Nucleotide-binding Site Leucine-rich Repeat Resistance Research Articles

An NBS-LRR protein in the Rpp1 locus negates the dominance of Rpp1-mediated resistance against Phakopsora pachyrhizi in soybean.

The soybean Rpp1 locus confers resistance to Phakopsora pachyrhizi, causal agent of rust, and resistance is usually dominant over susceptibility. However, dominance of Rpp1-mediated resistance is lost when a resistant genotype (Rpp1 or Rpp1b) is crossed with susceptible line TMG06_0011, and the mechanism of this dominant susceptibility (DS) is unknown. Sequencing the Rpp1 region reveals that the TMG06_0011 Rpp1 locus has a single nucleotide-binding site leucine-rich repeat (NBS-LRR) gene (DS-R), whereas resistant PI 594760B (Rpp1b) is similar to PI 200492 (Rpp1) and has three NBS-LRR resistance gene candidates. Evidence that DS-R is the cause of DS was reflected in virus-induced gene silencing of DS-R in Rpp1b/DS-R or Rpp1/DS-R heterozygous plants with resistance partially restored. In heterozygous Rpp1b/DS-R plants, expression of Rpp1b candidate genes was not significantly altered, indicating no effect of DS-R on transcription. Physical interaction of the DS-R protein with candidate Rpp1b resistance proteins was supported by yeast two-hybrid studies and in silico modeling. Thus, we conclude that suppression of resistance most likely does not occur at the transcript level, but instead probably at the protein level, possibly with Rpp1 function inhibited by binding to the DS-R protein. The DS-R gene was found in other soybean lines, with an estimated allele frequency of 6% in a diverse population, and also found in wild soybean (Glycine soja). The identification of a dominant susceptible NBS-LRR gene provides insight into the behavior of NBS-LRR proteins and serves as a reminder to breeders that the dominance of an R gene can be influenced by a susceptibility allele.

Genome wide identification and evolutionary analysis of vat like NBS-LRR genes potentially associated with resistance to aphids in cotton.

Nucleotide Binding Site - Leucine Rich Repeat (NBS-LRR) genes play a significant role in plant defense against biotic stresses and are an integral part of signal transduction pathways. Vat gene has been well reported for their role in resistance to Aphis gossypii and viruses transmitted by them. Despite their importance, Vat like NBS-LRR resistance genes have not yet been identified and studied in cotton species. This study report hundreds of orthologous Vat like NBS-LRR genes from the genomes of 18 cotton species through homology searches and the distribution of those identified genes were tend to be clustered on different chromosome. Especially, in a majority of the cases, Vat like genes were located on chromosome number 13 and they all shared two conserved NBS-LRR domains, one disease resistant domain and several repeats of LRR on the investigated cotton Vat like proteins. Gene ontology study on Vat like NBS-LRR genes revealed the molecular functions viz., ADP and protein binding. Phylogenetic analysis also revealed that Vat like sequences of two diploid species, viz., G. arboreum and G. anomalum, were closely related to the sequences of the tetraploids than all other diploids. The Vat like genes of G. aridum and G. schwendimanii were distantly related among diploids and tetraploids species. Various hormones and defense related cis-acting regulatory elements were identified from the 2kb upstream sequences of the Vat like genes implying their defensive response towards the biotic stresses. Interestingly, G. arboreum and G. trilobum were found to have more regulatory elements than larger genomes of tetraploid cotton species. Thus, the present study provides the evidence for the evolution of Vat like genes in defense mechanisms against aphids infestation in cotton genomes and allows further characterization of candidate genes for developing aphid and aphid transmitted viruses resistant crops through cotton breeding.

The Sm gene conferring resistance to gray leaf spot disease encodes an NBS-LRR (nucleotide-binding site-leucine-rich repeat) plant resistance protein in tomato

Gray leaf spot (GLS) resistance in tomato is controlled by one major dominant locus, Sm. Sm was fine mapped, and the nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene Solyc11g020100 was identified as a candidate gene for Sm. Further functional analysis indicated that this gene confers high resistance to Stemphylium lycopersici in tomato. Tomato (Solanum Lycopersicum) is widely consumed and cultivated in the world. Gray leaf spot (GLS), caused by Stemphylium lycopersici (S. lycopersici), is one of the most devastating diseases in tomato production. To date, only one resistance gene, Sm, which confers high resistance against GLS disease, has been identified in the wild tomato species Solanum pimpinellifolium. This resistance locus (comprising the Sm gene) has been transferred into the cultivated variety 'Motelle'. Although several studies have reported the mapping of the Sm gene, it has not been cloned, limiting the utilization in tomato breeding. Here, we cloned Sm using a map-based cloning strategy. The Sm gene was mapped in a region of 160kb at chromosome 11 between two markers, namely, M390 and M410, by using an F2 population from a cross between the resistant cultivar 'Motelle' (Mt) and susceptible line 'Moneymaker' (Mm). Three clustered NBS-LRR (nucleotide-binding site-leucine-rich repeat) resistance genes, namely, Solyc11g020080 (R1), Solyc11g020090 (R2), and Solyc11g020100 (R3) were identified in this interval. Nonsynonymous SNPs were identified in only the open reading frame (ORF) of R3, suggesting it as a strong candidate for the Sm gene. Furthermore, gene silencing of R3 abolished the high resistance to S. lycopersici in Motelle, demonstrating that this gene confers high resistance to S. lycopersici. The cloning of Sm may speed up its utilization for breeding resistant tomato varieties and represents an important step forward in our understanding of the mechanism underlying the resistance to GLS.

Colonisation of Oncidium orchid roots by the endophyte Piriformospora indica restricts Erwinia chrysanthemi infection, stimulates accumulation of NBS-LRR resistance gene transcripts and represses their targeting micro-RNAs in leaves

BackgroundErwinia chrysanthemi (Ec) is a destructive pathogen which causes soft-rot diseases in diverse plant species including orchids. We investigated whether colonization of Oncidium roots by the endophytic fungus Piriformospora indica (Pi) restricts Ec-induced disease development in leaves, and whether this might be related to the regulation of nucleotide binding site-leucine rich repeat (NBS-LRR) Resistance (R) genes.ResultsRoot colonization of Oncidium stackings by Pi restricts progression of Ec-induced disease development in the leaves. Since Pi does not inhibit Ec growth on agar plates, we tested whether NBS-LRR R gene transcripts and the levels of their potential target miRNAs in Oncidium leaves might be regulated by Pi. Using bioinformatic tools, we first identified NBS-LRR R gene sequences from Oncidium, which are predicted to be targets of miRNAs. Among them, the expression of two R genes was repressed and the accumulation of several regulatory miRNA stimulated by Ec in the leaves of Oncidium plants. This correlated with the progression of disease development, jasmonic and salicylic acid accumulation, ethylene synthesis and H2O2 production after Ec infection of Oncidium leaves. Interestingly, root colonization by Pi restricted disease development in the leaves, and this was accompanied by higher expression levels of several defense-related R genes and lower expression level of their target miRNA.ConclusionBased on these data we propose that Pi controls the levels of NBS-LRR R mRNAs and their target miRNAs in leaves. This regulatory circuit correlates with the protection of Oncidium plants against Ec infection, and molecular and biochemical investigations will demonstrate in the future whether, and if so, to what extent these two observations are related to each other.

Identification and Molecular Mapping of a Gummy Stem Blight Resistance Gene in Wild Watermelon (Citrullus amarus) Germplasm PI 189225.

Gummy stem blight (GSB), caused by Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), is a destructive foliar disease of watermelon in areas with hot and humid climates. The wild watermelon germplasm PI 189225 is a known source of resistance to GSB. The identification and use of molecular markers linked to resistance genes in the wild-type germplasm will speed up the introgression of GSB resistance into new watermelon varieties. An F2 segregating population was obtained from a cross between the resistant wild watermelon genotype PI 189225 and the susceptible genotype K3. The F2-derived F3 families were inoculated with a single isolate of S. cucurbitacearum (JS002) from Jiangsu Academy of Agricultural Sciences. The results of the genetic analysis demonstrated that GSB resistance in PI 189225 was controlled by a major quantitative trait locus (QTL), temporarily designated Qgsb8.1. Based on the results of bulk sergeant analysis and sequencing, one associated region spanning 5.7 Mb (10,358,659 to 16,101,517) on chromosome 8 was identified as responsible for the resistance to GSB using the Δ(single-nucleotide polymorphism [SNP]-index) method. The result of a QTL linkage analysis with Kompetitive allele-specific PCR (KASP) SNP markers further mapped the GSB resistance locus between the SNP markers KASP_JS9383 and KASP_JS9168 in a region of 571.27 kb on chromosome 8. According to the watermelon gene annotation database, the region contains approximately 19 annotated genes and, of these 19 genes, 2 are disease resistance gene analogs: Cla001017 (coiled-coil nucleotide-binding site leucine-rich repeat resistance protein) and Cla001019 (pathogenesis related). Reverse-transcription quantitative PCR demonstrated that the expression of the two genes changed following S. cucurbitacearum infection, suggesting that they play important roles in GSB resistance in watermelon. This result will facilitate fine mapping and cloning of the Qgsb8.1 locus, and the linked markers will further provide a useful tool for marker-assisted selection of this locus in watermelon breeding programs.

The Brassicaceae Family Displays Divergent, Shoot-Skewed NLR Resistance Gene Expression.

Nucleotide-binding site leucine-rich repeat resistance genes (NLRs) allow plants to detect microbial effectors. We hypothesized that NLR expression patterns could reflect organ-specific differences in effector challenge and tested this by carrying out a meta-analysis of expression data for 1,235 NLRs from nine plant species. We found stable NLR root/shoot expression ratios within species, suggesting organ-specific hardwiring of NLR expression patterns in anticipation of distinct challenges. Most monocot and dicot plant species preferentially expressed NLRs in roots. In contrast, Brassicaceae species, including oilseed rape (Brassica napus) and the model plant Arabidopsis (Arabidopsis thaliana), were unique in showing NLR expression skewed toward the shoot across multiple phylogenetically distinct groups of NLRs. The Brassicaceae are also outliers in the sense that they have lost the common symbiosis signaling pathway, which enables intracellular infection by root symbionts. While it is unclear if these two events are related, the NLR expression shift identified here suggests that the Brassicaceae may have evolved unique pattern-recognition receptors and antimicrobial root metabolites to substitute for NLR protection. Such innovations in root protection could potentially be exploited in crop rotation schemes or for enhancing root defense systems of non-Brassicaceae crops.

Identification and characterization of novel NBS-LRR resistance gene analogues from the pea.

Pea (Pisum sativum) is one of the most cultivated le-gumes in the world, and its yield and seed quality are affected by a variety of pathogens. In plants, NBS-LRR (nucleotide binding site-leucine-rich repeat) is the main class of disease resistance genes. Using degenerate primers deduced from conserved motifs in the NBS domain of known resistance genes, we identified 10 NBS sequences in three varieties of P. sativum. The deduced amino acid sequences of the iden-tified resistance gene analogues (RGAs) exhibited the typical motifs of the NBS domain (P-loop, kinase-2, kinase-3a, and the hydrophobic domain, GLPL) present in the majority of plant proteins belonging to the NBS-LRR class. Phylogenetic analysis showed that seven RGAs belonged to the non-TIR-NBS-LRR subclass and three to the TIR-NBS-LRR subclass. The results of this study provide insights into the structure of this class of resistance genes in the pea, and their evolution-ary relationships with those of other plant species.

Isolation and characterization of NBS–LRR resistance gene analogues from mango

The nucleotide-binding site (NBS)–leucine-rich repeat (LRR) gene family is a class of R genes in plants. NBS genes play a very important role in disease defence. To further study the variation and homology of mango NBS–LRR genes, 16 resistance gene analogues (RGAs) (GenBank accession number HM446507-22) were isolated from the polymerase chain reaction fragments and sequenced by using two degenerate primer sets. The total nucleotide diversity index Pi was 0.362, and 236 variation sites were found among 16 RGAs. The degree of homology between the RGAs varied from 44.4% to 98.5%. Sixteen RGAs could be translated into amino sequences. The high level of this homology in the protein sequences of the P-loop and kinase-2 of the NBS domain between the RGAs isolated in this study and previously characterized R genes indicated that these cloned sequences belonged to the NBS–LRR gene family. Moreover, these 16 RGAs could be classified into the non-TIR–NBS–LRR gene family because only tryptophan (W) could be claimed as the final residual of the kinase-2 domain of all RGAs isolated here. From our results, we concluded that our mango NBS–LRR genes possessed a high level of variation from the mango genome, which may allow mango to recognize many different pathogenic virulence factors.

Review Functional characterization and signal transduction ability of nucleotide-binding site-leucine-rich repeat resistance genes in plants

Pathogen infection in plants is often limited by a multifaceted defense response triggered by resistance genes. The most prevalent class of resistance proteins includes those that contain a nucleotide-binding site-leucine-rich repeat (NBS-LRR) domain. Over the past 15 years, more than 50 novel NBS-LRR class resistance genes have been isolated and characterized; they play a significant role in activating conserved defense-signaling networks. Recent molecular research on NBS-LRR resistance proteins and their signaling networks has the potential to broaden the use of resistance genes for disease control. Various transgenic approaches have been tested to broaden the disease resistance spectrum using NBS-LRR genes. This review highlights the recent progress in understanding the structure, function, signal transduction ability of NBS-LRR resistance genes in different host-pathogen systems and suggests new strategies for engineering pathogen resistance in crop plants.

Identification and Mapping of Nucleotide Binding Site–Leucine-rich Repeat Resistance Gene Analogs in Bermudagrass

Thirty-one partial bermudagrass (Cynodon spp.) disease-resistance gene analogs (BRGA) were cloned and sequenced from diploid, triploid, tetraploid, and hexaploid bermudagrass using degenerate primers to target the nucleotide binding site (NBS) of the NBS–leucine-rich repeat (LRR) resistance gene family. Alignment of deduced amino acid sequences revealed that the conserved motifs of the NBS are present and all sequences have non-Drosophila melanogaster Toll and mammalian interleukin-1 receptor (TIR) motifs. Using a neighbor-joining algorithm, a dendrogram was created and nine groups of deduced amino acid sequences from bermudagrass could be identified from those sequences that span the NBS. Four BRGA markers and 15 bermudagrass expressed sequence tags (ESTs) with similarity to resistance genes or resistance gene analogs were placed on a bermudagrass genetic map. Multiple BRGA and EST markers mapped on T89 linkage groups 1a and 5a and clusters were seen on T89 19 and two linkage groups previously unidentified. In addition, three primers made from BRGA groups and ESTs with similarity to NBS-LRR resistance genes amplify NBS-LRR analogs in zoysiagrass (Zoysia japonica or Z. matrella) or seashore paspalum (Paspalum vaginatum). This gives evidence of conservation of NBS-LRR analogs among the subfamilies Chloridoideae and Panicoideae. Once disease resistance genes are identified, these BRGA and EST markers may be useful in marker-assisted selection for the improvement of disease resistance in bermudagrass.

A Series of Novel rps2 Alleles Disrupt the RPS2-avrRpt2 Gene-for-gene Interaction

The Arabidopsis resistance (R) RPS2 gene confers resistance to Pseudomonas syringae (P. syringae) strains that express the avirulence (avr) avrRpt2 gene [Kunkel et al., 1993]. When Arabidopsis plants containing RPS2 are challenged with P. syringae bacteria carrying avrRpt2, the plants activate disease resistance responses, including a specialized type of programmed cell death known as the hypersensitive response (HR) [Kunkel et al., 1993; Hammond-Kosack and Jones, 1997]. The interaction between RPS2 and avrRpt2 products leads to an excellent model for pathogen-induced resistance responses and signal transduction events. As a typical resistance protein of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class, the RPS2 protein, consisting of 909 amino acids, contains several possible functional peptide motifs, including an Nterminal hydrophobic region, a putative leucine zipper (LZ), a NBS and a LRR (Fig. 1A) [Hammond-Kosack and Jones, 1997]. Additional motifs found in NBS-LRR resistance genes, such as GLPL and KMH motifs, are present in the RPS2 sequence [Traut, 1994]. The LZ and LRR domains have been implicated in regards to proteinprotein interactions (Alber, 1992; Kobe and Deisenhofer, 1994). The conserved central NBS domain is hypothesized to bind either to ATP or GTP involving kinase activities, containing 3 motifs: the P loop (or kninase-1a), kinase-2 and less conserved kinase-3a [Traut, 1994]. These structural features regarding the RPS2 protein suggest that it is involved in signal transduction processes for disease resistance responses. For studies of the molecular function of RPS2 and signaling by RPS2-avrRpt2 interaction, about 20 mutants including rps2-101C have so far been isolated [Kunkel et al., 1993; Mindrinos et al., 1994; Axtell et al., 2001]. In order to study on the spatial structure and function of RPS2, we tried to isolate numerous additional mutants, defective in RPS2-avrRpt2 dependent cell death, by using Axtell et al. [2001]’s method with minor modifications. Transgenic Col-0 Arabidopsis seeds, possessing a dexamethasone-inducible avrRpt2 transgene (pTA7001avrRpt2) [McNellis et al., 1998], were obtained from Dr. Brian J. Staskawicz (University of California, Berkeley, CA), and then mutagenized with 0.1% ethyl methane sulfonate (Sigma, St. Louis, MO). M2 plants based on the self-fertilization of M1 seeds were grown on soil. When dexamethasone (30 μM) was sprayed onto 2-week-old M2 plants, almost were yellowish in color, had aborted growth rates and eventually died a few days later (Fig. 1B). To identify the presence of the avrRpt2 transgene in the surviving plants, genomic DNAs were isolated from leaves, then the polymerase chain reaction (PCR) was performed by using two specific primers (data not shown). The upstream and downstream sequences were 5'-ATGAAAATTGCTCCAGTTGCCAT-3' and 5'-TTAG CGGTAGAGCATTGCGTGT-3', respectively. Twenty-six mutants out of the surviving plants were screened by PCR. Some mutations were expected to eliminate expression of the avrRpt2 transgene due to mutations in the dexamethasone-inducible expression system (pTA7001 expression cassette). To test this possibility, we performed a Northern blot analysis (Fig. 1C). Total RNA was isolated from leaves taken 1 day after application of dexamethasone. The accumulation of avrRpt2 transcripts was detected in 7 out of 26 mutants as well as rps2-101C as a positive control line. To examine whether 7 mutants showed the ability to mount an HR in response to avrRpt2, we performed a secondary screening procedure by inoculating mutant leaves with the P. syringae pv. maculicola strain DG6 (carrying avrRpt2) at OD600=0.1 [Guttman and Greenberg, 2001]. The typical resistance response to this interaction *Corresponding author Phone: +82-53-950-7753; Fax: +82-53-958-6880 E-mail: jtsong68@knu.ac.kr

Identification and characterisation of resistance gene analogues from wild Chinese Vitis species

SummaryGenerating a collection of resistance gene analogues (RGAs) is an initial step towards the isolation of plant disease resistance genes. Based on the two conserved motifs in the nucleotide-binding site leucine-rich repeat (NBS-LRR) domain from previously cloned plant resistance genes, degenerate oligonucleotide primers were designed and used to isolate RGAs from five wild Chinese Vitis species: V. quinquangularis ‘83-4-96’, V. bashanica ‘Xunyang-8’, V. davidii ‘Tangwei’, V. yeshanensis ‘Yanshan’, and V. bryoniifolia ‘Anlin-3’. A total of 55 RGAs with uninterrupted open reading frames were obtained and further assembled within each species into 26 distinct RGAs. These RGAs were classified into six distinct families. Their deduced amino acid sequences contained the conserved motifs characteristic of NBS-LRR resistance genes. However, the sequences of the identified RGAs were very divergent from each other. The low ratio of non-synonymous to synonymous substitutions, and non-detectable recombination events within families, indicate that the NBS regions of Vitis RGAs are under purifying selection rather than diversifying selection. Furthermore, all the RGAs identified in this study were grouped into the non-Toll/Interleukin 1 receptor (TIR)-type of NBS-LRR resistance genes, based on phylogenetic analysis with other known plant R genes.

Plant disease susceptibility conferred by a “resistance” gene

The molecular nature of many plant disease resistance (R) genes is known; the largest class encodes nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins that are structurally related to proteins involved in innate immunity in animals. Few genes conferring disease susceptibility, on the other hand, have been identified. Recent identification of susceptibility to the fungus Cochliobolus victoriae in Arabidopsis thaliana has enabled our cloning of LOV1, a disease susceptibility gene that, paradoxically, is a member of the NBS-LRR resistance gene family. We found LOV1 mediates responses associated with defense, but mutations in known defense response pathways do not prevent susceptibility to C. victoriae. These findings demonstrate that NBS-LRR genes can condition disease susceptibility and resistance and may have implications for R gene deployment.

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.

Physical and Genetic Mapping of Wheat Kinase Analogs and NBS-LRR Resistance Gene Analogs

Conserved motifs within resistance genes have been utilized in polymerase chain reaction (PCR)-based strategies to isolate resistance gene analogs (RGAs) and resistance gene-like (RGLs) sequences from many plant species. RGAs have the potential to serve as closely linked markers for marker-assisted breeding or even as resistance gene candidates. The objectives of this study were to clone, sequence, and map RGAs and kinase analogs (KA). Three motifs in nucleotide binding site-leucine-rich repeat (NBS-LRR) resistance genes and two conserved motifs within R-gene kinases were used to design degenerate primers and amplify RGAs and KAs from wheat (Triticum aestivum L.). Eight NBS-LRR and 26 KAs were isolated. The clones were physically mapped to chromosomes by means of wheat nulli-tetrasomic lines. The probes detected 137 fragments that could be assigned to 20 of the 21 chromosomes; nearly half of the fragments mapped in the B genome. None of the fragments mapped to chromosome 4D. Genetic mapping of clones showed simple and complex loci indicating both single and multigene families. The RGAs and KAs will be useful as markers for mapping resistance gene loci.

Diversity, distribution, and ancient taxonomic relationships within the TIR and non-TIR NBS-LRR resistance gene subfamilies.

Phylogenetic relationships among the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance gene homologues (RGHs) from 30 genera and nine families were evaluated relative to phylogenies for these taxa. More than 800 NBS-LRR RGHs were analyzed, primarily from Fabaceae, Brassicaceae, Poaceae, and Solanaceae species, but also from representatives of other angiosperm and gymnosperm families. Parsimony, maximum likelihood, and distance methods were used to classify these RGHs relative to previously observed gene subfamilies as well as within more closely related sequence clades. Grouping sequences using a distance cutoff of 250 PAM units (point accepted mutations per 100 residues) identified at least five ancient sequence clades with representatives from several plant families: the previously observed TIR gene subfamily and a minimum of four deep splits within the non-TIR gene subfamily. The deep splits in the non-TIR subfamily are also reflected in comparisons of amino acid substitution rates in various species and in ratios of nonsynonymous-to-synonymous nucleotide substitution rates ( K(A)/ K(S) values) in Arabidopsis thaliana. Lower K(A)/ K(S) values in the TIR than the non-TIR sequences suggest greater functional constraints in the TIR subfamily. At least three of the five identified ancient clades appear to predate the angiosperm-gymnosperm radiation. Monocot sequences are absent from the TIR subfamily, as observed in previous studies. In both subfamilies, clades with sequences separated by approximately 150 PAM units are family but not genus specific, providing a rough measure of minimum dates for the first diversification event within these clades. Within any one clade, particular taxa may be dramatically over- or underrepresented, suggesting preferential expansions or losses of certain RGH types within particular taxa and suggesting that no one species will provide models for all major sequence types in other taxa.

Mutational analysis of the Arabidopsis RPS2 disease resistance gene and the corresponding pseudomonas syringae avrRpt2 avirulence gene.

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.

Mutational analysis of the Arabidopsis nucleotide binding site-leucine-rich repeat resistance gene RPS2.

Disease resistance proteins containing a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region compose the largest class of disease resistance proteins. These so-called NBS-LRR proteins confer resistance against a wide variety of phytopathogens. To help elucidate the mechanism by which NBS-LRR proteins recognize and transmit pathogen-derived signals, we analyzed mutant versions of the Arabidopsis NBS-LRR protein RPS2. The RPS2 gene confers resistance against Pseudomonas syringae strains carrying the avirulence gene avrRpt2. The activity of RPS2 derivatives in response to AvrRpt2 was measured by using a functional transient expression assay or by expressing the mutant proteins in transgenic plants. Directed mutagenesis revealed that the NBS and an N-terminal leucine zipper (LZ) motif were critical for RPS2 function. Mutations near the N terminus, including an LZ mutation, resulted in proteins that exhibited a dominant negative effect on wild-type RPS2. Scanning the RPS2 molecule with a small in-frame internal deletion demonstrated that RPS2 does not have a large dispensable region. Overexpression of RPS2 in the transient assay in the absence of avrRpt2 also led to an apparent resistant response, presumably a consequence of a low basal activity of RPS2. The NBS and LZ were essential for this overdose effect, whereas the entire LRR was dispensable. RPS2 interaction with a 75-kD protein (p75) required an N-terminal portion of RPS2 that is smaller than the region required for the overdose effect. These findings illuminate the pathogen recognition mechanisms common among NBS-LRR proteins.