quantification of AWA expression of str-44::T2A-gfp in adult str-44(syb5943) hermaphrodites containing the str-44 genomic locus tagged with a gfp sequence when exposed to OP50, PA14, PA14-gacA(-) or P. fluorescens for 4–6 h. Intensity of fluorescence signals is normalized using average intensity of AWA expression of str-44::T2A-gfp in OP50-exposed worms measured in parallel. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values.  Numbers in parentheses, number of different neurons. Dots, signals of individual neurons. P values are derived from Kruskal-Wallis test with Dunn’s multiple comparisons test, asterisks indicate significant difference, **** P < 0.0001; ns, not significant. P values are shown in Source data. e, Schematic showing the deletion mutation in the str-44(syb4262) allele. f, Avoidance index in wild-type and str-44 mutant adult hermaphrodites, and in transgenic adult hermaphrodites expressing a wild-type str-44 DNA using either str-44 promoter or AWA-specific promoter gpa-4delta6p in str-44 mutant background exposed to OP50 or PA14. Pheromones, mixture of ascr#2, ascr#3 and ascr#5 (10 nM each at equilibrium). Positive avoidance index, avoidance. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different assays. Dots, avoidance indexes of individual assays. P values are derived from Two-way ANOVA with Tukey’s multiple comparisons test, asterisks indicate significant difference, *** P < 0.001, ** P < 0.01, * P < 0.05; ns, not significant. P values are shown in Source data.

Source data

Extended Data Fig. 8 AWA expression of str-44 is regulated by biological features associated with live PA14 cells and the function of zip-5, jmjd-3.1 and set-2 in AWA.

a–c, Quantification of str-44p::gfp signals in AWA in adult hermaphrodites exposed to OP50 or PA14 for 4–6 h, or to a heat shock at 32 °C for 8 h, or to 10 mM hydrogen peroxide for 24 h (a), or to a osmotic shock at 300 mOsm for 24 h (b), or to OP50 and PA14 cultivated under conditions that induce a higher level of virulence in PA14 (c, Methods). Intensity of fluorescence signals is normalized using average intensity of str-44p::gfp in OP50-exposed worms measured in parallel. d, Schematic of exposure to PA14 odorants by placing a lawn of PA14 on the lid. e,f, Traces of GCaMP6 signals in AWA neurons of wild-type adult hermaphrodites in response to pheromones after 4–6 h exposure to OP50, or PA14, or PA14 odorants (a lawn of PA14 on the lid) (e) and quantification of average GCaMP6 signals in AWA (f) during pheromone stimulation in e. Phe, pheromone mixture of ascr#2, ascr#3 and ascr#5 (1 μM each). Lines in traces, mean; shades, s.e.m. Fb is baseline, defined as average GCaMP6 signal in the first 30 s. g–j, Exposure to supernatant of PA14 culture does not induce AWA expression of str-44p::gfp (g), but exposure to PA14 cells with supernatant removed does (h); and heat-killing of PA14 cells abolishes the induction (i). Exposure to E. coli expressing the exotoxin ToxA of PA14 also does not induce str-44 expression (j). Intensity of fluorescence signals is normalized using average intensity of str-44p::gfp in OP50-exposed worms measured in parallel. pET100, cloning vector for ToxA. k,m,q,s,t, Avoidance of pheromones in wild-type, osm-9 mutant (k), zip-5 mutant (m), jmjd-3.1 mutant (s), or set-2 mutant (t) adult hermaphrodites, or in transgenic adult hermaphrodites specifically expressing in AWA a wild-type zip-5 DNA or a wild-type zip-5 DNA tagged with a sequence of 3xFLAG in zip-5 mutant background (m), or in transgenic adult hermaphrodites specifically expressing in AWA a wild-type zip-5 DNA tagged with a sequence of 3xFLAG in wild-type background (q), or in transgenic adult hermaphrodites expressing specifically in AWA a wild-type jmjd-3.1 DNA or a wild-type set-2 DNA in the respective mutant background (s,t) when exposed to OP50 or PA14 (avoidance indexes in q were measured during sample collection for ChIP-qPCR assays), and modulation of pheromone avoidance by PA14 (k). Pheromones, mixture of ascr#2, ascr#3 and ascr#5 (10 nM each at equilibrium). Positive avoidance index, avoidance. Positive modulation index, suppression of avoidance by PA14 exposure. l, Sample images of adult transgenic hermaphrodites expressing zip-5p::gfp exposed to OP50 or PA14 for 4–6 h. Dashed lines indicate enlarged views. Arrows indicate neuronal cell bodies. Scale bar (applicable to both images), 20 μm. A, anterior. D, dorsal. n,o,u,v, Quantification of str-44p::gfp signals in AWA in wild-type and zip-5 mutant adult hermaphrodites when exposed to OP50 or PA14 for 4–6 h (n), or in wild-type and zip-5 mutant adult hermaphrodites when exposed to OP50 or PA14 for 4–6 h or starved for 5 h (o), or in jmjd-3.1 mutant (u) or set-2 mutant (v) hermaphrodites exposed to OP50 or PA14 for 4-6 h. Intensity of fluorescence signals is normalized using average intensity of str-44p::gfp in OP50-exposed wild-type worms measured in parallel. p, Diagram of qPCR amplicon positions on str-44 genomic locus in ChIP assays for association of ZIP-5::3xFLAG with str-44 sequence. r, Ratio of ChIP signal (% of input) in PA14-exposed worms versus OP50-exposed animals. n = 3 independent assays, mean ± SD. Dots, ratios of individual assays. P values are derived from Two-way ANOVA with Bonferroni’s multiple comparisons test, asterisks indicate significant difference, **** P < 0.0001, *** P < 0.001, ** P < 0.01. P values are shown in Source data. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different neurons (a-c,g-j,n,o,u,v) or different worms (f) or different assays (k,m,q,s,t). Dots, signals of individual neurons (a-c,g-j,n,o,u,v) or individual worms (f), or avoidance indexes (k,m,q,s,t) or modulation indexes (k) of individual assays. P values are derived from Kruskal-Wallis test with Dunn’s multiple comparisons test (a,b,f–j) or two-tailed Mann-Whitney test (c) or Two-way ANOVA with Tukey’s multiple comparisons test [(avoidance index in k),n,o,q,u,v] or Two-way ANOVA with Bonferroni’s multiple comparisons test (m,s,t) or two-tailed unpaired t test (modulation index in k), asterisks indicate significant difference, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05; ns, not significant. P values are shown in Source data.

Source data

Extended Data Fig. 9 Inactivating npr-1 regulates pheromone response differently from pathogen exposure.

a, Avoidance of pheromones in wild-type and npr-1 mutant adult hermaphrodites. Pheromones, mixture of ascr#2, ascr#3 and ascr#5 (10 nM each at equilibrium). Positive avoidance index, avoidance. b–m, Traces of GCaMP signals in ASK (b,d,f) or ADL (h,j,l) neurons of wild-type adult hermaphrodites in response to ascaroside pheromones when exposed to OP50 or PA14 for 4-6 h and quantification of average GCaMP signals (c,e,g,i,k,m) during pheromone stimulation in b,d,f,h,j,l, respectively. Phe, pheromone mixture of ascr#2, ascr#3 and ascr#5 at indicated concentrations (10nM, 100nM or 1000nM each) (b,d,f,h,l) or 100nM ascr#3 (j). Lines in traces, mean; shades, s.e.m. (b,d,f,h,j,l). Fb is baseline, defined as average GCaMP signal in the first 30 s. n, Quantification of str-44p::gfp signals in AWA in wild-type and npr-1 mutant adult hermaphrodites. Intensity of fluorescence signals is normalized using average intensity of str-44p::gfp in OP50-exposed wild-type worms. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different assays (a) or different worms (c,e,g,i,k,m) or different neurons (n). Dots, Avoidance indexes of individual assays (a) or signals of individual worms (c,e,g,i,k,m) or signals of individual neurons (n). P values are derived from One-way ANOVA with Tukey’s multiple comparisons test (a) or two-tailed unpaired t test (c,g,i,k,m) or two-tailed Mann-Whitney test (e) or Kruskal-Wallis test with Dunn’s multiple comparisons test (n), asterisks indicate significant difference, **** P < 0.0001, *** P < 0.001; ns, not significant. P values are shown in Source data.

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Extended Data Fig. 10 Wild-type hermaphrodites exposed to OP50 or PA14 produce male progeny by selfing at a very low frequency, and mating on lawns of different sizes.

a, Schematic of control experiment. b, Percentage of male progeny produced by selfing of adult hermaphrodites exposed to OP50 or PA14 for 4 h. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different control assays (progeny of 10 hermaphrodites (P0) were scored for each assay). Dots, male progeny in F1 (%) of individual assays. Two-tailed Mann-Whitney test; ns, not significant. P values are shown in Source data. c, Schematic of mating assays on lawns of different sizes. d, Frequency of successful mating for adult wild-type hermaphrodites and males on lawns of different sizes. In box plots, the centre line shows the median, box edges delineate 1st and 3rd quartiles and whiskers extend to minimum and maximum values. Numbers in parentheses, number of different assays (each contains 10 hermaphrodites with their outcome of mating scored individually). Dots, mating frequency of individual assays. P values are derived from One-way ANOVA with Tukey’s multiple comparisons test, asterisks indicate significant difference, **** P < 0.0001, *** P < 0.001. P values are shown in Source data.

Source data

Supplementary information

Reporting Summary

Supplementary Data 1

Genes differentially expressed between AWA neurons of adult hermaphrodite worms exposed to PA14 and AWA neurons of adult hermaphrodite worms exposed to OP50 for 4-6 hours (FDR < 0.1). P values are derived from two-sided exact test used in the R package “edgeR” and the FDR values are Benjamini–Hochberg adjusted P values. CPM, counts per million. Average log2CPM values were calculated using samples in the comparison.

Supplementary Data 2

Genes differentially expressed between AWA neurons of adult hermaphrodite worms exposed to PA14-gacA(−) and AWA neurons of adult hermaphrodite worms exposed to OP50 for 4-6 hours (FDR < 0.1). P values are derived from two-sided exact test used in the R package “edgeR” and the FDR values are Benjamini–Hochberg adjusted P values. CPM, counts per million. Average log2CPM values were calculated using samples in the comparison.

Supplementary Data 3

Genes differentially expressed between AWA neurons of adult hermaphrodite worms exposed to P. fluorescens and AWA neurons of adult hermaphrodite worms exposed to OP50 for 4-6 hours (FDR < 0.1). P values are derived from two-sided exact test used in the R package “edgeR” and the FDR values are Benjamini–Hochberg adjusted P values. CPM, counts per million. Average log2CPM values were calculated using samples in the comparison.

Supplementary Data 4

GO term and KEGG pathways enriched in genes differentially expressed between AWA neurons of adult hermaphrodite worms exposed to PA14 and AWA neurons of adult hermaphrodite worms exposed to OP50 for 4-6 hours, and in genes differentially expressed between AWA neurons of adult hermaphrodite worms exposed to PA14-gacA(−) and AWA neurons of adult hermaphrodite worms exposed to OP50 for 4-6 hours (FDR < 0.1). P values are derived from one-sided Fisher’s Exact test implemented in DAVID Bioinformatics Resources 6.8.; Benjamini values are adjusted P values using the linear step-up method by Benjamini and Hochberg; FDR (false discovery rate) values are adjusted P values using adaptive linear step-up methodby Benjamini and Hochberg.

Supplementary Data 5

Genes differentially expressed between AWA neurons of adult hermaphrodite worms exposed to PA14 and AWA neurons of adult hermaphrodite worms exposed to OP50 for 4-6 hours, as well as between AWA neurons of adult hermaphrodite worms exposed to PA14-gacA(−) and AWA neurons of adult hermaphrodite worms exposed to OP50 for 4-6 hours, but not between P. fluorescens and OP50 (FDR < 0.1). P values are derived from two-sided exact test used in the R package “edgeR” and the FDR values are Benjamini–Hochberg adjusted P values. FC, fold change; CPM, counts per million. log2CPM values are average log2CPM calculated using samples in each comparison.

Supplementary Data 6

Expression levels measured in AWA TRAP–RNA-seq samples for previously identified receptors for pheromone sensing. CPM, counts per million. log2CPM values calculated using all samples are shown.

Supplementary Data 7

Transcription factors differentially expressed between AWA neurons of adult hermaphrodite worms exposed to PA14 and AWA neurons of adult hermaphrodite worms exposed to OP50 for 4-6 hours (FDR < 0.1) and their expression levels measured. P values are derived from two-sided exact test used in the R package “edgeR” and the FDR values are Benjamini–Hochberg adjusted P values. CPM, counts per million. log2FC (fold change) and average log2CPM values calculated using samples in the comparison are shown on sheet “TF_PA_FDR0.1”. log2CPM values calculated using all samples are shown on sheet “cpm”.

Source data

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Wu, T., Ge, M., Wu, M. et al. Pathogenic bacteria modulate pheromone response to promote mating. Nature 613, 324–331 (2023). https://doi.org/10.1038/s41586-022-05561-9

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