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The long-term maintenance of a resistance polymorphism through diffuse interactions

Abstract

Plant resistance (R) genes are a crucial component in plant defence against pathogens1. Although R genes often fail to provide durable resistance in an agricultural context, they frequently persist as long-lived balanced polymorphisms in nature2,3,4. Standard theory explains the maintenance of such polymorphisms through a balance of the costs and benefits of resistance and virulence in a tightly coevolving host–pathogen pair5,6. However, many plant–pathogen interactions lack such specificity7. Whether, and how, balanced polymorphisms are maintained in diffusely interacting species8 is unknown. Here we identify a naturally interacting R gene and effector pair in Arabidopsis thaliana and its facultative plant pathogen, Pseudomonas syringae. The protein encoded by the R gene RPS5 recognizes an AvrPphB homologue (AvrPphB2) and exhibits a balanced polymorphism that has been maintained for over 2 million years (ref. 3). Consistent with the presence of an ancient balanced polymorphism, the R gene confers a benefit when plants are infected with P. syringae carrying avrPphB2 but also incurs a large cost in the absence of infection. RPS5 alleles are maintained at intermediate frequencies in populations globally, suggesting ubiquitous selection for resistance. However, the presence of P. syringae carrying avrPphB is probably insufficient to explain the RPS5 polymorphism. First, avrPphB homologues occur at very low frequencies in P. syringae populations on A. thaliana. Second, AvrPphB only rarely confers a virulence benefit to P. syringae on A. thaliana. Instead, we find evidence that selection for RPS5 involves multiple non-homologous effectors and multiple pathogen species. These results and an associated model suggest that the R gene polymorphism in A. thaliana may not be maintained through a tightly coupled interaction involving a single coevolved R gene and effector pair. More likely, the stable polymorphism is maintained through complex and diffuse community-wide interactions.

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Figure 1: Identification of RPS5 and AvrPphB2 as a naturally interacting R-gene–effector pair in A. thaliana and P. syringae populations.
Figure 2: The distribution of RPS5 and avrPphB homologues in co-occurring A. thaliana and P. syringae populations.
Figure 3: The cost of RPS5-mediated resistance in the absence of AvrPphB homologues.
Figure 4: The maintenance of a balanced polymorphism in a diffuse interaction.

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Acknowledgements

We thank G. Dwyer, M. Kreitman, L. Merwin, C. Morris and M. Nordborg for discussions, D. Baltrus, D. Dahlbeck, J. Greenberg and B. Vinatzer for supplying plasmids, N. Faure for help collecting the French isolates of P. syringae, C. Godé, J. Higgins and A. Stathos for contributions to genotyping RPS5 presence/absence, G. Sperone for mapping, and T. Morton and the staff of the University of Chicago greenhouse for planting and maintaining A. thaliana. T.L.K. was supported by a Department of Education GAANN fellowship, J.M.K. and L.G.B. were supported by postdoctoral fellowships from the Dropkin Foundation, and R.L. was supported by a National Institutes of Health (NIH) Postbaccalaureate Research Education Program award. F.R. was supported by the Laboratory of Excellence (Labex) TULIP (ANR-10-LABX-41; ANR-11-IDEX-0002-02). This work was supported by grants NIH-NIGMS R01 GM046451 to R.W.I., and grants NSF MCB0603515, NIH-NIGMS R01 GM057994 and NIH-NIGMS R01 GM083068 to J.B.

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Authors and Affiliations

Authors

Contributions

J.B. conceived the project and organized components; J.M.K. and R.L. cloned AvrPphB2 and performed functional analyses, J.M.K. mapped and cloned RPS5; L.G. performed fitness trials; B.J.D. and U.D. performed effector biochemical analyses; T.L.K. performed sequence divergence analyses; J.M.K., F.R. and J.D. assayed RPS5 frequencies in A. thaliana; J.M.K., S.N. and T.L.K. performed P. syringae fitness assays; T.L.K. cloned AvrPphB homologues; T.L.K. and R.R.H. designed the population genetics analyses and R.W.I. designed the biochemical analyses; T.L.K. performed the modelling; J.B., L.B., T.L.K. and J.M.K. were involved in the study design, the experiments and the analyses; T.L.K. and J.B. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Joy Bergelson.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Recognition of AvrPphB/2 by RPS5 reduces bacterial growth.

Growth of DC3000(avrPphB2) in planta in Ga-0 is reduced by the presence of RPS5. In contrast, growth of DC3000 containing the empty vector pME6010 is unaffected by the presence of RPS5. The star denotes P < 0.05 in a Wilcoxon rank-sum test. Growth is measured in colony-forming units per square centimetre. Eight biological replicates were performed per genotype. Results are presented as the mean ± one s.e.m.

Extended Data Figure 2 Detection of RPS5 in global populations.

PCR was used to test for the frequency of RPS5 in six populations of A. thaliana in the Midwestern USA. The RPS5 locus was polymorphic in all Midwestern populations. RPS5 alleles were present at a frequency of 11–32%.

Extended Data Figure 3 Distribution of synonymous divergence in genes orthologous between P. syringae isolates Pan (kiwi pathovar) and PNA29.1a (A. thaliana pathovar).

The red arrow indicates the level of synonymous divergence between the homologues avrPphB and avrPphB2. The extreme synonymous divergence between avrPphB homologues suggests that one of the homologues has undergone horizontal gene transfer from a distantly related bacterium (empirical P = 0.003).

Extended Data Figure 4 AvrPphB homologues from several crop pathovars are recognized by RPS5.

AvrPphB homologues found in crop pathovars were tested for the ability to elicit RPS5-mediated hypersensitive response. A maximum likelihood phylogeny of avrPphB homologues from crop pathovars and A. thaliana isolate PNA29.1a is presented here. The majority of homologues induced hypersensitive response. Homologues from 302460, 301436, PTBR2004 and ES4326 each encode homologues with truncated alleles. B5 encodes a full transcript. Recognition was determined by a Fisher’s exact test comparison of hypersensitive response frequency upon infection of RPS5+ with a homologue versus an empty vector (see Supplementary Information). The result for 302091 was marginally significant (P = 0.02, but after adjusting for multiple testing P = 0.14).

Extended Data Figure 5 AvrPphB2 enhances the proliferation of DC3000 in planta in the Ga-0 background.

Growth of DC3000 is augmented in RPS5 plants by the presence of AvrPphB2. The star denotes P < 0.05 in a Wilcoxon rank-sum test. Results are presented as the mean ± one s.e.m. (calculated with seven biological replicates per genotype).

Extended Data Figure 6 The increase in virulence conferred by AvrPphB2 is genotype dependent.

AvrPphB2 increases the virulence of one of three P. syringae isolates from A. thaliana populations on RPS5 Ga-0 plants. The star denotes P < 0.0167 (multiple-test corrected P value corresponding to α = 0.05) in a Wilcoxon rank-sum test. Results are presented as the mean ± one s.e.m. The P values corresponding to KN843.1a, LP217a and ME880.1a are 0.401, 0.838 and 0.014 respectively (calculated with 32 biological replicates for both constructs in the KN843.1a background, 30 empty vector and 32 avrPphB2-containing replicates in the LP217a background and 30 empty vector, 29 avrPphB2-containing replicates in the ME880.1a background).

Extended Data Figure 7 Conditions for a stable polymorphism that is robust to changes in the initial frequency of the resistance allele.

To determine the stability of the R gene polymorphism independent of the initial frequency of the R gene, we determined the parameters for the cost of infection and the probability of infection for which the R allele increases when at low frequencies but decreases at high frequencies (described in Supplementary Information). The model included frequency dependence, similar to the model used to generate Fig. 4b. The black shading signifies the conditions for which the polymorphism is robustly maintained irrespective of the starting frequency of the R allele.

Supplementary information

Supplementary Information

This file contains Supplementary Materials and Methods, Supplementary Tables 1-2 and Supplementary References. (PDF 254 kb)

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Karasov, T., Kniskern, J., Gao, L. et al. The long-term maintenance of a resistance polymorphism through diffuse interactions. Nature 512, 436–440 (2014). https://doi.org/10.1038/nature13439

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