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Accumulation of genetic incompatibilities within species can lead to reproductive isolation and, potentially, speciation. In this study, we show that allelic variation at SRF3 (Strubbelig Receptor Family 3), encoding a receptor-like kinase, conditions the occurrence of incompatibility between Arabidopsis thaliana accessions. The geographical distribution of SRF3 alleles reveals that allelic forms causing epistatic incompatibility with a Landsberg erecta allele at the RPP1 resistance locus are present in A. thaliana accessions in central Asia. Incompatible SRF3 alleles condition for an enhanced early immune response to pathogens as compared to the resistance-dampening effect of compatible SRF3 forms in isogenic backgrounds. Variation in disease susceptibility suggests a basis for the molecular patterns of a recent selective sweep detected at the SRF3 locus in central Asian populations.

Reproductive isolation barriers in plants are documented between individuals that have already departed from an interbreeding situa-tion1,2. The Dobzhansky-Muller model of genetic incompatibilities postulates that reproductive isolation is driven by deleterious epistatic interactions between alleles arising from evolutionarily divergent populations3,4. In plants, Arabidopsis and its relatives are useful models for the study of genetic incompatibilities5. In Arabidopsis ­thaliana, known mechanisms underlying post-zygotic incompatibilities involve loss of essential duplicated genes or inappropriate activation of

immune responses6–8. Previously, we reported genetic epistatic inter-actions between the Arabidopsis accession Landsberg erecta (Ler) from northern Europe and the central Asian accessions Kashmir-2 (Kas-2) and Kondara (Kond), which result in severe growth defects at low tem-perature (14 °C)6. One of the loci conditioning for these incompatible interactions mapped to within the RPP1 cluster of the TIR-NB-LRR (Toll-Interleukin1, ­Receptor-Nucleotide ­Binding, ­Leucine-Rich ­Repeat) genes on chromosome 3, whose recessive Ler alleles were incompat-ible with Kas-2 or Kond alleles at an undetermined recessive locus on

Natural variation at Strubbelig Receptor Kinase 3 drives immune-triggered incompatibilities between Arabidopsis ­thaliana accessionsRubén Alcázar1, Ana V García2, Ilkka Kronholm1, Juliette de Meaux1, Maarten Koornneef1, Jane E Parker2 & Matthieu Reymond1

1Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, Cologne, Germany. 2Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany. Present addresses: Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Institut National de la Recherche Agronomique (INRA) Centre de Versailles-Grignon, Route de St-Cyr (RD10), Versailles Cedex, France (M.R.) and Unité de Recherche en Génomique Végétale, INRA-Centre National de la Recherche Scientifique-Université Evry Val d’Essonne (CNRS-UEVE), Evry CEdex, France (A.V.G.). Correspondence should be addressed to M.R. (reymond@mpiz-koeln.mpg.de).

Received 30 March; accepted 7 October; published online 31 October 2010; doi:10.1038/ng.704

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chromosome 4. We performed fine mapping to identify the causal gene for incompatibil-ity on chromosome 4. We genotyped plants in generation F2 segregating at this locus (Supplementary Table 1) to identify recom-binants, and we determined the genotypes associated with dwarfism at low temperature. The incompatible locus on chromosome 4 was fine-mapped to a 30 Kb region spanning eight annotated genes in the Columbia (Col) reference sequence (Supplementary Fig. 1). Sequencing this interval in Kas-2 revealed a similar genomic organization as in Col (Supplementary Fig. 1), which includes the receptor-like kinase encoded by At4g03390, annotated as a member of the SRF3 family (Strubbelig ­ Receptor ­ Family), whose bio-logical function is unknown9. Genetic proof that SRF3 underlies the incompatible locus on chromosome 4 was provided by comple-mentation of the SRF3 Kas-2 allele with the dominant SRF3 Ler allele, which suppresses incompatibility. As a recipient for the SRF3 Ler allele, we used the incompatible Ler–Kas-2 near isogenic line (NIL) carrying a single Ler introgression spanning the RPP1 locus in an otherwise homogeneous Kas-2 background6. Multiple independent NIL transformants of the compatible SRF3 Ler allele (termed here NILSRF3Ler) showed complete suppression of dwarfism, delay in flowering, and spontaneous cell death, which are all features of incompatible Ler–Kas-2 lines at low temperature (Supplementary Fig. 2). Presence of the compatible SRF3 Ler allele in NILSRF3Ler suppressed expression of ~80% of the genes that were deregulated in the NIL (Supplementary Fig. 3). A high proportion of these genes are associated with protein kinase cascades and/or biotic stress responses (Supplementary Fig. 3 and Supplementary Table 2). Importantly, deregulated expression of salicylic acid pathway genes and repression of jasmonic acid responses reported in NIL6 were suppressed in NILSRF3Ler (Supplementary Table 2), suggesting a restoration of a stress-hormone pathway balance. We established that SRF3 also underlies the incompatible locus on chromosome 4 in Ler-Kond by allelism tests between recessive Ler-Kond incompatible lines, NIL and NILSRF3Ler (Supplementary Fig. 4). Sequencing of Kas-2 and Kond SRF3 alleles identified non-synonymous substitutions within the ectodomain of the Kond allele and the juxtamembrane domain of the Kas-2 allele, which associate with incompatibility (Fig. 1a). Hence, two different SRF3 alleles are incompatible with Ler alleles at the RPP1 cluster, consistent with two independent mutations having

arisen that cause the same phenotype. Increased or strongly attenu-ated expression of SRF3 in Col transfer DNA (T-DNA) insertion lines did not cause dwarfism, spontaneous cell death or altered responses to pathogen infection (Supplementary Fig. 5a–c). Also, introgres-sion of Ler alleles at the RPP1 cluster in these lines failed to produce hallmarks of incompatibility (Supplementary Fig. 5d,e). Based on these observations, we reasoned that SRF3 gene expression differences are unlikely to account for the incompatibility which, instead, may be conditioned for post-transcriptionally.

We studied the distribution of Kas-2 and Kond incompatible SRF3 alleles in the wild by genotyping 603 accessions from 37 countries in Europe and central Asia (Fig. 1b and Supplementary Table 3) with markers that differentiate between Kas-2, Kond and other alleles (Supplementary Table 1). Notably, Kas-2 and Kond incompatible alleles were only found in central Asia, whereas other allele forms were uniformly distributed over Europe and central Asia (Fig. 1b). We inves-tigated whether the observed geographical pattern corresponds to the distribution of incompatibilities with Ler. We selected accessions from central Asia (n = 33; Supplementary Tables 4 and 5), north Europe

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and south Europe (n = 30; Supplementary Table 5), and we identified three genetic clusters (north Europe, south Europe and central Asia) by applying STRUCTURE (see URLs) analysis to 139 genome-wide SNP markers10 (Fig. 2a). These accessions were then sequenced at the polymorphic region spanning the SRF3 exons 8–12. Most of the sequences from central Asian accessions fell into two distinct clades containing either Kas-2 or Kond alleles (Fig. 2b). Notably, the two incompatible SRF3 allelic groups (Fig. 2b) were not related to obvious population structure subdivisions in Central Asia (Fig. 2a and Supplementary Fig. 6). All selected accessions from north Europe, south Europe and central Asia were crossed to Ler to generate F2 populations which were scored for incompatible phenotypes at 14 °C. Only compatible phenotypes were found in populations derived from crosses between the north European Ler and other European accessions (Supplementary Table 5), consistent with the absence of incompatible SRF3 alleles in European populations (Fig. 1b). By con-trast, 24 F2 populations tested from crosses between Ler and central Asian accessions, whose alleles belonged to the Kas-2 or Kond clades within the SRF3 phylogeny (Fig. 2b), exhibited segregation of dwarfism (Supplementary Fig. 7 and Supplementary Table 4). Segregation anal-yses revealed involvement of at least two recessive loci in most of the incompatible combinations detected, and in all cases, stunted F2 plants were homozygous for Kas-2 or Kond alleles at SRF3 and for Ler alleles at the RPP1 locus (Supplementary Table 4). All tested dwarf lines had spontaneous cell death phenotypes, as previously found in Ler– Kas-2 and Ler-Kond incompatibilities (Supplementary Fig. 8). Results of allelism tests were consistent with involvement of the same recessive alleles as in Ler–Kas-2 and Ler-Kond (Supplementary Fig. 9). Also, the introduction of Ler ­SRF3 forms to incompatible F2 plants by crossing them with NILSRF3Ler plants restored compatibility (Supplementary Fig. 9). Hence, the geographical distribution of incompatible SRF3 alle-les correlates with the occurrence of incompatibilities to Ler, and this is not a rare event between Ler and central Asian accessions.

We similarly monitored the distribution of Ler incompatible alleles at the RPP1 locus in Europe by crossing Kas-2 with European accessions. A total of 21 F2 populations were scored for dwarfism at 14 °C (Supplementary Table 5). This survey revealed

compatibility between Kas-2 and all European accessions tested (Supplementary Table 5). Therefore, incompatible alleles at the RPP1 locus on chromosome 3 are rare in Europe, contrasting with a high frequency of incompatible Kas-2 and Kond alleles on chromosome 4 (SRF3) in central Asia (Fig. 1b).

Previously, we found that incompatibility between Ler and Kas-2 or Kond is caused by inappropriate activation of the salicylic acid pathway leading to autoimmunity and cell death at low temperature6. To gain an insight to the function of SRF3, we examined innate immune responses driven by compatible and incompatible SRF3 alleles. Compared to the NIL or either parent (that is Ler or Kas-2), the compatible NILSRF3Ler exhibited enhanced susceptibility to infection by a viru-lent isolate of the adapted downy mildew pathogen Hyaloperonospora ­ ­arabidopsidis (Hpa; Supplementary Fig. 10a) or virulent Pseudomonas ­ ­syringae ­pv. ­tomato DC3000 bacteria (Pst DC3000; Fig. 3a). Similarly, transformants of Kas-2 expressing the SRF3 Ler allele (termed here Kas-2SRF3Ler) and recombinant inbred lines (RILs) carrying the Ler ­SRF3 allele in Kond backgrounds11 exhibited enhanced susceptibility to Pst DC3000 (Fig. 3a and Supplementary Fig. 10b). Dampening of immune responses by the Ler ­SRF3 allele in the Kas-2, NIL and Kond backgrounds suggests that the Asian SRF3 alleles contribute positively to resistance. Also, hybrids derived from a cross between Ler–Kas-2 NIL and Ler-Kond incompatible RILs displayed similar levels of resistance as their parents (Supplementary Fig. 10c), suggesting that both Kas-2 and Kond SRF3 alleles contribute to the pathogen resistance of incompatible hybrids. An early barrier to infection is conferred by membrane receptor recognition of microbe-associated molecular patterns (MAMPs)12. SRF3 is differentially phosphorylated in response to the bacterial MAMP flg22 (a 22-amino–acid epitope of flagellin), which is recognized by the plasma membrane receptor-like kinase FLS213,14. We found that YFP fusions of Ler, Kas-2 and Kond SRF3 variants also localize to the plasma membrane when transiently expressed in Nicotiana ­ benthamiana (Supplementary Fig. 11), indicating that SRF3 might act at the level of MAMP rec-ognition or signaling. We therefore tested whether the compatible and incompatible SRF3 alleles differentially affect early responses to flg22. One rapid change was activation of the mitogen-activated

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protein kinases (MAPKs) MPK4, MPK3 and MPK6, which mediate phosphorelay and lead to a balanced immune response15,16. Loss-of-function mutations in Arabidopsis ­MPK4 cause severe dwarfism and constitutive salicylic-acid–pathway activation resembling those phenotypes present in the NIL (Supplementary Fig. 2a), consistent with MPK4 normally limiting salicylic-acid–induced defenses6,15–18. We measured flg22-induced MPK3, MPK4 and MPK6 activities in genetic backgrounds with compatible and incompatible allelic combi-nations (Fig. 3b). Presence of the compatible SRF3 Ler allele in Kas-2 (Kas-2SRF3Ler) or the NIL (NILSRF3Ler) reduced flg22-triggered activa-tion of MPK4 and MPK6 compared to Kas-2 without the SRF3 Ler allele (Fig. 3b), consistent with an activity of Ler SRF3 in restricting early MAMP-triggered immunity (MTI). Decreased flg22-triggered MPK4, but not MPK6 activation, was observed in the incompatible NIL compared to the Kas-2 parent (Fig. 3b). The altered kinetics of MPK4 and MPK6 activation suggest that compatible and incompatible SRF3 alleles differ in their modulation of MAMP-induced defenses. We tested this idea further by inoculating plants with a disabled hrcC mutant of Pst DC3000 bacteria, which induces a strong MAMP- triggered response by failing to secrete defense-suppressing effectors into host cells19,20. Both Kas-2SRF3Ler and NILSRF3Ler compatible lines displayed increased susceptibility to Pst DC3000 hrcC compared to their near-isogenic backgrounds (Fig. 3a). These data support a poten-tially beneficial role of the incompatible SRF3 Kas-2 allele in Kas-2 backgrounds by increasing early responsiveness to pathogens.

Genetic differences between partially isolated populations can emerge over time as a result of genetic drift or natural selection. To test whether SRF3 incompatible alleles were spread in central Asia by drift or by selection, we looked for molecular patterns of a recent selective sweep in central Asian and European accessions. Values of Fay and Wu’s Hn statistic were calculated over a region spanning ~150 kb on each side of the SRF3 locus (Fig. 4 and Supplementary Table 6). The negative Fay and Wu’s Hn values detected at SRF3 locus in central Asia indicate an excess of derived alleles, which was a signa-ture expected after a recent selective sweep21,22. This pattern was not present in Europe, where only compatible alleles are present (Fig. 4). These observations suggest that natural selection rather than drift is likely to underlie the high frequency of SRF3 incompatible alleles in central Asia.

Here we present evidence that SRF3 drives incompatibility between the European A. ­thaliana Ler and several central Asian accessions. The epistatic interaction between TIR-NB-LRR Ler alleles at the RPP1 cluster and Kas-2 or Kond alleles at SRF3 is shared among all incompatible cases reported here. Different allelic variants at the same RPP1 cluster seem to participate in the dominant Uk-1–Uk-3 hybrid incompatibility5,6,8, which might account for the different

epistatic networks involved there. The geographical partitioning of incompatible SRF3 alleles correlates with the occurrence of incompatibilities (Fig. 1b). Notably, the SRF3 locus shows signa-tures of selection in central Asia (Fig. 4), suggesting that incompat-ibility has spread as a byproduct of selection. The different levels of disease susceptibility associated with compatible and incompatible SRF3 alleles appear to reflect differences in MTI responses (Fig. 3b). Reducing the threshold for activation of MTI by incompatible SRF3 alleles may create a robust selective advantage under conditions of high pathogen pressure23. Current evidence suggests that MTI serves to boost salicylic-acid–mediated defense against further pathogen attack19,24. Accordingly, Ler–Kas-2 and Ler-Kond incompatibilities were also found to require salicylic acid signaling6,25,26. A possible scenario in these epistatic interactions is that one or more TIR-NB-LRR proteins encoded within the RPP1 cluster guards SRF3 against effectors targeting MTI and involving SRF3. The recessivity of the incompatible interaction might be due to restoration of normal guard-guardee interactions by compatible SRF3 forms. These observations are consistent with a guard model of pathogen recognition12 in which allelic divergence at incompatible loci would impair normal guard-guardee relationships leading to stress signaling imbalances and autoactivation of immunity.

URLs. STRUCTURE, http://pritch.bsd.uchicago.edu/structure.html.

MeThodsMethods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/.

Accession codes. Sequences reported in this paper have been depo-sited in the GenBank database under accession numbers GU570412 (SRF3 Ler, genomic DNA); GU570413 (SRF3 Kond, genomic DNA); GU571158 (BAC Kas-2 incompatible locus); HM538833-HM539307 (At4g03020, At4g03290, At4g03320, At4g03330, At4g03340, At4g03410, At4g03420, At4g03430, At4g03460, At4g03510 and At4g03390). Transcriptome data (CEL and CHP files) have been sub-mitted to the public repository database ArrayExpress under acces-sion number E-MEXP-2569.

Note: ­Supplementary ­information ­is ­available ­on ­the ­Nature ­Genetics ­website.

AcKnowlEdGMEntsWe thank C. Alonso-Blanco, V. le Corre, M.H. Hoffmann, K. Schmid, O.A. Rognli and O. Loudet for providing seed materials. We thank B. Huettel for microarray hybridizations. This work was funded by Deutsche Forschungsgemeinschaft SFB 680 grants (to M.R., J.E.P. and J.d.M.). The authors also acknowledge the Max Planck Society and an International Max Planck Research School fellowship to A.V.G.

AUtHoR contRIBUtIonsR.A., M.K., J.E.P. and M.R. conceived the study. R.A. performed most of the experimental work with contributions from A.V.G. in the pathogen infection assays. I.K. and J.d.M. performed the computer analysis and interpretation of Fay and Wu’s Hn statistics. M.K. provided accessions and European F2 populations. J.E.P. provided materials for immune analyses. M.R. performed all statistical analyses. All authors analyzed the data. R.A., J.E.P and M.R. wrote the paper with contributions from all authors.

coMPEtInG FInAncIAl IntEREstsThe authors declare no competing financial interests.

Published online at http://www.nature.com/naturegenetics/. Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

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24. Mishina, T.E. & Zeier, J. Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis. Plant J. 50, 500–513 (2007).

25. Aarts, N. et al. Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis. Proc. Natl. Acad. Sci. USA 95, 10306–10311 (1998).

26. Feys, B.J., Moisan, L.J., Newman, M.A. & Parker, J.E. Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J. 20, 5400–5411 (2001).

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oNLINe MeThodsPlant materials and growth conditions. Stock numbers for the accessions used in this study are listed in Supplementary Table 3. Plants were germi-nated and grown on soil in growth chambers (Percival Scientific) under 12 h dark and 12 h light cycles at 14 °C and 16 °C or 20 °C and 22 °C and 70% relative humidity.

Fine mapping and bacterial artificial chromosome (BAC) sequencing. Fine mapping of the incompatible locus on chromosome 4 was performed in 1,344 F2 Ler–Kas-2 plants segregating for this locus with fixed Ler alleles at the RPP1 locus on chromosome 3 and Kas-2 alleles on chromosome 5 (ref. 6). These lines were generated from crosses between the incompatible Ler–Kas-2 RILs 24 × 126 and 129 × 15 (ref. 11). Markers used for genotyping are listed in Supplementary Table 1. BAC clones spanning the fine-mapped region were isolated from a Kas-2 BAC library in pINDIGOBAC-5 vector (Epicentre Inc.) by DNA blot hybridization using PCR-amplified flanking markers as probes. BAC sequencing was performed as described6.

Generation of NILSRF3Ler and Kas-2SRF3Ler transgenic lines. SRF3 with its own promoter was amplified by PCR from genomic DNA of Ler (Supplementary Table 1), cloned in pGEM T-easy (Promega), and different clones were sequenced (Supplementary Table 1). SRF3 Ler was then cloned in pCambia2300 binary vector. The generated construct was used for transformation of NIL and Kas-2 (ref. 27) using the Agrobacterium ­tumefaciens GV3101 pMP90 strain28. Selection of transformants was carried out in MS agar plates in the presence of 50 μg/ml of kanamycin. T1-resistant plants were checked by PCR for the pres-ence of the nptII gene, which confers kanamycin resistance (Supplementary Table 1). Homozygous lines with single T-DNA insertions were isolated by segregation analyses.

Microarray analysis. Three-week-old plants of the NIL, Kas-2 and an individual NILSRF3Ler line grown at 14 °C were used for microarray analysis. RNA samples were extracted in triplicate from pooled samples of five plants per genotype. The entire aerial rosette was used for RNA extraction as described6. Transcriptomes were analyzed using 1 μg of total RNA as starting material. Targets were prepared with MessageAmp II-Biotin Enhanced Single Round aRNA Amplification Kit (Ambion) and hybridized to ATH1 gene chips (Affymetrix). Microarray data and gene ontology classification were analyzed using GeneSpring GX 10 software (Agilent Technologies). A threshold of significance at P = 0.05 and a cutoff value of ≥ ± twofold change was used for the identification of genes that were differen-tially expressed between genotypes. The validation of the microarray data was checked by analysis of EDS1, PDF1.2 and PR-1 expression by real time PCR6 in the NIL, Kas-2 and two independent NILSRF3Ler lines (data not shown).

Isolation of Col srf3 mutants and Col srf3 × Ler lines. The srf3 T-DNA insertion mutants in the Col SALK_029908, SALK_057621 and SALK_001389 lines were obtained from Nottingham Arabidopsis Stock Centre (NASC)29. The position of the T-DNA insertion was checked by PCR amplification and sequencing of the T-DNA flanking sequences (Supplementary Table 1). Homozygous lines were identified by PCR (Supplementary Table 1). Expression analyses were performed by RT-PCR using specific primer combinations (Supplementary Table 1) and standard procedures for cDNA synthesis6. The SALK_029908 and SALK_001389 mutants were crossed to Ler and F2 plants genotyped to isolate those with homozygous Ler alleles at the RPP1 locus and homozygous SRF3 mutation (Supplementary Table 1).

STRUCTURE analysis. The underlying population structure of accessions used for the crosses was inferred using the model-based clustering algorithms implemented in the software STRUCTURE using the haploid setting and run-ning 20 replicates with 50,000 and 20,000 MCMC iterations of burn-in and after-burning length, respectively30. SNP multilocus genotypes were deter-mined as in a previous study10 (Sequenom Inc.). To adjudicate the correct number of genetic clusters K, we applied the ΔK method31 in combination with the absolute value of ln P(X | K).

Histochemical analyses and pathogen infection assays. Infections with H. ­arabidopsidis Cala2 were performed as previously described6. Plant cell

death and pathogen growth structures were revealed by staining leaves with lactophenol trypan blue6. Infections with Pseudomonas ­syringae ­pv. tomato DC3000 (Pst DC3000) and Pst DC3000 hrcC mutants19 were performed at 20 °C in 5-week-old plants grown at 14 °C unless otherwise indicated by spray inoculation with a bacterial suspension of 1 × 108 cfu/ml in 10 mM MgCl2 with 0.04% (v/v) Silwet L-77 (Lehle Seeds). In ­planta bacterial titers were determined at the indicated time points after inoculation as described32. These experiments were performed in two different transgenic NILSRF3Ler and Kas-2SRF3Ler lines with similar results. Ler-Kond RILs used for bacterial inoculation have been previously described11.

Subcellular localization of SRF3 variants. The SRF3 Ler cDNA clone was obtained by RT-PCR (Supplementary Table 1). SRF3 Kas-2 and Kond cDNA clones were generated by site-directed mutagenesis (QuickChange II XL, Stratagene) from the Ler ­SRF3 cDNA template (Supplementary Table 1). cDNA clones were fused to YFP by cloning in pXCSG-YFP binary vector33, transformed into Agrobacterium ­tumefaciens strain GV3101 with helper plas-mid pMP90RK28 and infiltrated into Nicotiana ­bethamiana (310A) leaves as previously described34. Agroinfiltrated leaves were imaged with a confocal laser-scanning microscope, the Leica TCS 4D.

Flg22 treatments and immunocomplex kinase assays. Seedlings from each genotype (Ler, Kas-2, NIL, NILSRF3Ler and Kas-2SRF3Ler) were grown under sterile conditions in one-half MS agar plates sealed with leukopor tape to allow aeration. Two weeks after germination, seedlings were transferred to one-half MS liquid media and kept there for 24 h to stabilize under the same condi-tions. Seedlings were treated with 10 μM flg22, and samples were harvested and frozen immediately in liquid nitrogen after 0, 15 and 30 min of treatment. Protein extracts were obtained by homogenization in extraction buffer (50 mM HEPES KOH (pH 7.4), 50 mM NaCl, 10 mM EDTA, 5 mM NaF, 1 mM Na3VO4, 0.1% Triton-X 100, 1 mM DTT, 1 mM PMSF, and ×1 protease inhibi-tor cocktail from Sigma) and centrifuged at 14,000g at 4 °C. The supernatants were used for immunoprecipitation of 100 μg of protein extracts quantified by Bradford (Bio-Rad). Five micrograms of MPK3, MPK4 and MPK6 antibodies (Sigma) were added to each sample and incubated at 4 °C for 1 h. Following incubation, 20 μl of protein A-Sepharose (GE) was added and incubated for an additional 3 h at 4 °C. The resin was collected by centrifugation and washed three times with elution buffer and once with kinase buffer without ATP and resuspended in kinase buffer (50 mM HEPES KOH pH 7.4, 1 mM DTT, 10 mM MgCl2, and 10 μM ATP). The immunoprecipitated MPK3, MPK4 and MPK6 samples were incubated in kinase buffer containing 0.5 mg/ml myelin basic protein (MBP, Sigma) as an artificial substrate and 2.5 μCi of [γ-32P] ATP for 30 min at 30 °C. Reactions were ended by adding SDS-PAGE loading buffer and phosphorylated myelin basic protein (MBP) revealed by autoradiography after SDS-PAGE. For signal normalization, immunodetection of MPK4 protein was performed on a protein blot using equal amounts of total protein extract. Similar results were obtained in two independent experiments.

Fay and Wu’s Hn statistics. The normalized Fay and Wu’s H was calculated as described previously22 in a set of 22 accessions in central Asia and 24 acces-sions in Europe. Different individuals in central Asia were selected to represent a uniform sampling of one individual per population. Accessions from north Europe (from Germany, Belgium and The Netherlands) were selected following the same premises as described above. By sampling single accessions, our sample represents coalescing lineages from different populations (collecting phase of a coalescent metapopulation) and can be analyzed as a single standard popula-tion35. The sequences of SRF3 (At4g03390) and flanking genes (At4g03020, At4g03290, At4g03320, At4g03330, At4g0340, At4g03410, At4g03420, At4g03430, At4g03460, At4g03510 and At4g03620) in central Asian accessions (Bas-1, Cha-2, Dja-1, Hog, Kar-1, Kas-2, Kly-1, Kond, Koz-1, Kyr-1, Leb-3, Mas, Neo-6, Nov-2, Pan-4, Rak-1, Sev-1, Sha, Sij-1, Sorbo, Sus-1 and Zal-1) and European accessions (Aa-0, Amrum, Appt-2, Bay-0, Benk, Berlin, Dune, Fran-0, Föhr, Ga-0, Gu-1, Je-0, Kl-pw-1, Leiden, Li-1, Ma-0, Nd-1, No-0, Ob-2, Oerd-7, Uk-2, Ven-1, Wag-4 and Werl-1) were obtained after PCR amplification from genomic DNA (Supplementary Table 1) and sequencing in ABI 3730XL automated sequencers (Applied Biosystems). Sequences were aligned with ClustalW36 and inspected manually. Arabidopsis ­lyrata sequences were obtained by BLAST search and

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used as an outgroup to assign ancestral and derived states to SNP variants. To assess the significance of the Hn statistic for SRF3, coalescent simulations were used to generate distributions (5,000 replicates) for the test statistic under different demographic models using the program ms37. The observed SRF3 value of Hn was tested against the standard neutral model and three alternative demographic models (Supplementary Table 6).

27. Bechtold, N. & Pelletier, G. In planta Agrobacterium mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259–266 (1998).

28. Koncz, C. & Schell, J. The promoter of Tl-DNA Gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383–396 (1986).

29. Alonso, J.M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003).

30. Pritchard, J.K., Stephens, M. & Donnelly, P. Inference of population structure using multilocus genotype data. Genetics 155, 945–959 (2000).

31. Evanno, G., Regnaut, S. & Goudet, J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–2620 (2005).

32. Tornero, P. & Dangl, J.L. A high-throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana. Plant J. 28, 475–481 (2001).

33. Feys, B.J. et al. Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant innate immunity. Plant Cell 17, 2601–2613 (2005).

34. Häweker, H. et al. Pattern recognition receptors require N-glycosylation to mediate plant immunity. J. Biol. Chem. 285, 4629–4636 (2010).

35. Wakeley, J. & Aliacar, N. Gene genealogies in a metapopulation. Genetics 159, 893–905 (2001).

36. Thompson, J.D., Higgins, D.G. & Gibson, T.J. Clustal-W–Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

37. Hudson, R.R. Generating samples under a Wright-Fisher neutral model of genetic variation. Bioinformatics 18, 337–338 (2002).