Specific resistances against Pseudomonas syringae effectors AvrB and AvrRpm1 have evolved differently in common bean (Phaseolus vulgaris), soybean (Glycine max), and Arabidopsis thaliana

Abstract

*In plants, the evolution of specific resistance is poorly understood. Pseudomonas syringae effectors AvrB and AvrRpm1 are recognized by phylogenetically distinct resistance (R) proteins in Arabidopsis thaliana (Brassicaceae) and soybean (Glycine max, Fabaceae). In soybean, these resistances are encoded by two tightly linked R genes, Rpg1-b and Rpg1-r. To study the evolution of these specific resistances, we investigated AvrB- and AvrRpm1-induced responses in common bean (Phaseolus vulgaris, Fabaceae). *Common bean genotypes of various geographical origins were inoculated with P. syringae strains expressing AvrB or AvrRpm1. A common bean recombinant inbred line (RIL) population was used to map R genes to AvrRpm1. *No common bean genotypes recognized AvrB. By contrast, multiple genotypes responded to AvrRpm1, and two independent R genes conferring AvrRpm1-specific resistance were mapped to the ends of linkage group B11 (Rpsar-1, for resistance to Pseudomonas syringae effector AvrRpm1 number 1) and B8 (Rpsar-2). Rpsar-1 is located in a region syntenic with the soybean Rpg1 cluster. However, mapping of specific Rpg1 homologous genes suggests that AvrRpm1 recognition evolved independently in common bean and soybean. *The conservation of the genomic position of AvrRpm1-specific genes between soybean and common bean suggests a model whereby specific clusters of R genes are predisposed to evolve recognition of the same effector molecules.

Fig. 1

Genetic mapping of Rpg1 syntenic regions, Rpg1-related CNL, and Rpsar-1 and Rpsar-2 resistance genes in common bean (Phaseolus vulgaris). (a) Linkage groups (LG) according to Hougaard et al. (2008). Green vertical bars indicate regions magnified in (b) and (c). Green text indicates markers used in (b) and (c). The genomic location of homoeologous region 3 (H3) is indicated by an arrow at the end of LG B5. (b) Genetic mapping of the sequenced common bean contigs (Innes et al., 2008) and of CNL from the Rpg1-b syntenic region to LG B11, and mapping of the Rpsar-1 gene. Vertical black lines on the left represent sequenced BAC contigs. Arrows represent predicted CNL genes and their orientation. Fill and text colors correspond to the colors of the CNL clades defined in Innes et al. (2008) (CNL1, blue; CNL2, absent; CNL3, red, CNL4, purple). The LOD score values, which indicate the most probable genomic location of Rpsar-1, are presented on the right of B11. (c) Mapping of the Rpsar-2 gene to the end of LG B8. SAS13 and Dj1kSCAR are two SCAR markers linked to the anthracnose Co-4 R gene (Adam-Blondon, 1994; Young et al., 1998). Oval symbols correspond to R genes. Genetic distances between each marker are indicated on the left in Kosambi cM for (b) and (c).

Fig. 2

Phylogenetic analysis and physical map of CNL genes. (a) Bayesian tree of CNL genes from the Rpg1 syntenic region in comparison to all Arabidopsis CNL genes. This tree was constructed using just the NB domains (from the P-loop to the MHD motif). Clades of CNL from the Rpg1 locus are colored according to Innes et al. (2008). Green text indicates CNL from the RPM1 clade. Gene names are abbreviated as follows: At, Arabidopsis thaliana; Gm, Glycine max; Mt, Medicago truncatula; Pv, Phaseolus vulgaris. (b) Physical map of CNL genes of the Rpg1-b syntenic region. Vertical black lines represent sequenced BAC contigs from common bean, or chromosome regions from soybean or Arabidopsis with the following coordinates (from top to bottom): AtChr3 (from 4880000bp to 2145000bp), AtChr5 (from 3010000bp to 275000bp), GmChr13 (from 28650000bp to 31385000bp), and GmChr6 (from 50600000bp to 47865000bp). Arrows represent predicted CNL genes and their orientation. Filled colors correspond to the colors of the clades defined in (a). White CNL are pseudogenes with partial or total deletion of the NB region, that were not included in the phylogenetic analysis (Innes et al., 2008). Horizontal lines represent low copy genes conserved between species or homoeologues (in black for genes from side-by-side regions in the Figure (e.g. PvChr11 and GmChr13) and in grey for genes from two non-side-by-side regions (e.g. GmChr6 and AtChr3). The Rpg1-b co-linear region in PvChr11 is indicated by a black horizontal arrow. In common bean, LG B11 and LG B5 corresponds to PvChr11 and PvChr5, respectively (Pedrosa-Harand et al., 2009; Fonsêca et al. 2010).

Fig. 3

(a, b) Psp 1448AN race 6 strains carrying avrRpm1 or not, were inoculated into near-isogenic lines (NILs) diverging for the Co-2 gene (P12S and P12R), and into Cornell49242 (abbreviated as Cornell), the Co-2 donor parent. (a) Phenotypes in leaves 3 d after inoculation. (b) Bacterial titres determined immediately after inoculation (dark grey bars) and 3 d later (light gray bars). Each bar represents the average of three independent samples, ± 1 SD. This experiment was performed twice with similar results. (c) Hybridization of CNL1 or CNL3/4 probes on restricted DNA with EcoRI and HaeIII of P12R, P12S, and Cornell49242. CNL1 bands that differ between P12R and Cornell49242 are indicated by a red asterisk. Molecular weights in kilobases are indicated to the left.

Fig. 4

Reaction phenotypes in pods of common bean (Phaseolus vulgaris) genotypes after inoculation with Psp 1448AN race 6 strains expressing avrRpm1 or avrRpt2 type III effectors. B76 and B87 are two recombinant inbred lines (RILs; derived from a cross between BAT93 and JaloEEP558) displaying an intermediate and a full resistance phenotype after inoculation with Psp(avrRpm1) in leaves, respectively (data not shown). +, a 1:1 mixture of two strains was inoculated simultaneously.→, the second strain was inoculated 2 h after the first strain: 1, Psp; 2, Psp(avrRpm1); 3, Psp(avrRpt2::Ω); 4, Psp(avrRpt2); 5, 1:1 mix of Psp(avrRpm1) + Psp(avrRpt2::Ω); 6, 1:1 mix of Psp(avrRpt2) + Psp(avrRpt2::Ω); 7, 1:1 mix of Psp(avrRpm1) + Psp(avrRpt2); 8, Psp(avrRpt2) followed by Psp(avrRpm1) 2 h later; 9, Psp(avrRpt2::Ω) followed by Psp(avrRpm1) 2 h later.

Fig. 5

Phylogenic analysis and alignment of RIN4 homologs from various plant species. Species names are abbreviated as follows: Al, Arabidopsis lyrata; At, Arabidopsis thaliana; Bd, Brachypodium distachyon; Gm, Glycine max; Os, Oryza sativa; Pt, Populus trichocarpa; Pv, Phaseolus vulgaris; Sb, Sorghum bicolor; Vv, Vitis vinifera. (a) Bayesian tree of RIN4 homologs. The first polyploïdy event (Ks = 0.64 ± 0.06) is indicated by an open circle, and the second polyploïdy event (Ks = 0.16 ± 0.06) is indicated by closed circles. (b) Alignment of complete amino acid sequences of RIN4 homologs. Two highly conserved domains (1 and 2) are indicated. The two AvrRpt2 cleavage sites (RCS1 and RCS2), and the AvrB binding site (BBS) are boxed (Kim et al., 2005). The region that has been co-crystallized with AvrB (RIN4_142–176) is underlined (Desveaux et al., 2007).

Fig. 6

Hypothetical models for the evolution of AvrB and AvrRpm1 recognition specificities in plants. Leg, legumes; At, Arabidopsis thaliana. Only models implying the most parcimonious series of events are shown. In the recombination and classical models, the ability to interact with RIN4 is included with the ability to recognize AvrRpm1 and/or AvrB. Time is not drawn to scale. Mya, million yr ago.