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Cooperative virulence via the collective action of secreted pathogen effectors

Nature Microbiology volume 8, pages 640–650 (2023)Cite this article

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Abstract

Although virulence is typically attributed to single pathogenic strains, here we investigated whether effectors secreted by a population of non-virulent strains could function as public goods to enable the emergence of collective virulence. We disaggregated the 36 type III effectors of the phytopathogenic bacterium Pseudomonas syringae strain PtoDC3000 into a ‘metaclone’ of 36 coisogenic strains, each carrying a single effector in an effectorless background. Each coisogenic strain was individually unfit, but the metaclone was collectively as virulent as the wild-type strain on Arabidopsis thaliana, suggesting that effectors can drive the emergence of cooperation-based virulence through their public action. We show that independently evolved effector suits can equally drive this cooperative behaviour by transferring the effector alleles native to the strain PmaES4326 into the conspecific but divergent strain PtoDC3000. Finally, we transferred the disaggregated PtoDC3000 effector arsenal into Pseudomonas fluorescens and show that their cooperative action was sufficient to convert this rhizosphere-inhabiting beneficial bacterium into a phyllosphere pathogen. These results emphasize the importance of microbial community interactions and expand the ecological scale at which disease may be attributed.

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Fig. 1: Metaclones of P. syringae facilitate the emergence of population virulence.
Fig. 2: Collective action of the PtoDC3000 metaclone supports growth of laboratory and natural freeloader strains.
Fig. 3: Portability of pathogenic effector repertoires.
Fig. 4: Effectors are sufficient for converting a beneficial strain into a pathogen.

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Data availability

Metaclone data are available in Supplementary Data 1. Barcode sequence data are available at https://doi.org/10.5281/zenodo.7254975.

Code availability

The script used for reading metaclone barcodes is available at https://doi.org/10.5281/zenodo.7249118 (https://zenodo.org/record/7249118).

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Acknowledgements

We thank the members of the Desveaux and Guttman laboratories for their advice and feedback throughout this project, with particular thanks to A. Martel for his assistance with strain transformations and to M. Dillon for his insights into effector extraction and construct synthesis. We would also like to thank and acknowledge A. Collmer and J. Chang for providing us with the strains PtoDC3000D36E (that is, D36E, CUCPB6119) and PfEtHAn, which were invaluable to this study. This project is supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants (to D.S.G. and D.D.).

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Author notes

  1. These authors contributed equally: Darrell Desveaux, David S. Guttman.

Authors and Affiliations

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

    Tatiana Ruiz-Bedoya, Pauline W. Wang, Darrell Desveaux & David S. Guttman

  2. Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario, Canada

    Pauline W. Wang, Darrell Desveaux & David S. Guttman

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Contributions

T.R.-B., D.D. and D.S.G. designed the project. T.R.-B. performed transformations, tri-parental matings, metaclone constructions, spray infections, in planta and in vitro growth assays, primer design, barcode quantification and analysed the data. P.W.W. generated sequence data and assisted with many aspects of the project. T.R.-B., D.D. and D.S.G. wrote the paper. All authors reviewed and agreed on the manuscript.

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Correspondence to Darrell Desveaux or David S. Guttman.

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Nature Microbiology thanks Maren Friesen, John Mansfield, Hai-Lei Wei and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 In-planta fitness of individual effector clones.

Bacterial growth was quantified on day 6 post spray-infection for at least 4 independent plant replicates infected with each of the 36 effector clones that comprise the well-expressed effector arsenal of the pathogen PtoDC3000. All infections were performed at the same time and controls were placed in each flat. D36E and PtoDC3000, each harboring an empty vector (EV), were used as negative and positive virulence controls, respectively. No individual effector clone deviated significantly from the mean of the effector-less control strain. Elements in boxplots represent the median, 25th and 75th percentiles, and 1.5 * inter-quantile ranges. All comparisons were performed via one-way ANOVA with post-hoc Tukey-Kramer HSD using a 95% confidence interval (Source data and p-values provided in Supplementary Data 1).

Extended Data Fig. 2 In-vitro fitness of individual effector clones.

Growth curves of individual effector clones composing the PtoDC3000 metaclone D36E::McDC[36], and control strains harboring empty vectors (EV). Harboring any of the individual effector plasmids did not substantially burden or benefit the growth of D36E in vitro. Data points represent the average optical density (OD) measured at 600 nm across 3 technical replicates grown in rich media (KB) with kanamycin. Time measures with SE > 0.07 were excluded as the high variability was likely due to condensation, which was noticeable during lag phase, predominantly over the first 10 hours. This, however, did not impact the overall trajectory of the growth curves and there were no overall substantial differences between effector clones. NC: negative buffer control.

Extended Data Fig. 3 Collective virulence emerges when using syringe pressure infiltration assays on A. thaliana.

a. Endophytic bacterial growth at 0-, 3-, 5- and 7-days post infection using syringe pressure infiltration. Diagrams on the right represent the corresponding population and genetic makeup of each infection. Error bars correspond to standard deviation from the mean of at least 4 biological replicates per treatment at each timepoint. b-d. Host symptoms shown in photos taken of 4 representative leaves per bacterial treatment, and 2 plants for MgSO4 controls at b, 3-, c, 5- and d, 7- days post infection.

Extended Data Fig. 4 Mean Shannon’s diversity index for metaclones does not deviate significantly throughout disease progression.

Diversity calculations were based on the relative abundance of each D36E::effector barcode composing the PtoDC3000 metaclones D36E::McDC[36] (ETI-eliciting) and D36E::McDC[35] (not ETI-eliciting). Barcode sequence diversity is reported as the mean Shannon index across four biological replicates + /- SE for each metaclone. No significant differences were found between any stage of infection and the mean barcode diversity of the original inoculum (Bonferroni-corrected two-sided Student’s t-tests. p-values: D36E::McDC[35]: D0pi-D3pi = 0.66, D0pi-D5pi = 0.41, D0pi-D7pi = 0.89; D36E::McDC[36]: D0pi-D3pi = 0.56, D0pi-D5pi = 0.57, D0pi-D7pi = 0.35)). Error bars represent standard error across 4 biological replicates. Higher variance across replicates is observed in the ETI-eliciting metaclone D36E::McDC[36], particularly on the latter stages of the infection process, which can be attributed to smaller population sizes and increased drift in the metaclone composition.

Extended Data Fig. 5 Freeloading strains benefit from collective virulence and coexist with cooperating portion of the metaclone.

The virulent PtoDC3000 metaclone D36E::McDC[35] or mock population (D36E::EV) were mixed with strains unfit for multiplication in the plant apoplastic space (that is. freeloaders). Bacterial endophytic growth is reported at 7 dpi for cooperator and freeloader, separately and together, following standard spray infection of mixed populations and controls (Fig2). Differential antibiotic selection shown was used to measure fitness of the two genotypes present in each population, together (Total Growth –Rifampicin only -grey) and separately (Cooperator -rifampicin and tetracycline -blue; Freeloader- rifampicin and kanamycin -orange) a. The effectorless polymutant strain D36E, and b. the natural soil-associated sister species Pf0-1 are rescued to significantly higher load when in the presence of public goods provided cooperatively by the metaclone. Elements in boxplots represent the median of 6 biological replicates per treatment per selection category, the 25th and 75th percentiles, and 1.5 * inter-quantile ranges. Letters represent significant groups estimated via one-way ANOVA with post-hoc Tukey-Kramer HSD using a 95% confidence interval (Source data and p-values in in Supplementary Data 1).

Extended Data Fig. 6 Metaclone representing the effector allele overlap between PtoDC3000 and PmaES4326, McDC&ES[7], fails to match fitness of PtoDC3000 and the non-ETI-eliciting metaclone D36E::McDC[35].

D36E::McDC&ES[7] shows a fitness significantly closer to that of the effectorless polymutant D36E::EV and does not restore the growth levels typical of their wildtype parental strain PtoDC3000 or its non-ETI eliciting metaclone D36E::McDC[35]. In-planta fitness was quantified 6 dpi for 5 biological replicates per treatment and 4 independent infection replicates. Elements in boxplots represent the median, 25th and 75th percentiles, and 1.5 * inter-quantile ranges. Comparisons were performed via one-way ANOVA and post-hoc Tukey-Kramer HSD using a 95% confidence interval was performed separately for 4 independent experiments (Source data and p-values in Supplementary Data 1).

Extended Data Fig. 7 Representative plant photos show symptom development for the metaclones D36E::McDC[35] and PfEtHAn::McDC[35] compared to their effector-less background and the pathogen PtoDC3000.

For each treatment, a total of 8 leaves from 2 plants show host symptoms at a. 3, b. 5, and c. 7 days post bacterial pressure infiltration. The progression of symptom development is visible in photos taken with a no-flash standard settings (left panels) and with a camera green-pass effect filter setting which facilitates the inspection of lesions (grey) and viable (green) tissue (right panels). The MgS04 control, D36E::EV and PfEtHAn::EV treatments are presented on the left (top to bottom), and PtoDC3000::EV, D36E::McDC[35] and PfEtHAn::McDC[35] are presented on the right (top to bottom).

Extended Data Fig. 8 Dose response of the PtoDC3000 metaclone D36E::McDC[35].

A range of inoculum concentrations (OD600 0.5, 1, 2 and 4) was tested on 8 plants and visual symptoms were followed at 3, 5, 6 and 12 dpi. Photos were taken with a green-only pass filter, showing in grey any chlorotic yellow or brown tissue. Plant fitness (ie. green) detectably declines more when infected with the pathogen PtoDC3000 and the D36E::McDC[35] than it does for the effectorless control D36E::EV. No visual differences are observed across the range of concentrations tested for the metaclone D36E::McDC[35] and plant fitness is visually reduced at an optical density as low as 0.5.

Supplementary information

Reporting Summary

Supplementary Tables 1–3.

Supplementary Data 1

Source data and statistical analyses for Figs. 1–4 and Extended Data Figs. 1, 5 and 6.

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Ruiz-Bedoya, T., Wang, P.W., Desveaux, D. et al. Cooperative virulence via the collective action of secreted pathogen effectors. Nat Microbiol 8, 640–650 (2023). https://doi.org/10.1038/s41564-023-01328-8

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