Fine genetic mapping of the <Emphasis Type="Italic">TuNI</Emphasis> locus causing systemic veinal necrosis by turnip mosaic virus infection in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In the pathosystem of turnip mosaic virus (TuMV) and Arabidopsis thaliana, two distinct symptoms (mosaic symptom and veinal necrosis) were observed that were dependent upon the combination of the TuMV isolate and the Arabidopsis ecotype. The Col-0 ecotype developed mosaic symptoms after infection with the TuMV isolate Azu while the Ler ecotype developed veinal necrosis after infection with the same TuMV isolate. The Ler phenotype is controlled by a single dominant gene TuNI (TuMV necrosis inducer) which is located on chromosome 1. The TuNI gene was precisely mapped to the ~105 kb interval between the two markers of mXF41 and mRF28 by using several types of DNA polymorphism markers. Within this region, which included largely duplicated sequences, a total of 19 putative genes were predicted and 15 of these were classified into five gene families. The genes belonging to the gene families At1g58480 and At1g58602 may function in response to infection by pathogens. The gene family At1g58480 encodes lipase-like proteins, which might be involved in the induction of defence responses that are mediated by salicylic acid. The gene family At1g58602 encodes the CC-NBS-LRR (CNL) proteins, which are known to function as one of the plant resistance (R) proteins against pathogens. In the present study, the possibility that TuNI might function as an R gene was discussed.Y.K. and T.I. contributed equally to this study     

The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana

We have used the naturally occurring plant-parasite system of Arabidopsis thaliana and its common parasite Peronospora parasitica (downy mildew) to study the evolution of resistance specificity in the host population. DNA sequence of the resistance gene, RPP13, from 24 accessions, including 20 from the United Kingdom, revealed amino acid sequence diversity higher than that of any protein coding gene reported so far in A. thaliana. A significant excess of amino acid polymorphism segregating within this species is localized within the leucine-rich repeat (LRR) domain of RPP13. These results indicate that single alleles of the gene have not swept through the population, but instead, a diverse collection of alleles have been maintained. Transgenic complementation experiments demonstrate functional differences among alleles in their resistance to various pathogen isolates, suggesting that the extreme amino acid polymorphism in RPP13 is maintained through continual reciprocal selection between host and pathogen.     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean

A new disease resistance locus in Arabidopsis, RPS3 , was identified using a previously cloned avirulence gene from a non- Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis , and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB , and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2. In F₂ plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3 . Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and ≤ 1 cM from another disease resistance locus, RPM1 . In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1 . Thus, RPG1 and RPS3 both confer avrB -specific disease resistance, suggesting that these genes may be homologs.     

A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes.

Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance (R) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways.     

The tomato Cf-5 disease resistance gene and six homologs show pronounced allelic variation in leucine-rich repeat copy number.

The tomato Cf-2 and Cf-5 genes confer resistance to Cladosporium fulvum and map to a complex locus on chromosome 6. The Cf-5 gene has been isolated and is predicted to encode a largely extracytoplasmic protein containing 32 leucine-rich repeats (LRRs), resembling the previously isolated Cf-2 gene, which has 38 LRRs. Three haplotypes of this locus from Lycopersicon esculentum, L. pimpinellifolium, and L. esculentum var cerasiforme were compared, and five additional homologs of Cf-5 were sequenced. All share extensive sequence identity, particularly within the C-terminal portions of the predicted proteins. In striking contrast to the Cf-9 gene family, six of seven homologs in the Cf-2/Cf-5 gene family vary in LRR copy number, ranging from 25 to 38 LRRs. Cf-5 and one adjacent homolog differ by only two LRRs. Recombination events that vary the LRR copy number in this region could provide a mechanism for the generation of new specificities for recognition of different ligands. A recombination breakpoint between the Cf-2 and Cf-5 loci was fully characterized and shown to be intragenic.     

Genetic analysis of developmentally regulated resistance to downy mildew (Hyaloperonospora parasitica) in Arabidopsis thaliana

Although developmentally regulated disease resistance has been observed in a variety of plant-pathogen interactions, the molecular basis of this phenomenon is not well understood. Arabidopsis thaliana ecotype Columbia-0 (Col-0) expresses a developmentally regulated resistance to Hyaloperonospora parasitica isolate Emco5. Col-0 seedlings support profuse mycelial growth and asexual spore formation in the cotyledons. In contrast, Emco5 growth and reproduction is dramatically (but not completely) restricted in the first set of true leaves. Subsequent leaves exhibit progresssively increased resistance. This adult resistance is strongly suppressed by expression of the salicylic acid-degrading transgene NahG and by loss-of-function mutations in the defense-response regulators PAD4, NDR1, RAR1, PBS3, and NPR1. In contrast to Col-0, the Wassilewskija-0 (Ws-0) ecotype supports profuse growth of Emco5 at all stages of development. Gene-dosage experiments and segregation patterns indicate that adult susceptibility in Ws-0 is incomepletely dominant to adult resistance in Col-0. Genetic mapping in a Col x Ws F2 population revealed a major locus on the bottom arm of chromosome 5, which we named RPP31. Analysis of T-DNA insertion lines indicated that the Columbia allele of RPP8, though tightly linked to RPP31, is not necessary for adult resistance.     

Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

Background  

Nucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes.     

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.     

A genome-wide survey of R gene polymorphisms in Arabidopsis

We used polymorphism analysis to study the evolutionary dynamics of 27 disease resistance (R) genes by resequencing the leucine-rich repeat (LRR) region in 96 Arabidopsis thaliana accessions. We compared single nucleotide polymorphisms (SNPs) in these R genes to an empirical distribution of SNP in the same sample based on 876 fragments selected to sample the entire genome. LRR regions are highly polymorphic for protein variants but not for synonymous changes, suggesting that they generate many alleles maintained for short time periods. Recombination is also relatively common and important for generating protein variants. Although none of the genes is nearly as polymorphic as RPP13, a locus previously shown to have strong signatures of balancing selection, seven genes show weaker indications of balancing selection. Five R genes are relatively invariant, indicating young alleles, but all contain segregating protein variants. Polymorphism analysis in neighboring fragments yielded inconclusive evidence for recent selective sweeps at these loci. In addition, few alleles are candidates for rapid increases in frequency expected under directional selection. Haplotype sharing analysis revealed significant underrepresentation of R gene alleles with extended haplotypes compared with 1102 random genomic fragments. Lack of convincing evidence for directional selection or selective sweeps argues against an arms race driving R gene evolution. Instead, the data support transient or frequency-dependent selection maintaining protein variants at a locus for variable time periods.     

Two TIR:NB:LRR genes are required to specify resistance to Peronospora parasitica isolate Cala2 in Arabidopsis

Resistance responses that plants deploy in defence against pathogens are often triggered following a recognition event mediated by resistance (R) genes. The encoded R proteins usually contain a nucleotide-binding site (NB) and a leucine-rich repeat (LRR) domain. They are further classified into those that contain an N-terminal coiled coil (CC) motif or a Toll interleukin receptor (TIR) domain. Such R genes, when transferred into a susceptible plant of the same or closely related species, usually impart full resistance capability. We have used map-based cloning and mutation analysis to study the recognition of Peronospora parasitica (RPP)2 (At) locus in Arabidopsis accession Columbia (Col-0), which is a determinant of specific recognition of P. parasitica (At) isolate Cala2. Genetic mapping located RPP2 to a 200-kb interval on chromosome 4, which contained four adjacent TIR:NB:LRR genes. Mutational analysis revealed three classes of genes involved in specifying resistance to Cala2. One class, which resulted in pleiotropic effects on resistance to other P. parasitica (At) isolates, was unlinked to the RPP2 locus; this class included AtSGT1b. The other two classes were mapped within the interval and were specific to Cala2 resistance. Representatives of each of these classes were sequenced, and mutations were found in one or the other of two (RPP2A and RPP2B) of the four TIR:NB:LRR genes. RPP2A and RPP2B complemented their specific mutations, but failed to impart resistance when present alone, and it is concluded that both genes are essential determinants for isolate-specific recognition of Cala2. RPP2A has an unusual structure with a short LRR domain at the C-terminus, preceded by two potential but incomplete TIR:NB domains. In addition, the RPP2A LRR domain lacks conserved motifs found in all but three other TIR:NB:LRR class proteins. In contrast, RPP2B has a complete TIR:NB:LRR structure. It is concluded that RPP2A and RPP2B cooperate to specify Cala2 resistance by providing recognition or signalling functions lacked by either partner protein.     

A new Ac-like transposon of Arabidopsis is associated with a deletion of the RPS5 disease resistance gene

The RPS5 and RFL1 disease resistance genes of Arabidopsis ecotype Col-0 are oriented in tandem and are separated by 1.4 kb. The Ler-0 ecotype contains RFL1, but lacks RPS5. Sequence analysis of the RPS5 deletion region in Ler-0 revealed the presence of an Ac-like transposable element, which we have designated Tag2. Southern hybridization analysis of six Arabidopsis ecotypes revealed 4-11 Tag2-homologous sequences in each, indicating that this element is ubiquitous in Arabidopsis and has been active in recent evolutionary time. The Tag2 insertion adjacent to RFL1 was unique to the Ler-0 ecotype, however, and was not present in two other ecotypes that lack RPS5. DNA sequence from the latter ecotypes lacked a transposon footprint, suggesting that insertion of Tag2 occurred after the initial deletion of RPS5. The deletion breakpoint contained a 192-bp insertion that displayed hallmarks of a nonhomologous DNA end-joining event. We conclude that loss of RPS5 was caused by a double-strand break and subsequent repair, and cannot be attributed to unequal crossing over between resistance gene homologs.     

Arabidopsis downy mildew resistance gene RPP27 encodes a receptor-like protein similar to CLAVATA2 and tomato Cf-9

The Arabidopsis Ler-RPP27 gene confers AtSgt1b-independent resistance to downy mildew (Peronospora parasitica) isolate Hiks1. The RPP27 locus was mapped to a four-bacterial artificial chromosome interval on chromosome 1 from genetic analysis of a cross between the enhanced susceptibility mutant Col-edm1 (Col-sgt1) and Landsberg erecta (Ler-0). A Cf-like candidate gene in this interval was PCR amplified from Ler-0 and transformed into mutant Col-rpp7.1 plants. Homozygous transgenic lines conferred resistance to Hiks1 and at least four Ler-0 avirulent/Columbia-0 (Col-0) virulent isolates of downy mildew pathogen. A full-length RPP27 cDNA was isolated, and analysis of the deduced amino acid sequences showed that the gene encodes a receptor-like protein (RLP) with a distinct domain structure, composed of a signal peptide followed by extracellular Leu-rich repeats, a membrane spanning region, and a short cytoplasmic carboxyl domain. RPP27 is the first RLP-encoding gene to be implicated in disease resistance in Arabidopsis, enabling the deployment of Arabidopsis techniques to investigate the mechanisms of RLP function. Homology searches of the Arabidopsis genome, using the RPP27, Cf-9, and Cf-2 protein sequences as a starting point, identify 59 RLPs, including the already known CLAVATA2 and TOO MANY MOUTHS genes. A combination of sequence and phylogenetic analysis of these predicted RLPs reveals conserved structural features of the family.     

The leucine-rich repeat domain can determine effective interaction between RPS2 and other host factors in arabidopsis RPS2-mediated disease resistance

Like many other plant disease resistance genes, Arabidopsis thaliana RPS2 encodes a product with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. This study explored the hypothesized interaction of RPS2 with other host factors that may be required for perception of Pseudomonas syringae pathogens that express avrRpt2 and/or for the subsequent induction of plant defense responses. Crosses between Arabidopsis ecotypes Col-0 (resistant) and Po-1 (susceptible) revealed segregation of more than one gene that controls resistance to P. syringae that express avrRpt2. Many F(2) and F(3) progeny exhibited intermediate resistance phenotypes. In addition to RPS2, at least one additional genetic interval associated with this defense response was identified and mapped using quantitative genetic methods. Further genetic and molecular genetic complementation experiments with cloned RPS2 alleles revealed that the Po-1 allele of RPS2 can function in a Col-0 genetic background, but not in a Po-1 background. The other resistance-determining genes of Po-1 can function, however, as they successfully conferred resistance in combination with the Col-0 allele of RPS2. Domain-swap experiments revealed that in RPS2, a polymorphism at six amino acids in the LRR region is responsible for this allele-specific ability to function with other host factors.     

Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines

The perception of downy mildew avirulence (Arabidopsis thaliana Recognized [ATR]) gene products by matching Arabidopsis thaliana resistance (Recognition of Peronospora parasitica [RPP]) gene products triggers localized cell death (a hypersensitive response) in the host plant, and this inhibits pathogen development. The oomycete pathogen, therefore, is under selection pressure to alter the form of these gene products to prevent detection. That the pathogen maintains these genes indicates that they play a positive role in pathogen survival. Despite significant progress in cloning plant RPP genes and characterizing essential plant components of resistance signaling pathways, little progress has been made in identifying the oomycete molecules that trigger them. Concluding a map-based cloning effort, we have identified an avirulence gene, ATR1NdWsB, that is detected by RPP1 from the Arabidopsis accession Niederzenz in the cytoplasm of host plant cells. We report the cloning of six highly divergent alleles of ATR1NdWsB from eight downy mildew isolates and demonstrate that the ATR1NdWsB alleles are differentially recognized by RPP1 genes from two Arabidopsis accessions (Niederzenz and Wassilewskija). RPP1-Nd recognizes a single allele of ATR1NdWsB; RPP1-WsB also detects this allele plus three additional alleles with divergent sequences. The Emco5 isolate expresses an allele of ATR1NdWsB that is recognized by RPP1-WsB, but the isolate evades detection in planta. Although the Cala2 isolate is recognized by RPP1-WsA, the ATR1NdWsB allele from Cala2 is not, demonstrating that RPP1-WsA detects a novel ATR gene product. Cloning of ATR1NdWsB has highlighted the presence of a highly conserved novel amino acid motif in avirulence proteins from three different oomycetes. The presence of the motif in additional secreted proteins from plant pathogenic oomycetes and its similarity to a host-targeting signal from malaria parasites suggest a conserved role in pathogenicity.     

ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt

Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species. We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana. One of these strains, 14.25, causes wilting symptoms in A. thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0). Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity. Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R. solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5. These loci explain about 90% of the resistance carried by the Col-0 accession. The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1. Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R. solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

TIR-X and TIR-NBS proteins: two new families related to disease resistance TIR-NBS-LRR proteins encoded in Arabidopsis and other plant genomes

The Toll/interleukin-1 receptor (TIR) domain is found in one of the two large families of homologues of plant disease resistance proteins (R proteins) in Arabidopsis and other dicotyledonous plants. In addition to these TIR-NBS-LRR (TNL) R proteins, we identified two families of TIR-containing proteins encoded in the Arabidopsis Col-0 genome. The TIR-X (TX) family of proteins lacks both the nucleotide-binding site (NBS) and the leucine rich repeats (LRRs) that are characteristic of the R proteins, while the TIR-NBS (TN) proteins contain much of the NBS, but lack the LRR. In Col-0, the TX family is encoded by 27 genes and three pseudogenes; the TN family is encoded by 20 genes and one pseudogene. Using massively parallel signature sequencing (MPSS), expression was detected at low levels for approximately 85% of the TN-encoding genes. Expression was detected for only approximately 40% of the TX-encoding genes, again at low levels. Physical map data and phylogenetic analysis indicated that multiple genomic duplication events have increased the numbers of TX and TN genes in Arabidopsis. Genes encoding TX, TN and TNL proteins were demonstrated in conifers; TX and TN genes are present in very low numbers in grass genomes. The expression, prevalence, and diversity of TX and TN genes suggests that these genes encode functional proteins rather than resulting from degradation or deletions of TNL genes. These TX and TN proteins could be plant analogues of small TIR-adapter proteins that function in mammalian innate immune responses such as MyD88 and Mal.     

RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica

Disease resistance (R) genes are found in plants as either simple (single allelic series) loci, or more frequently as complex loci of tandemly repeated genes. These different loci are likely to be under similar evolutionary forces from pathogens, but the contrast between them suggests important differences in mechanisms associated with DNA structure and recombination that generate and maintain R gene diversity. The RPP13 locus in Arabidopsis represents an important paradigm for studying the evolution of an R gene at a simple locus. The RPP13 allele from the accession Nd-1, designated RPP13-Nd, confers resistance to five different isolates of the biotrophic oomycete, Peronospora parasitica (causal agent of downy mildew), and encodes an NBS-LRR type R protein with a putative amino-terminal leucine zipper. The RPP13-Rld allele, cloned from the accession Rld-2, encodes a different specificity. Comparison of three RPP13 alleles revealed a high rate of amino acid divergence within the LRR domain, less than 80% identity overall, compared to the remainder of the protein (> 95% identity). We also found evidence for positive selection in the LRR domain for amino acid diversification outside the core conserved beta-strand/beta-turn motif, suggesting that more of the LRR structure is available for interaction with target molecules than has previously been reported for other R gene products. Furthermore, an amino acid sequence (LLRVLDL) identical in an LRR among RPP13 alleles is conserved in other LZ NBS-LRR type R proteins, suggesting functional significance.