RESULTS

Intraspecific variation at RPP13: The length of the complete alignment of the 24 alleles was 2652 nucleotides. The predicted proteins encoded by individual alleles were between 820 and 843 amino acids in length. All alleles had the same overall domain structure and there were no obviously truncated genes (Bittner-Eddyet al. 2000). The alleles showed numerous indel and nucleotide polymorphisms. In this 2.6-kb region, there were 32 indel polymorphisms and 365 nucleotide polymorphisms (see supplemental Figure 1 at http://www.genetics.org/supplemental/). One-half of all the indel polymorphisms occurred in the LRR region of the gene. Within the LRR, the indels were located most frequently in the putative β-α-connecting loop of the LRR, also described as the third subdomain in the repeat (Bittner-Eddyet al. 2000).

The RPP13 gene shows the greatest nucleotide polymorphism (π= 0.043; θ= 0.040) of any gene surveyed to date from A. thaliana (Table 2; Figure 2). The average value of θ from A. thaliana across a sample of 17 other genes taken from the literature is 0.0085 (ranging from 0.0026 to 0.0206). Assuming even the highest of these θ-values (θ= 0.0206 for ChiA; Kawabeet al. 1997), coalescent simulations with no recombination (the most conservative test) indicate that observing values of π or θ as high as those for the RPP13 locus is unlikely (P = 0.04 and P = 0.021, respectively). The converse is also true. Assuming a genome-wide value of θ equal to that of RPP13 (θ= 0.04), even the highest levels of polymorphism in this sample from published surveys is improbable (θ≤ 0.0206, P = 0.026; π≤ 0.0109, P = 0.003).

Not only is the synonymous and nonsynonymous polymorphism high across the entire gene (π_syn = 0.049; π_non = 0.041) compared to other genes in A. thaliana, but also the ratio of π_non/π_syn = 1.5 in the LRR. There is a significant excess of nonsynonymous polymorphisms per nonsynonymous site relative to synonymous polymorphisms per synonymous site (χ² = 3.92, P = 0.048) in the LRR, suggesting balancing selection favoring amino acid variation in the LRR. In the non-LRR region, π_non/π_syn = 0.44 and there is a significant excess of synonymous polymorphisms per synonymous site (χ² = 13.6, P = 0.0002). This suggests purifying selection against amino acid variants outside of the LRR.

While the number of segregating sites is 324, the estimated minimum number of mutations is 403, indicating that multiple hits have occurred at some positions. In some cases, three or more different amino acid residues were observed at a single codon position. In the first two-thirds of the gene, the region encoding the coiled-coil domain and the nucleotide-binding site, 71/542 (13%) of the codons exhibited nonsynonymous polymorphisms. Three or more amino acids were encoded at 6/71 of these polymorphic codons (8.4%). In contrast, 124/282 (43%) of the codons in the LRR exhibited nonsynonymous polymorphisms. Not only was the level of polymorphism higher, but also 55% of the polymorphic codons encoded three or more amino acids and more than one-quarter encoded four or more amino acids. These codons with greater than two amino acids segregating are concentrated in the junctions between the β-strand, β-turn motif and the connecting β-α-loop of the individual LRRs (see supplemental Figure 1 at http://www.genetics.org/supplemental/).

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Figure 2.

—The ratio of π_non to π_syn across 18 genes of A. thaliana. The sequence data for the interlocus comparisons were obtained from GenBank and the literature (Hanfstinglet al. 1994; Kawabe et al. 1997, 2000; Henikoff and Comai 1998; Purugganan and Suddith 1998, 1999; Caicedoet al. 1999; Kawabe and Miyashita 1999; Stahlet al. 1999; Kamiyaet al. 2000; Kuittinen and Aguadé 2000; Aguadé 2001; Hauseret al. 2001; Kliebensteinet al. 2001).

Frequency spectrum of variation: The Tajima's D (Tajima 1989) value was negative, but did not differ significantly from zero. Partitioning the gene into two regions (exclusively the LRR vs. the rest of the gene) to test if the pattern of evolution differed between these two functionally differentiated parts did not result in a significant deviation from zero (Table 2). The frequency of singletons (polymorphic sites where the rarest variant is present only once in the sample) in this sample is 27%. An excess of singleton mutations relative to the neutral expectation has been observed at multiple A. thaliana loci including Adh1, AP3, CAL, CHI, ChiA, and PI (Innanet al. 1996; Kawabeet al. 1997; Purugganan and Suddith 1998, 1999; Kuittinen and Aguadé 2000). To examine the difference between RPP13 and samples from other loci, the frequency spectra of polymorphism were compared across 14 loci (see supplemental Figure 2 at http://www.genetics.org/supplemental/). In this comparison, over half of the non-RPP13 genes show a high proportion of singletons (>50%). Furthermore, singletons make up a large proportion (60%) of the polymorphic amino acids at these other loci, while singletons make up a smaller proportion (26%) of the polymorphic amino acids at the RPP13 locus.

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Figure 3.

—Neighbor-joining tree of alleles of RPP13, ortholog from A. arenosa, and paralogs from A. thaliana. A neighbor-joining tree was inferred on the basis of the nucleotide sequences of the RPP13 alleles from A. thaliana and A. arenosa using PAUP*4.0b8 (Swofford 1999). The tree was rooted using the sequence of two paralogs from the accession Columbia (GenBank accession nos. At3g46730 and At3g46710). The HKY85 substitution model was used. Bootstrap proportions of 100 bootstrap replicates >50 are indicated on the branches. Shaded area highlights the cluster of A. thaliana RPP13 alleles.

Haplotype structure: Parsimony and neighbor-joining trees were inferred on the basis of the nucleotide sequences of the RPP13 alleles from A. thaliana and A. arenosa (Figure 3; see also supplemental Figure 3 at http://www.genetics.org/supplemental/). High boot-strap values support the monophyly of the clade composed of the RPP13 alleles from A. thaliana, as well as the larger clade containing the RPP13 alleles from A. thaliana plus the ortholog from A. arenosa. Nineteen different RPP13 alleles were detected in the 24 accessions. One allele was found in four accessions (i.e., Hil-1, Duc-1, Leg-1, and Lha-1) and two alleles were found in two accessions (Ci-1 and Ti; Crl-1 and Bra-1). While multiple clades within A. thaliana were well supported in both analyses, the alleles in clade A and clade B are the most differentiated; alleles from these two clades show 66 fixed differences distributed across the entire RPP13 coding region. Clade A shows a low level of within-clade variation (π= 0.0019). Among the five alleles in this clade, 14 of 15 total polymorphisms are singletons and nearly all of these result in an amino acid difference. Variation in clade B is much greater (π= 0.04) and the overall proportion of singletons is much lower within this haplotype (25.7%). Within clade B, there is some evidence for further divisions among alleles. The three pairs of alleles (Nd-1 and Frd-1, Ws-2 and Coc-1, and God-1 and Edi-2) each emerge in both neighbor-joining and parsimony analyses with high bootstrap support. These three allele pairs are diverged relative to all others in their sequences, despite evidence for recombination at this locus (see below). Another cluster of seven alleles (Rld-2, Poo-1, Sna-1, Ci-1, Ty-0, Pet-1, and Asp-1) also share distinct substitutions with one another relative to all other alleles. However, a straightforward haplotype tree could not be constructed due to recombination.

Evidence for recombination: Although estimates of outcrossing rates in A. thaliana are very low (Abbott and Gomes 1989), there was abundant evidence for recombination at the RPP13 locus. Given such high levels of polymorphism at RPP13, there was even greater power to detect recombinants at this locus than at other loci in A. thaliana. The four-gamete test (Hudson and Kaplan 1985) indicated that a minimum of 38 recombination events are necessary to explain the pattern of variation at this locus (Figure 4). The program Geneconv (Sawyer 1999) was used to infer recombination or gene conversion events in the genealogy of the alleles. Over 50 putative gene conversion/recombination tracks were identified in this sample, reinforcing the observation of extensive recombination at this locus (Figure 4). In some places the two analyses identified the same endpoints of recombinational events, while in other regions, the two analyses were not in agreement (Figure 4).

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Figure 4.

—Length and position of putative gene conversion/recombination tracks ordered by decreasing P value. The P values are Karlin-Altschul P values, based on the BLAST method for finding sequence matches in DNA or protein databases (Altschulet al. 1990) and are Bonferroni corrected. The names of the alleles involved in these gene conversion/recombination events are separated by a slash. Three sets of alleles are identical in sequence and are separated by a comma. The tick marks along the x-axis are the midpoints of the 38 minimum recombination intervals based on Hudson's four-gamete test. The structure of the RPP13 gene is indicated at the top of the figure.

Only one putative recombinant between haplotype clades A and B was detected. This allele, Ksk-2, shared 82 unique polymorphisms and only two differences with Sco-1 between sites 1 and 1286; whereas Ksk-2, Lha-1, Leg-1, Duc-1, and Hil-1 shared 300 polymorphisms and no differences between sites 1243 and 2652. The Ksk-2 allele may have originated fairly recently because only one event is needed to infer the origin of this allele and the potential donor sequences found among other alleles in this study. The recent origin of this allele is further supported by the high sequence identity shared between each “recombinant” portion of the Ksk-2 allele with the inferred donor sequences.

The program Geneconv was also used to evaluate whether recombination or gene conversion events involving alleles of RPP13 and RPP13 paralogs had occurred. No evidence of conversion or recombination was found between these alleles and the two paralog sequences available from the Columbia ecotype.

Interspecific comparisons: Joint analyses of within- and between-species divergence can increase the statistical power to detect deviations from neutral evolution (Akashi 1999). To identify orthologous sequences from close relatives of A. thaliana, sequences were PCR amplified with RPP13-specific A. thaliana primers. Phylogenetic analyses revealed that one of these amplified sequences, Aren1 from A. arenosa, was sister to the clade of RPP13 alleles from A. thaliana (Figure 3; see also supplemental Figure 3 at http://www.genetics.org/supplemental/). Many other sequences amplified from A. arenosa and A. lyrata cluster with either one of the two previously described paralogs of A. thaliana (e.g., Lyr3, Aren3, and Aren4). A number of studies use A. lyrata for interspecific sequence comparisons to A. thaliana (e.g., Wrightet al. 2002; Barrieret al. 2003). However, none of our amplification products from A. lyrata appeared to be orthologous to RPP13. Two sequences, Lyr1 and Lyr2, possess the greatest nucleotide identity to RPP13 alleles among the collection of A. lyrata sequences. However, frameshift mutations within both of these sequences prevent their use in population genetic analyses (i.e., calculations of K_s and K_a).

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Figure 5.

—Sliding window analyses: (a) average number of differences per site between RPP13 alleles within A. thaliana and (b) between A. thaliana and A. arenosa. Dashed lines are synonymous variation, while solid lines are nonsynonymous. The sliding window analysis was conducted as in Aguadé et al. (1992) on the basis of the estimation of silent and replacement substitutions proposed by Nei and Gojobori (1986). Values are midpoints of 36-bp windows. The positions of the coiled coil (CC), the nucleotide-binding and ARC domains (NB-ARC), and the leucine-rich repeats (LRR) are indicated below the plots.

Average divergence at synonymous sites between Aren1 and the RPP13 alleles from A. thaliana is 0.15. This value is close to the estimated divergence at synonymous sites across several loci between A. thaliana and A. lyrata: Wright et al. (2002) report an average K_s = 0.126 (range 0.027–0.23) for 24 loci; Barrier et al. (2003) report an average K_s = 0.119 (range 0.0–0.55) for 304 expressed sequence tags. However, nonsynonymous divergence (K_a) between A. thaliana and A. arenosa at RPP13 (K_a = 0.089) exceeds the estimates reported for the A. thaliana/A. lyrata comparisons: K_a = 0.0211 (range 0.0–0.092) from Wright et al. (2002) and K_a = 0.025 (range 0.0–0.16) from Barrier et al. (2003). Amino acid divergence reaches 16% in the LRR, at least six times greater than the average amino acid divergence in the A. thaliana/A. lyrata gene comparisons.

Distribution of variation across the gene: Sliding window analyses were used to characterize the pattern of polymorphism and divergence across the RPP13 gene (Figure 5). The pattern of nonsynonymous and synonymous polymorphism and divergence differs across the gene with nonsynonymous polymorphism and divergence peaking in the LRR. For the interspecific comparison of RPP13 in A. thaliana and A. arenosa, the level of synonymous divergence (K_s) exceeded nonsynonymous divergence (K_a) in the first two-thirds of the gene (Figure 5). The K_a/K_s ratio of 0.389 in this region is comparable to π_non/π_syn = 0.44 for the intraspecific comparison. The pattern of sequence divergence also mirrors polymorphism in the LRR region, showing greater amino acid divergence relative to silent divergence. K_a/K_s is ∼1 in the LRR and exceeds 1 at the junctions between β-strand, β-turn motif and the connecting β-α-loop of the individual LRRs. Both the interspecific and the intraspecific comparisons indicate that RPP13 has a rate of amino acid evolution higher than that of any gene studied in A. thaliana, especially in the LRR.

The McDonald-Kreitman test was also used to determine if the level of nonsynonymous polymorphism observed at the RPP13 locus exceeded that expected under neutrality. Under neutrality, the levels of intraspecific polymorphism and interspecific divergence are expected to be correlated (McDonald and Kreitman 1991). At the RPP13 locus, we detected a significant departure from the null expectation (i.e., the ratios of polymorphism and divergence differed between synonymous and nonsynonymous mutations, P < 0.0005; Table 3). Since studies of other R genes revealed a pattern of diversifying selection acting on the LRR region and functional studies indicate that pathogen-recognition specificity is encoded by this region, we tested whether this region alone was responsible for the deviation from the neutral expectation. Indeed, an analysis of the first half of the gene, which does not contain the LRR, does not show a deviation from neutrality, while the analysis of the LRR region alone does (Table 3). These results indicate an excess of amino acid polymorphisms segregating in the LRR region relative to the neutral expectation.

Correlation with resistance phenotype: The resistance responses to three isolates of P. parasitica, Maks9, Emco5, and Wela3, were determined for the 24 accessions of A. thaliana (Table 1). The genetic basis of resistance to these isolates of P. parasitica was investigated in greater depth by transgenic complementation (Table 1; Bittner-Eddyet al. 2000). The Nd-RPP13 allele encodes specific recognition to both the Maks9 and the Emco5 isolates of P. parasitica, while the Col and Rld alleles do not encode specific resistance to these two isolates (Bittner-Eddyet al. 2000). Furthermore, the Rld-RPP13 gene encodes resistance to a third isolate, Wela3, while Col and Nd alleles do not encode resistance to this isolate (Bittner-Eddyet al. 2000). Recently a third allele, Frd, has been transformed into the susceptible Col ecotype; this allele encodes resistance to Emco5, but not to Maks9 (P. Bittner-Eddy, unpublished data). Although the RPP13 allele isolated from the Col individual does not confer resistance to any of the three P. parasitica isolates used in this study, the Col-RPP13 allele does not contain any frameshift mutations nor are there any other indications that this allele is nonfunctional. Downstream genes in the RPP13 pathway of Col are not compromised, as this genotype showed isolate-specific resistance when transformed with each of the Rld, Frd, or Nd alleles.

The Nd, Frd, Rld, and Col alleles differ from each other by a large number of amino acids, so identifying residues that may be involved in pathogen recognition was not possible through simple pairwise comparisons. However, all of the accessions in this study were phenotyped for resistance to Maks9 and Emco5. Since resistance due specifically to the RPP13 gene has been demonstrated only for the Nd and Frd alleles, resistance in other plants may not be encoded by the RPP13 locus. The Rld accession is resistant to Maks9 and Emco5 but this resistance maps to other position(s) in the genome (Bittner-Eddyet al. 2000). Furthermore, some accessions had the same RPP13 allele but differed in their reaction to P. parasitica (i.e., Bra and Crl). Therefore the only informative comparison to identify amino acid differences that are associated with resistance or susceptibility was between the alleles from the susceptible accessions and those that have been shown by transformation to confer resistance.

Over half of the accessions studied were susceptible to one of the two isolates. The subset of 10 accessions that were resistant to the Maks9 isolate was different from the subset of 11 accessions that were resistant to Emco5. Nine were susceptible to both isolates. The “susceptible” alleles to each pathogen isolate are found in all the major clusters of the gene tree, indicating that a large diversity in amino acid sequence was found among alleles from susceptible plants; i.e., susceptibility alleles are not more similar in sequence to one another than they are to alleles from resistant plants.

Alleles of RPP13 from the Nd and Frd accessions conferred resistance to Emco5. There are 13 amino acid positions in which Nd and Frd have the same amino acid, but all of the susceptible alleles have a different amino acid (see supplemental Table 1 at http://www.genetics.org/supplemental/). Twelve of 13 of these polymorphisms are located in the LRR. Since the amino acid residues associated with resistance to Emco5 reside predominantly in the LRR, and the LRR regions of the Nd and Frd alleles are substantially differentiated from other alleles in the sample, it is likely that at least some of the recognition determinants of Emco5 are localized to the LRR region. The Nd allele has been demonstrated to confer resistance to Maks9 (Bittner-Eddyet al. 2000). A comparison of the Nd sequence to the alleles from accessions susceptible to Maks9 revealed that five amino acid residues were unique to Nd (see supplemental Table 2 at http://www.genetics.org/supplemental/). These five amino acids are located between sites 167 and 313 of the amino acid alignment and were restricted to the NBS. One difference at site 167 is found within the conserved motif 1 as described in van der Biezen and Jones (1998). None of the other four differences are located in conserved regions or regions for which functions have been ascribed such as kinase domains of the NBS.