Tom Ashfield

Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance

Plants can recognize pathogens through the action of disease resistance (R) genes, which confer resistance to pathogens expressing unique corresponding avirulence (avr) genes. The molecular basis of this gene-for-gene specificity is unknown. The Arabidopsis thaliana RPM1 gene enables dual specificity to pathogens expressing either of two unrelated Pseudomonas syringae avr genes. Despite this function, RPM1 encodes a protein sharing molecular features with recently described single-specificity R genes. Surprisingly, RPM1 is lacking from naturally occurring, disease-susceptible Arabidopsis accessions.

Convergent Evolution of Disease Resistance Gene Specificity in Two Flowering Plant Families

Plant disease resistance (R) genes that mediate recognition of the same pathogen determinant sometimes can be found in distantly related plant families. This observation implies that some R gene alleles may have been conserved throughout the diversification of land plants. To address this question, we have compared R genes from Glycine max (soybean), Rpg1-b, and Arabidopsis thaliana, RPM1, that mediate recognition of the same type III effector protein from Pseudomonas syringae, AvrB. RPM1 has been cloned previously, and here, we describe the isolation of Rpg1-b. Although RPM1 and Rpg1-b both belong to the coiled-coil nucleotide binding site (NBS) Leu-rich repeat (LRR) class of R genes, they share only limited sequence similarity outside the conserved domains characteristic of this class. Phylogenetic analyses of A. thaliana and legume NBS-LRR sequences demonstrate that Rpg1-b and RPM1 are not orthologous. We conclude that convergent evolution, rather than the conservation of an ancient specificity, is responsible for the generation of these AvrB-specific genes.

Differential Accumulation of Retroelements and Diversification of NB-LRR Disease Resistance Genes in Duplicated Regions following Polyploidy in the Ancestor of Soybean

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.

Rpg1, a soybean gene effective against races of bacterial blight, maps to a cluster of previously identified disease resistance genes

Abstract Alleles, or tightly linked genes, at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to races of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. In this study we demonstrate that Rpg1 maps to a cluster of previously identified resistance genes, including those effective against fungal, viral and nematode pathogens. Rpg1 is in molecular linkage group (MLG) F, flanked by the markers K644 and B212. The RFLP markers R45, php2265 and php2385 cosegregated with Rpg1, as did the marker nbs61, which encodes a protein related to previously isolated resistance genes.

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.

Using decoys to expand the recognition specificity of a plant disease resistance protein.

Improving plant disease responses Disease resistance in plants depends on genes that allow them to recognize when they are infected by a pathogen so that they can mount a timely defense response. Unfortunately, pathogens can often overcome endogenous disease resistance genes by evolving new virulence strategies that escape detection. Kim et al. modified the pathogen recognition systems in the model plant Arabidopsis thaliana to widen its reach. The approach should enable the development of crops with more durable disease resistance and hence reduce pesticide use and increase crop yields. Science, this issue p. 684 Swapping proteolytic cleavage sites in a plant immune response pathway enables defense against a new suite of pathogens. Maintaining high crop yields in an environmentally sustainable manner requires the development of disease-resistant crop varieties. We describe a method to engineer disease resistance in plants by means of an endogenous disease resistance gene from Arabidopsis thaliana named RPS5, which encodes a nucleotide-binding leucine-rich repeat (NLR) protein. RPS5 is normally activated when a second host protein, PBS1, is cleaved by the pathogen-secreted protease AvrPphB. We show that the AvrPphB cleavage site within PBS1 can be substituted with cleavage sites for other pathogen proteases, which then enables RPS5 to be activated by these proteases, thereby conferring resistance to new pathogens. This “decoy” approach may be applicable to other NLR proteins and should enable engineering of resistance in plants to diseases for which we currently lack robust genetic resistance.

Replication of Nonautonomous Retroelements in Soybean Appears to Be Both Recent and Common

Retrotransposons and their remnants often constitute more than 50% of higher plant genomes. Although extensively studied in monocot crops such as maize (Zea mays) and rice (Oryza sativa), the impact of retrotransposons on dicot crop genomes is not well documented. Here, we present an analysis of retrotransposons in soybean (Glycine max). Analysis of approximately 3.7 megabases (Mb) of genomic sequence, including 0.87 Mb of pericentromeric sequence, uncovered 45 intact long terminal repeat (LTR)-retrotransposons. The ratio of intact elements to solo LTRs was 8:1, one of the highest reported to date in plants, suggesting that removal of retrotransposons by homologous recombination between LTRs is occurring more slowly in soybean than in previously characterized plant species. Analysis of paired LTR sequences uncovered a low frequency of deletions relative to base substitutions, indicating that removal of retrotransposon sequences by illegitimate recombination is also operating more slowly. Significantly, we identified three subfamilies of nonautonomous elements that have replicated in the recent past, suggesting that retrotransposition can be catalyzed in trans by autonomous elements elsewhere in the genome. Analysis of 1.6 Mb of sequence from Glycine tomentella, a wild perennial relative of soybean, uncovered 23 intact retroelements, two of which had accumulated no mutations in their LTRs, indicating very recent insertion. A similar pattern was found in 0.94 Mb of sequence from Phaseolus vulgaris (common bean). Thus, autonomous and nonautonomous retrotransposons appear to be both abundant and active in Glycine and Phaseolus. The impact of nonautonomous retrotransposon replication on genome size appears to be much greater than previously appreciated.

Evolution of a Complex Disease Resistance Gene Cluster in Diploid Phaseolus and Tetraploid Glycine

We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.

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

*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.

Diversification of Non-TIR Class NB-LRR Genes in Relation to Whole-Genome Duplication Events in Arabidopsis

Arabidopsis thaliana is believed to have experienced at least two and possibly three whole-genome duplication events in its evolutionary history. In order to investigate the evolutionary relationships between these duplication events and diversification of disease resistance (R) genes, segmental-duplication events containing R genes belonging to the nucleotide binding-leucine rich repeat (NB-LRR) class were identified. Of 153 segmental-duplication events containing NB-LRR genes, only 22 contained NB-LRR genes in both members of the duplication pair, indicating a high frequency of NB-LRR gene loss after whole-genome duplication. The relative age of the duplication events was estimated based on the average synonymous substitution rate of the duplicated gene pairs in the segments. These data were combined with phylogenetic analyses. NB-LRR genes present in segment pairs derived from the most recent whole-genome duplication event, estimated to have occurred only 20 to 40 million years ago, occupy very distant branches of the NB-LRR phylogenetic tree. These data suggest that when NB-LRR clusters are duplicated as part of a whole-genome duplication, homoeologous NB-LRR genes are preferentially lost, either by eliminating one copy of the cluster or by eliminating individual genes such that only paralogous NB-LRR genes are maintained.