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

Abstract

Plant resistance (R) genes are a crucial component in plant defence against pathogens. Although R genes often fail to provide durable resistance in an agricultural context, they frequently persist as long-lived balanced polymorphisms in nature. 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 pair. However, many plant-pathogen interactions lack such specificity. Whether, and how, balanced polymorphisms are maintained in diffusely interacting species 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.

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.

Figure 1. Identification of RPS5 and AvrPphB2 as a naturally interacting R-gene–effector pair in A. thaliana and P. syringae populations

a, Hypersensitive response in response to infection by DC3000(avrPphB2) was scored in 75 worldwide A. thaliana accessions that were previously genotyped at ~250,000 single nucleotide polymorphisms. EMMAX was used to perform genome-wide association mapping of differential hypersensitive response. The Manhattan plot illustrates the P values associated with each of the single nucleotide polymorphisms, and the dotted line signifies the Bonferroni correction threshold for significance (P = 2.33 × 10⁻²⁷). The top three single nucleotide polymorphisms, each with P values 1.27 × 10⁻¹², lie within 3.5 kb of RPS5 on chromosome (Chr) 1. b, AvrPphB2 cleaves PBS1. Immunoblot of PBS1 tagged with haemagglutinin (PBS1-HA) expressed alone or co-expressed with AvrPphB, AvrPphB C98S (non-active mutant) or AvrPphB2-myc in Nicotiana benthamiana. AvrPphB and AvrPphB2 both cleave the full-length (FL) PBS1 whereas AvrPphB C98S does not. The upper panel shows the immunoblotting results for the HA-tagged PBS1 and the bottom panel shows the results for immunoblotting for AvrPphB variants.

Figure 2. The distribution of RPS5 and avrPphB homologues in co-occurring A. thaliana and P. syringae populations

PCR was used to test for the frequency of RPS5 in A. thaliana populations across Europe (a) and the Midwestern USA (Extended Data Fig. 3). The RPS5 locus was polymorphic in more than 90% (36 out of 39) of these populations (one population in eastern Asia is not illustrated). Dot-blot assays tested for the presence of AvrPphB homologues in isolates of P. syringae from the Midwestern USA (b) and from France (c). avrPphB homologues are found at 3.27% frequency in the Midwestern USA (RPS5 frequency ~ 24%) and at 2.29% frequency in France (RPS5 frequency ~ 81%).

Figure 3. The cost of RPS5-mediated resistance in the absence of AvrPphB homologues

Field trials of paired isogenic lines of A. thaliana that differed only in the presence or absence of RPS5 driven by its natural promoter. Lines C1 and C2 were generated by the insertion of RPS5 and its flanking regions into the susceptible Ga-0 background and subsequent excision of RPS5 using a Cre-lox system. Lines E1–E4 were generated by random point mutation in the native RPS5 gene to generate a null allele in the resistant Col-0 background (described in Supplementary Information). In all pairings, RPS5⁺ plants exhibited reduced fitness relative to RPS5⁻ plants, ranging in magnitude from 5.0 to 10.2%. The percentage indicates the percentage decrease in seed production in the RPS5⁺ line relative to the RPS5⁻ line, and the stars denote P < 0.05 in a paired t-test (with 51 and 56 plant pairings in the two susceptible backgrounds, and 59, 52, 66 and 62 plant pairings in the resistant backgrounds). Results are presented as the mean ± one s.e.m.

Figure 4. The maintenance of a balanced polymorphism in a diffuse interaction

To ascertain the conditions sufficient to maintain an R gene polymorphism stably in a diffuse interaction, we modelled the dynamics of an R gene polymorphism with a range of parameters for the cost of infection (y axis) and probability of infection with bacteria containing a recognized effector (x axis). With a starting frequency of 0.5 for the R gene and a cost of resistance of 0.08 (a value consistent with our observations in Fig. 3), we recursively simulated the frequency of the R gene to determine the number of generations that the R gene remained at a putatively detectable frequency (between 0.01 and 0.99). The simulation was run for 100,000 generations. The colour bar signifies the number of generations, up to 100,000, that the polymorphism is maintained. For a, the dynamics were modelled without frequency dependence. The R gene polymorphism is stably maintained for a narrow range of parameters. With the incorporation of negative frequency dependence into the model (described in Supplementary Information), as illustrated in b, the parameter space allowing a stable polymorphism expands.