The Leucine-Rich Repeat Domain Can Determine Effective Interaction Between RPS2 and Other Host Factors in Arabidopsis RPS2-Mediated Disease Resistance

Corresponding author: Andrew F. Bent, Department of Plant Pathology, Russell Laboratories, University of Wisconsin, 1630 Linden Dr., Madison, WI 53706-1598., afb{at}plantpath.wisc.edu (E-mail)

Communicating editor: B. S. GILL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 F₂ and F₃ 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.

PLANT disease resistance is often controlled by gene-for-gene interaction between plant resistance (R) genes and pathogen avirulence (avr) genes (CRUTE and PINK 1996 ; HAMMOND-KOSACK and JONES 1997 ). When R and avr alleles of matched specificity are present, the plant induces strong defense responses that restrict pathogen growth. This defense-inducing capacity is likely to require the action of many host factors in addition to the R gene product.

The interaction between R and avr gene products has often been modeled as a receptor-ligand interaction, and a small number of examples provide support for direct physical interaction (SCOFIELD et al. 1996 ; TANG et al. 1996 ; JIA et al. 2000 ; LEISTER and KATAGIRI 2000 ). To date, new pathogen recognition specificities have most often been traced to variation within the leucine-rich repeat (LRR)-encoding domain of R genes, reinforcing the concept that the LRR is primarily a pathogen recognition domain (PARNISKE et al. 1997 ; THOMAS et al. 1997 ; MCDOWELL et al. 1998 ; MEYERS et al. 1998 ; ELLIS et al. 1999 ; NOEL et al. 1999 ; BITTNER-EDDY et al. 2000 ; LUCK et al. 2000 ). A similar paradigm is well developed for LRR receptor proteins from mammals and other organisms (e.g., BRAUN et al. 1991 ; KOBE and DEISENHOFER 1994 ; MARINO et al. 2000 ). Individual plants carry hundreds of apparent R genes and substantial allelic diversity can exist among the LRR-encoding domains of R genes, giving rise to a wide array of pathogen recognition specificities (ELLIS et al. 2000 ; YOUNG 2000 ). Structural variation within other R gene domains and within pathogen avr alleles can also contribute to new pathogen recognition specificities (HERBERS et al. 1992 ; ELLIS et al. 2000 ; WHITE et al. 2000 ).

A simple receptor-ligand model for the interaction of R and avr gene products does not preclude a requirement for additional host factors in defense signaling. These other host factors may act upstream, downstream, in parallel, or in concert with an interaction between R and avr gene products. In one example, the Rar1 gene is required for the function of some Mla R gene alleles in barley (SHIRASU et al. 1999 ). Two tomato R gene products, the Pto kinase and the Prf nucleotide-binding site (NBS)-LRR protein, are both required for the resistance response against P. syringae pathogens that express avrPto (MARTIN et al. 1993 ; SALMERON et al. 1996 ), but physical interaction between the Pto and Prf proteins has not been reported. The presence of a high-affinity binding site for Avr9 peptide in both Cf-9⁺ and Cf-9^- tomato cell extracts suggests that other gene products are required for a defense-inducing interaction to take place between Cf-9 and Avr9 (KOOMAN-GERSMANN et al. 1996 ).

In some cases, genes have been identified that contribute to defense signaling in multiple R/avr gene pathways. Prf of tomato is required for the function of both Pto and the Pto homolog Fen, and thus is shared between two separate pathways that mediate responses to different ligands (SALMERON et al. 1994 ). EDS1, NDR1, PBS2, and PBS3 provide examples of Arabidopsis genes for which mutations disrupt multiple, but not all, gene-for-gene interactions (INNES 1998 ). Rcr loci are required for the function of tomato Cf-9 and Cf-2 R genes (HAMMOND-KOSACK et al. 1994 ). The literature on classical resistance genetics and breeding contains many additional examples of "modifier" loci that alter the activity or quantitative strength of one or more resistance loci (MICHELMORE 1995 ; CRUTE and PINK 1996 ; HAMMOND-KOSACK and JONES 1997 ). Hence the presence and strength of the defense response in a given gene-for-gene resistance pathway can be modulated by variation at avr genes, R genes, or accessory plant loci. However, the molecular basis of these defense-determining interactions remains poorly understood.

The disease resistance gene RPS2 of Arabidopsis thaliana blocks infection by Pseudomonas syringae pathogens that express the avirulence gene avrRpt2 (KUNKEL et al. 1993 ; YU et al. 1993 ). As part of this response, resistant plants develop the hypersensitive response, (HR), a programmed cell death process that arises within hours at and around the site of infection. The HR is associated with disease resistance in many gene-for-gene systems (GOODMAN and NOVACKY 1994 ; GREENBERG 1997 ). Like many other R genes, RPS2 encodes an NBS-LRR protein (BENT et al. 1994 ; MINDRINOS et al. 1994 ; YOUNG 2000 ). The present study was initially designed to identify additional host genes that function with RPS2 in defense activation. Ecotype Col-0 is RPS2/RPS2 and responds to P. syringae expressing avrRpt2 by inducing defense responses and limiting bacterial growth (KUNKEL et al. 1993 ; YU et al. 1993 ). The Arabidopsis ecotype Po-1 was previously identified as susceptible to P. syringae expressing avrRpt2 (WHALEN et al. 1991 ), but the cause of susceptibility was not determined. Here we use genetic and molecular genetic analysis of Col-0 and Po-1 to show the involvement of one or more loci other than RPS2 in controlling the avrRpt2-specific resistance response. Allele-specific interactions were observed. We discovered that the LRR-encoding domain is the RPS2 determinant of allele-specific interactions between RPS2 and one or more of the other loci that participate in RPS2-mediated resistance.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant and bacterial strains: growth, inoculation, and transformation procedures:
P. syringae pv. tomato (Pst) DC3000 and P. syringae pv. glycinea Race 4 (Psg) carrying pVSP61 (empty vector, no avr gene) or pV288 (pVSP61 + avrRpt2) were constructed and used as described (KUNKEL et al. 1993 ). Arabidopsis ecotype Col-0 was originally obtained from S. Somerville (Stanford University, Stanford, CA) and Po-1 was obtained from the former Arabidopsis Information Service seed bank (now available from ABRC, Columbus, OH; http://www.aims.cps.msu.edu/aims/). The Po-1 lines used in this study were derived from a line produced by two generations of single-seed descent. Arabidopsis plants were grown from seed in growth chambers under a 9-hr photoperiod at 22° and were moved after inoculation and scoring to a 24-hr photoperiod for flowering and seed production.

To assay for the HR, bacterial suspensions of 2 x 10⁸ cfu/ml of Psg strains carrying pVSP61 or pV288 were infiltrated into leaf mesophyll tissue by vacuum infiltration, with a disposable plastic Pasteur pipette, or with a 1.0-ml syringe applied to the undersurface of healthy, fully expanded Arabidopsis leaves (KUNKEL et al. 1993 ; YU et al. 1993 ). Leaves were scored for HR symptoms at 24–48 hr after inoculation. To assay for disease, Pst bacterial suspensions of 5 x 10⁵ or 1 x 10⁶ cfu/ml in 10 mM MgCl₂ were inoculated into plant leaves as described for the HR assay above (WHALEN et al. 1991 ). The inoculated leaves were scored for disease symptoms (necrosis and yellowing) 4 days after inoculation. To determine levels of bacterial growth in the leaves of Arabidopsis, leaves of at least six plants per bacterial strain were vacuum infiltrated with bacterial suspensions of 2 x 10⁴ cfu/ml or 5 x 10⁴ cfu/ml. Bacterial growth was monitored by dilution plating of leaf samples at various time points between days 0 and 4 after inoculation as described previously (WHALEN et al. 1991 ).

A modified vacuum infiltration procedure was used for transformation of Arabidopsis with constructs delivered by Agrobacterium tumefaciens strain GV3101(pMP90) (BECHTOLD et al. 1993 ; CLOUGH and BENT 1998 ). Controls for experiments with transgenic plants included Po-1 and Col-0 ecotypes either grown on 0.5x MS/0.8% agarose media without antibiotics and transplanted to soil or transformed with the parent binary cosmid pCLD04541 (BANCROFT et al. 1997 ), selected on antibiotic media, and transplanted to soil.

Genetic linkage analysis:
Genetic mapping with Po-1 x Col-0 F₂ individuals and F₃ families was performed using the indicated cleaved amplified polymorphic sequence (CAPS), simple sequence length polymorphism (SSLP) markers (Research Genetics, Huntsville, AL), and restriction fragment length polymorphism (RFLP) markers (ABRC) that map throughout the Arabidopsis genome (NAM et al. 1989 ; KONIECZNY and AUSUBEL 1993 ; BELL and ECKER 1994 ; RHEE et al. 1998 ; http://www.arabidopsis.org/). For plant genomic DNA, one to two inner rosette leaves from F₂ plants, or 1 g fresh weight of leaves from 30 F₃ plants, were collected after testing plants for the HR phenotype, immediately frozen in liquid N₂, and stored at -70°. Genomic DNA was isolated using a CTAB-based protocol (ROGERS and BENDICH 1988 ). PCR for genetic mapping was essentially as described (KONIECZNY and AUSUBEL 1993 ; BELL and ECKER 1994 ). For the RPS2 CAPS, a portion of RPS2 was amplified using primers 53 (5'-CAG AGC TTT GAG ACA G-3') and 54 (5'-GTA CTC CAA GTC ATG-3'), and an aliquot of the PCR product was digested with restriction enzyme EcoRI and resolved by agarose gel electrophoresis. The 16 individuals mentioned as the "biased mapping set" were selected by screening a total of 785 Po-1 x Col-0 F₂ individuals by hand inoculation with Psg avrRpt2⁺ to test for the HR and by genotyping at RPS2 using the EcoRI-based CAPS marker. Unless otherwise noted, molecular biological methods used in these and other experiments were essentially as described (AUSUBEL et al. 1997 ).

In initial mapping studies, significant associations between marker and defense phenotype were assessed using 57 susceptible F₂ individuals using the chi-square statistic to test for deviation from a 3:1 or 1:2:1 ratio (P < 0.05). Statistically significant associations were observed between the resistance phenotype and the three markers nga8, RPS2, and DHS1A.

More detailed genetic mapping was performed using "set I" (131 F₃ families derived by self-fertilization from randomly chosen Po-1 x Col-0 F₂ individuals from 5 different F₁ plants) separately or with "set II" (16 F₃ lines from the biased mapping set described above and 53 F₃ families derived from other Po-1 x Col-0 F₂ individuals homozygous at RPS2). Phenotypes of the F₃ families were determined using at least two separate pots, each containing 9 and typically 14 or more plants from each F₃ family. Plants were inoculated with Psg avrRpt2⁺ by vacuum infiltration and before viewing of labels the set of F₃ plants in a pot were assigned a single group score for severity of the HR on a scale of 0–4. Each infiltration set included one pot each of Col-0 and Po-1 as controls. The following categories were used: (1) no HR, all leaves on all plants show no HR or at most HR1; (2) rare and/or weak HR, most leaves do not show extensive tissue collapse, a few leaves may show HR3, with most leaves showing HR1–2; (3) intermediate HR, most leaves on all plants show an intermediate HR2 or HR3, with some leaves showing HR4; (4) full HR, all leaves on all plants show extensive tissue collapse (HR4–5); (segregating) majority of plants show HR4–5 but some plants show no HR or intermediate HR (HR0–3). After being placed into these categories without reference to labels, variation of HR within an F₃ family was evaluated by comparing the response of the plants between duplicate pots of the same F₃ family. As a check, independent scoring of selected experiments by other laboratory personnel produced consistent categorization of F₃ families.

F₃ mapping data were analyzed using QGene v3.06 (NELSON 1997 ), with map distances for molecular marker maps obtained from the Lister and Dean RI map (RHEE et al. 1998 ; http://www.arabidopsis.org). Single interval mapping protocols were used and significance of association between marker and phenotype was determined using a cutoff LOD value of 3.0.

DNA sequencing:
The DNA sequence of Po-1 RPS2 was determined for both strands using dideoxy sequencing methods and RPS2 internal primers. One PCR product amplified from genomic Po-1 DNA and cloned into pBluescript II SK(+) was used for initial sequencing. This PCR product was generated using the primers aa#1 (5'-CGGGATCCATGGATTTCATCTCATCTCTT-3') and 46S (5'-ACAGAGTGCTCTTAGC-3'). Any deviations from the known Col-0 RPS2 sequence were then checked using independent Po-1 RPS2 PCR products. Note that no introns are present in Col-0 or Po-1 RPS2. The promoter region of Col-0 RPS2 was cloned from a genomic subclone (BENT et al. 1994 ) as a 1.3-kb SalI-BamHI fragment into pBluescript II SK(+); the promoter region of Po-1 RPS2 was cloned from a PCR product generated using RPS2-P1K-Cla (5'-CGGCATCGATAGACAGGTCCCCCTTTTA-3') and RPS2#60 (5'-CTCCGTTACTTGCAC-3'), and multiple cloned independent PCR products were pooled for sequencing. Sequence comparisons were made using SeqApp v1.9a169 (D. GILBERT, Bloomington, IN; http://www.ftp.bio.indiana.edu).

Construction of RPS2 + 1.0-kb native promoter constructs:
For complementation experiments, Col-0 and Po-1 alleles of RPS2 were cloned together with their native promoter sequences into the binary vector pCLD04541 (BANCROFT et al. 1997 ). PCR products were amplified from genomic DNA using high-fidelity Pfu DNA polymerase (Stratagene, La Jolla, CA) and the primers RPS2-P1k-Cla (see above) and RPS2-Sal46 (5'-GGAATTCGTCGACACAGAGTGCTCTTAGCTC-3'), giving a product spanning from -980 bp upstream from the start of the RPS2 open reading frame to +30 bp downstream from the stop codon. Products from at least two independent PCR reactions were separately cloned into the relevant vectors and tested in plants. Products were restricted with ClaI and SalI and cloned into pBluescript II SK(+) and then into ClaI/XhoI-restricted pCLD04541. Constructs were then transferred into the Agrobacterium strain GV3101 (pMP90) (KONCZ and SCHELL 1986 ) by triparental mating.

Generation of RPS2 promoter-swap and LRR-swap constructs:
The 980-bp segments of the RPS2 promoter immediately upstream of the RPS2 open reading frame (ORF) were amplified by PCR from Po-1 and Col-0 genomic DNA using high-fidelity Pfu DNA polymerase and the primers RPS2-P1k-Sac (5'-GCACGAGCTCAGACAGGTCCCCCTTTTA-3') and RPS2-1Cla-R (5'-AATCCATATCGATGATTTCTCGCTC-3'). RPS2-1Cla-R incorporates a single base change (underlined) 1 bp upstream from the ATG start codon that creates a ClaI restriction site (boldface letters). Products were restricted with SacI and ClaI and cloned into SacI/ClaI-restricted pBluescript II SK(+). The RPS2 open reading frame was similarly amplified and cloned using RPS2-1Cla-F2 (5'-CGGCATCGATATGGATTTCATCTCATCTCTT-3') and RPS2-Sal46 (described above). RPS2-1Cla-F2 also creates a ClaI restriction site (boldface letters) one base upstream of the ATG start codon (underlined). Purified PCR products were digested with ClaI, blunted with mung bean nuclease (New England Biolabs, Beverly, MA), digested with SalI, ligated with the pBluescript II SK(+)/RPS2 promoter constructs (described above) that had been digested with EcoRI, and then blunt ended and digested with SalI. Each type of RPS2 promoter construct was ligated with each of two RPS2 ORF sequences (from the same RPS2 allele) that were products of separate PCR reactions. The resulting RPS2 promoter + ORF constructs were restricted out of pBluescript using SacI and SalI restriction enzymes, ligated into SacI/XhoI-digested pCLD04541, transformed into Escherichia coli, and then transferred into Agrobacterium for plant transformation as described above. Products from at least two independent PCR reactions were used to create separate constructs that were independently tested in plants.

To generate the RPS2 LRR-swap constructs, the pBluescript RPS2 + 1.0-kb native promoter constructs described in the previous paragraph were used. A 1.35-kb HindIII fragment encoding the LRR domain from the Po-1 construct was replaced with the corresponding fragment from the Col-0 construct and vice versa. Products from at least two independent PCR reactions were used to create separate constructs. RPS2 LRR-swap constructs were transferred into pCLD04541 as ClaI/SalI fragments and used as described above.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Response of Po-1 to P. syringae expressing avrRpt2:
To investigate the response of Po-1 to infection by P. syringae expressing avrRpt2, leaves of Po-1 and Col-0 were inoculated by syringe or by vacuum infiltration with the virulent Pst strain DC3000 or with Pst DC3000 expressing avrRpt2 (DC3000avrRpt2⁺). Wild-type Col-0, which is resistant to avrRpt2, developed few or no disease symptoms when inoculated with Pst DC3000avrRpt2⁺ at a titer of 10⁶ colony-forming units (cfu)/ml (Table 1). In confirmation of previous work (WHALEN et al. 1991 ), Po-1 plants developed necrotic lesions and pronounced chlorosis 4 days after inoculation with DC3000avrRpt2⁺, which are similar to the symptoms observed on susceptible Col-0 rps2/rps2 mutants or on wild-type Col-0 inoculated with DC3000 (Table 1).

The lack of a resistance response in Po-1 was quantified by measuring the extent of pathogen growth within the plant. In Po-1 inoculated with either DC3000 or DC3000avrRpt2⁺, bacteria grew to high levels (Fig 1). These levels were similar to those attained by DC3000avrRpt2⁺ in susceptible rps2 mutants of Col-0 or by DC3000 (no avr) in wild-type Col-0. In contrast, growth of DC3000avrRpt2⁺ in wild-type Col-0 plants was restricted, reaching maximum levels of 10⁴–10⁵ cfu/cm² (Fig 1).

View larger version (31K): In this window In a new window Download PPT slide   Figure 1. Growth of virulent and avirulent P. syringae pv. tomato within Arabidopsis leaves. A and B are from the same experiment; C and D are from a separate single experiment. Plants were all inoculated with the indicated bacterial strain. D203, Col-0 rps2-201/rps2-201; F1, F1 progeny from Po-1 x Col-0. Two leaves from each of six plants were sampled for each data point; values shown are mean ± SE.

The hypersensitive response (HR) is a programmed cell death response that develops within hours at and around the site of infection. The ability of Po-1 to develop an HR in response to P. syringae pv. glycinea (Psg) expressing avrRpt2 was tested by syringe or vacuum infiltration with a high titer of bacteria (10⁸ cfu/ml; KLEMENT et al. 1964 ). While Col-0 plants exhibited a strong, visible HR within 24 hr of inoculation, Po-1 plants did not manifest an HR at the macroscopic level in response to Psg avrRpt2⁺ (Table 1). Po-1 plants do have the capacity to induce gene-for-gene defenses and the HR in response to P. syringae pathogens, however, as Po-1 activates these responses when inoculated with P. syringae that express avrRps4 (HINSCH and STASKAWICZ 1996 ; data not shown). Although the HR is not always required for an effective resistance response (YU et al. 1998 ; BENDAHMANE et al. 1999 ), it is closely associated with the disease resistance response mediated by RPS2 and most other R genes (KUNKEL et al. 1993 ; YU et al. 1993 ; GOODMAN and NOVACKY 1994 ; GREENBERG 1997 ). In this study the level of the HR was frequently used as an indicator of the avrRpt2-RPS2 resistance response of the plant.

To summarize, Col-0 plants inoculated with P. syringae expressing avrRpt2 developed an HR, restricted pathogen growth, and did not develop disease. In response to the same bacteria Po-1 plants did not manifest an HR, limited pathogen growth poorly, and developed disease. The simplest explanation for Po-1 susceptibility to P. syringae that express avrRpt2 would be that Po-1 carries a nonfunctional allele of RPS2.

Multigenic control of RPS2-mediated defense:
The genetic basis of susceptibility in Po-1 was investigated by crossing ecotypes Po-1 and Col-0. Po-1 x Col-0 F₁ individuals and those from reciprocal crosses exhibited a strong disease-resistant phenotype and a full HR in response to avrRpt2 infection, indicating dominance of the Col-0 genotype in determining resistance (Fig 1B; Table 2). However, in the F₂ of reciprocal crosses, intermediate phenotypes were consistently observed in addition to the two parental phenotypes. These were grouped into intermediate-resistant (moderate HR) and intermediate-susceptible (rare and/or weak HR) classes (Fig 2). The presence of the intermediate phenotypes was also observed using disease assays rather than HR assays (Fig 2B), was confirmed in separate HR and disease assays with other F₂ populations (data not shown), and was confirmed with F₃ families derived from individual F₂ plants (Fig 2C). If all but the most disease-susceptible or HR^- class of F₂ individuals were grouped together as "resistant," F₂ segregation ratios were in some cases consistent with a 3:1 ratio. However, grouping individuals with such different phenotypes into a single class seemed inappropriate, especially given the much clearer bimodal phenotypic groupings obtained in other studies with the same pathosystem but with different parents (e.g., KUNKEL et al. 1993 ). F₂ and F₃ data also did not fit a 1:2:1 ratio for segregation of a single gene with incomplete dominance.

View larger version (26K): In this window In a new window Download PPT slide   Figure 2. Phenotypic segregation in F2 and F3 progeny of Po-1 crossed to various genotypes, as indicated. A and B represent different F2 populations, with A subjected to the HR assay and B subjected to the disease assay (see MATERIALS AND METHODS). C–E also report results of HR assays; C reports data for F3 families rather than for F2 individuals.

To test whether susceptibility in Po-1 segregated as a multigenic trait in combination with genetic backgrounds other than Col-0, Po-1 was crossed to the ecotype No-0, which like Col-0 is resistant to P. syringae that express avrRpt2. The Po-1 x No-0 F₁ were resistant (Table 2), indicating dominance, but as was the case in the Po-1 x Col-0 populations, F₂ phenotype distribution revealed intermediate phenotypes in addition to the parental phenotypes (Fig 2D) and F₂ segregation patterns did not fit single-gene models. These findings again suggested the involvement of multiple genes in specifying avrRpt2-specific resistance.

While the avrRpt2-specific resistance response data were not consistent with the segregation of a single dominant R gene or with standard ratios for digenic inheritance, such as 9:7 or 9:3:4, the data also did not resemble the bell-shaped curves that are often observed in F₂ populations segregating for a quantitative trait controlled by a large number of genes displaying small additive effects (FALCONER and MACKAY 1996 ). Instead, the bimodal distribution of Po-1 x Col-0 and Po-1 x No-0 F₂ and F₃ phenotypes indicated that resistance segregates as a multigenic trait controlled by a small number of major-effect genes or by a single dominant gene and a small number of "modifier" genes.

Genetic evidence for Po-1 RPS2 functionality:
To determine whether the RPS2 allele of Po-1 is compromised for response to avrRpt2, the RPS2 genotype was determined for F₂ lines that were also scored for resistance phenotype (Table 3). A single-base pair EcoRI CAPS within RPS2 was identified that differentiates the RPS2 alleles of Col-0 and Po-1. F₂ individuals were identified that are homozygous for the Po-1 RPS2 allele, yet they showed a partially or fully disease-resistant phenotype (Table 3). These F₂ individuals suggested that, despite the lack of avrRpt2-specific resistance in wild-type Po-1, the Po-1 RPS2 allele can function in a partial Col-0 background. Another class of F₂ individuals was homozygous for the Col-0 RPS2 allele but showed little or no disease resistance (Table 3). These individuals indicated that other Po-1 loci can cause functional RPS2 alleles to be ineffective for resistance signaling in response to avrRpt2. Results consistent with these F₂ data were obtained in repeat assays with F₃ families derived from the key F₂ lines and in 16 additional F₂ individuals identified among 785 Po-1 x Col-0 F₂ (see MATERIALS AND METHODS). To reiterate, these classes of F₂ RPS2 homozygotes indicated that the Po-1 allele of RPS2 can be functional and/or that the progeny of Po-1 x Col-0 crosses segregate for genes other than RPS2 that control disease resistance.

In Po-1 x Col-0 F₂ populations, the defense phenotype did not segregate independently of the RPS2 genotype (Table 3). F₂ plants homozygous for the Col-0 RPS2 allele were most frequently resistant and F₂ plants homozygous for the Po-1 RPS2 allele were most frequently susceptible. Because resistance/susceptibility did not segregate entirely independently of the RPS2 genotype, we hypothesized that one or more of the other resistance-modifying genes is linked to RPS2. A separate but not mutually exclusive hypothesis was that RPS2 is one of the genes contributing to the avrRpt2-specific resistance response, with allele-specific interactions causing the presence or absence of resistance.

Mapping of RPS2-pathway loci:
An approximate map position for one or more other RPS2-pathway loci that alter the defense response against P. syringae that express avrRpt2 was determined using a population of 131 random F₂-derived F₃ families from crosses between Po-1 and Col-0. A second population of 69 F₃ families contained a small biased population of 16 lines in which the resistance phenotype was the opposite of that predicted by the RPS2 genotype (e.g., underlined classes in Table 3), as well as 53 other F₃ families not from set I and chosen due to homozygosity at RPS2. Plants were inoculated with Psg avrRpt2⁺ by vacuum infiltration and scored for the HR. Previously mapped CAPS or RFLP markers were used to determine genotype across the Arabidopsis genome with genetic intervals of 50 cM or less.

Analysis of the initial marker data set revealed linkage of the avrRpt2-specific response to at least two regions on chromosome 4, near markers nga8, RPS2, and DHS1A, and detected no linkage to chromosomes 1–3 or 5 (data not shown). The additional F₃ lines and additional chromosome 4 markers were subsequently used for higher resolution mapping. Quantitative trait statistical analysis of the marker data, using single-interval mapping methods, localized genetic determination of the avrRpt2-specific response to two discrete genetic intervals (Fig 3). The strongest effect was at the RPS2 locus. A second locus that contributed to the avrRpt2-specific response was linked to marker DHS1, roughly 33 cM away from RPS2. The possibility that additional loci linked to RPS2 on chromosome 4 also contribute to this phenotype cannot be excluded. No linkage association was detected between the defense trait and any markers on chromosomes 1–3 or 5 (Fig 3).

View larger version (24K): In this window In a new window Download PPT slide   Figure 3. Significance of association between genetic intervals and phenotype (response to P. syringae that express avrRpt2). Output from single-interval mapping performed using the QGene computer program is shown with HR scores and genotypic data for 131 randomly chosen Po-1 x Col-0 F3 families as input (see MATERIALS AND METHODS). Trace shows LOD score; maximum LOD score for a given chromosome is noted on the x-axis. Centimorgan scale (y-axis) shows relative map position along chromosome of molecular markers. Patterned bar represents significance scores as P values. Note that no significant associations were observed for markers on chromosomes 1–3 or 5 (data not shown).

Allele-specific functionality of RPS2:
The discovery of Po-1 x Col-0 F₂ individuals that are homozygous for the Po-1 allele of the RPS2 allele but that show a resistant phenotype suggested that the Po-1 RPS2 allele can be functional when it is in a partially Col-0 background. Functionality of Po-1 RPS2 was investigated further by testing for the resistance response of plants carrying the Po-1 RPS2 allele in a Col-0 rps2/rps2 background. In a genetic approach, Po-1 was reciprocally crossed with Col-0 mutants rps2-201/rps2-201 (D203) and rps2-101C/rps2-101C (101C). The rps2-201 allele carries a point mutation that causes a single-amino-acid change in the LRR and creates a nonfunctional RPS2 protein, while the rps2-101C allele contains a frame-shift mutation that causes a premature stop codon at the front of RPS2 (BENT et al. 1994 ; MINDRINOS et al. 1994 ). In the progeny of D203 or 101C crosses to Po-1, all F₁ were HR^- (Table 2). However, 70% of the F₂ showed an intermediate or strong HR (Fig 2E; data not shown). These results again suggested (see also Table 3) that the Po-1 allele of RPS2 is functional when moved into a partially Col-0 genetic background but cannot signal for resistance in conjunction with Po-1 alleles of these resistance-modifying loci.

An alternative hypothesis to explain these results was that Po-1 genes other than RPS2 are capable of mediating the HR in conjunction with Col-0 genes other than RPS2. To test this hypothesis, we investigated whether any HR⁺ individuals were homozygous for the nonfunctional Col-0 rps2-201 or rps2-101C mutant alleles of RPS2. The RPS2 genotype was determined for all F₂ progeny that showed an intermediate or strong HR, and all 56 HR⁺ F₂ individuals carried at least one copy of the Po-1 RPS2 allele (data not shown). This suggested that Po-1 RPS2 is the cause of avrRpt2-specific resistance signaling in these lines. However, because of possible contributions from loci tightly linked to RPS2, this result still did not conclusively rule out the possibility that resistance is mediated by interaction among genes other than RPS2.

Functionality of Po-1 RPS2 was investigated more precisely by molecular complementation. The Po-1 RPS2 allele under 1.0 kb of native Po-1 RPS2 promoter was cloned into a binary cosmid and transferred by Agrobacterium-mediated transformation into the Col-0 rps2/rps2 mutants D203 and 101C. Transformants were found to produce a resistance response upon challenge with Psg avrRpt2, indicating that the Po-1 RPS2 allele can be functional in a Col-0 genetic background (Fig 4A). It was noted, however, that the HR in these lines was intermediate in intensity.

View larger version (29K): In this window In a new window Download PPT slide   Figure 4. Molecular complementation experiments using cloned RPS2 constructs. Values shown are mean ± SE for severity of HR in multiple T1 transformants tested for their response to Psg R4 avrRpt2+. Plants were transformed with the following: (A) an intact RPS2 gene driven by 1.0 kb of native RPS2 promoter from the genotype indicated; (B) an intact RPS2 open reading frame driven by 1.0 kb of native promoter or by heterologous RPS2 promoter from a different genotype, as indicated; (C) RPS2 LRR-swap constructs fusing promoter and amino-terminus-encoding region from one RPS2 allele with the LRR-encoding region from a heterologous RPS2 allele, as indicated. Ø, plants transformed with vector (no RPS2 insert), or, in some cases, nontransformed plants carried through growth and transplanting in parallel with transformants but on nonselective media.

In a reciprocal experiment, the Col-0 RPS2 allele under 1.6 or 1.0 kb of native promoter was transformed into Po-1 plants. The Col-0 RPS2 allele complemented Po-1 to resistance in response to Psg avrRpt2⁺ (Fig 4A). This complementation result was significant, as it indicated that the absence of avrRpt2-specific resistance in Po-1 is due not only to defects at other loci, but also to the Po-1 allele of RPS2.

To summarize the above genetic and molecular genetic complementation experiments, allele-specific interactions were observed between RPS2 and one or more other loci. Col-0 RPS2 could function with the Po-1 allele of one or more genes other than RPS2 that control avrRpt2-specific disease resistance, while RPS2 from Po-1 did not function with the Po-1 alleles of these other genes. Po-1 RPS2 did function with the Col-0 alleles of these other genes, as did Col-0 RPS2. The Po-1 alleles of RPS2 and this other gene or genes are each capable of disease resistance function, but they cannot function with each other.

Sequence of Po-1 RPS2 allele:
To investigate possible structural differences between the Po-1 and Col-0 RPS2 alleles that might account for their differences in resistance signaling, the Po-1 allele of RPS2 was cloned and sequenced (GenBank accession no. AF368301). The derived amino acid sequence revealed a substantial number of differences—11 amino acid changes—between the Po-1 and Col-0 RPS2 alleles (Fig 5). Many of the nonconservative amino acid changes are located in the leucine-rich repeat (LRR) region, but residue changes are scattered over much of the RPS2 ORF. The derived amino acid sequence did not reveal obvious structural features that might suggest that the Po-1 allele of RPS2 is nonfunctional.

View larger version (59K): In this window In a new window Download PPT slide   Figure 5. Derived amino acid sequence encoded by RPS2 of Po-1. Differences with Col-0 RPS2 are in boldface type and the Col-0 amino acid is shown directly above. Lines indicate the approximate extent of putative functional domains; broken lines for the leucine-rich repeat reflect the imperfect nature of the LRR in the RPS2 gene product. ***, HindIII site that formed junction for LRR-swap alleles (see Fig 4).

No transcriptional differences between Col-0 and Po-1 RPS2 alleles:
Previous Northern analysis of RPS2 mRNA from noninoculated Po-1 and Col-0 leaf tissue did not show a discernible difference in expression between the Po-1 and Col-0 RPS2 transcripts (BENT et al. 1994 ), suggesting that differences in the level of transcription do not account for the difference in resistance signaling activity between Po-1 and Col-0 RPS2 alleles.

To further investigate whether transcriptional differences between Po-1 and Col-0 RPS2 transcripts are responsible for the difference in defense signaling activity of the two alleles, the RPS2 promoter sequences were investigated. Approximately 1.0 kb of genomic DNA immediately upstream of the Col-0 and Po-1 RPS2 open reading frames was cloned, sequenced, and compared. Across this 986-bp sequence, the Po-1 RPS2 promoter differed from the Col-0 RPS2 promoter at only 7 bp positions, none obviously disrupting a promoter motif (see GenBank accession nos. AL049483 and AF368301).

To directly test for differences in the Po-1 and Col-0 RPS2 promoters that might effect disease resistance, a "promoter-swap" molecular complementation strategy was pursued. PCR primers at -1 and -986 relative to the ATG start of RPS2 were used to amplify and clone the native RPS2 promoters of the Po-1 and Col-0 alleles. Heterologous combinations of promoter and RPS2 alleles in the binary vector pCLD04541 were used to transform Po-1 and rps2/rps2 mutants of Col-0. The ability of the chimeric transgenes to signal for resistance in response to Psg avrRpt2⁺ was assayed by inoculating leaves of T₁ transformant plants and monitoring the HR. In both Col-0 and Po-1 genetic backgrounds, the resistance response to avrRpt2 by the Col-0 RPS2 transgene driven by the Po-1 RPS2 promoter was indistinguishable from the resistance response of the Col-0 RPS2 transgene under its native promoter (Fig 4B). The Po-1 RPS2 transgene driven by the Col-0 RPS2 promoter behaved like the Po-1 RPS2 transgene under its own promoter in the Col-0 or Po-1 backgrounds (Fig 4B). These results provided functional evidence that Po-1 and Col-0 RPS2 promoters do not differ in any appreciable manner that might account for differences in the phenotypic expression of RPS2-mediated defense responses.

Differences responsible for allele-specific interaction are in the LRR domain:
R gene products contain identifiable motifs such as a coiled-coil domain, NBS, and LRR (HAMMOND-KOSACK and JONES 1997 ; YOUNG 2000 ). We pursued further domain-swap experiments to determine if functional differences betweeen the Po-1 and Col-0 alleles of RPS2 could be assigned to amino acid differences in a given domain.

The Po-1 and Col-0 alleles of RPS2 under the control of 1.0 kb of native promoter in a binary vector were used as the parent constructs. From the parent constructs, the 3' 1.35-kb fragment of Po-1 RPS2 encoding the LRR was cloned out and replaced with the 3' 1.35-kb fragment of Col-0 RPS2 and vice versa. The chimeric LRR-swap constructs were transformed into Po-1 and into the Col-0 rps2/rps2 mutants D203 and 101C by Agrobacterium-mediated transformation, and transformants were tested for their HR in response to Psg avrRpt2. We found that the Po-1 amino terminus + Col-0 LRR constructs could mediate an intermediate level of HR in Po-1 and in Col-0 rps2/rps2 genetic backgrounds, indicating that the amino terminus of Po-1 RPS2 can function even in a Po-1 genetic background (Fig 4C). The Col-0 amino terminus + Po-1 LRR constructs, on the other hand, mimicked the results obtained with intact Po-1 RPS2: an intermediate HR was observed in Col-0 rps2/rps2 genetic backgrounds, and no HR was conferred in a Po-1 genetic background (Fig 4C). The Col-0 RPS2 LRR domain corrected the nonfunctional Po-1 RPS2 LRR domain for resistance in a Po-1 genetic background. This indicated that the LRR domain is the key structural determinant for allele-specific interactions between RPS2 and other host loci that modify the avrRpt2/RPS2 pathway in this Col-0/Po-1 system.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study explored the interaction of RPS2 with other hypothesized host factors required for the perception of P. syringae pathogens that express avrRpt2 and/or for the subsequent induction of plant defense responses. Progeny of crosses between a resistant and a susceptible ecotype of Arabidopsis revealed segregation of more than one gene controlling this defense response. Polymorphism between the Po-1 and Col-0 alleles of RPS2 was a major factor determining the strength of the avrRpt2-specific resistance response, but it was not the only factor. At least one additional genetic interval that contributes to this phenotype was identified and mapped. We discovered that Po-1 RPS2 can function in a Col-0 genetic background, but not in Po-1. In RPS2, the LRR domain was responsible for ineffective interaction between Po-1 RPS2 and one or more of the other Po-1 loci.

Roles of the LRR:
LRR domains are found in a wide array of proteins from all taxa and are present in almost all structural classes of plant R genes that mediate gene-for-gene disease resistance (KOBE and DEISENHOFER 1994 ; HAMMOND-KOSACK and JONES 1997 ; MARINO et al. 2000 ). LRRs are involved in the perception of protein or peptide ligands in a number of systems, including interactions between the Drosophila Toll receptor and the dorsal/ventral patterning factor Spatzle; human follicle stimulating hormone and its receptor; and among plant development proteins such as CLAVATA1, 2, and 3 (KOBE and DEISENHOFER 1994 ; FLETCHER et al. 1999 ; MARINO et al. 2000 ). However, LRRs have also been shown to mediate intracellular interactions among proteins not thought of as "receptors" and "ligands," such as yeast adenylate cyclase and Ras (e.g., SUZUKI et al. 1990 ).

In plant R gene products, studies suggest that the LRR domains are major determinants of recognitional specificity for Avr factors (ELLIS et al. 2000 ). Evolution of new pathogen specificity has been traced to shifts in solvent-exposed LRR residues that are caused by single-base changes, insertion or deletion events, and by equal- or unequal-exchange meiotic recombination events within R genes or between closely linked homologous R genes in a cluster (ELLIS et al. 2000 ).

Roles other than pathogen recognition have also been hypothesized for the LRR of R gene products, but these have been less clearly demonstrated. In this study we obtained evidence that the LRR region can influence effective interaction with host factors. Consistent with our results, a study with the Arabidopsis R gene RPS5 also suggested a role for the LRR domain in interaction with other host factors (WARREN et al. 1998 ). A nonfunctional RPS5 allele containing a mutation in the third repeat of the LRR blocked the resistance conferred by other R genes, and overexpression of wild-type RPS5 did not suppress the dominant-negative phenotype of the mutant allele (WARREN et al. 1998 ). This RPS5 mutation of the third LRR might have caused increased binding to a pathway component(s) shared by multiple R genes and thereby interfered with essential downstream signaling. In our study, the difference in interaction between Col-0 and Po-1 RPS2 and other host loci was attributed to six amino acid differences between the RPS2 LRR domains. In the future, it will be interesting to see whether amino acid polymorphisms within the LRR of RPS2 alleles from other ecotypes correlate with the level of the resistance response.

The RPS2 and RPS5 examples fit into a generalized model proposed by GRANT and MANSFIELD 1999 to account for the involvement of additional loci in R-Avr interactions. In their model, the LRR protein only indirectly matches the Avr protein and is involved in interpreting signals generated from other cellular proteins, designated signaling linker proteins (SLIKs), which directly interface with the Avr peptide. The presence of the elicitor or Avr factor, or its activity, may alter the normal configuration of the SLIK or SLIK complex, leading to functional interaction with the R gene product and subsequent resistance pathway activation (GRANT and MANSFIELD 1999 ). The interactions that we observed involving avrRpt2, RPS2, and other host factors may, upon further investigation, form one example of this type of SLIK interaction.

RPS2-interacting loci:
As an initial step toward isolation of the RPS2-interacting host factors predicted by our genetic studies, quantitative trait methods were used to map genetic intervals associated with the avrRpt2-specific response. The bimodal distribution of resistance phenotypes among Po-1 x Col-0 and Po-1 x No-0 F₂ (Fig 2) classically would indicate that the phenotype, in this case resistance in response to avrRpt2, is controlled by a small number of major-effect genes or a single dominant gene and a small number of "modifier" genes. The observed bias toward defense phenotypes that correlated with the RPS2 genotype (HR⁺ if homozygous for Col-0 RPS2, HR^- if homozygous for Po-1 RPS2; see Table 3) had suggested that RPS2 would have a significant phenotypic effect and/or that other relevant loci would be linked to RPS2. Mapping supported both hypotheses. The defense phenotype associated most strongly with the RPS2 locus, which was also shown by other means to have a major effect on resistance phenotypes (Fig 3). The other genetic interval associated with the response to P. syringae that express avrRpt2 also mapped to chromosome 4, 33 cM away from RPS2. As mentioned previously, the possibility that additional loci linked to RPS2 on chromosome 4 also contribute to this phenotype could not be excluded.

Reports of multigenic control of resistance are gaining relevance in research on the molecular basis of defense signal transduction as resources improve for the mapping and cloning genes known only by phenotype. A number of other Arabidopsis genes have been identified for which mutant alleles disrupt defense pathways (GLAZEBROOK 1999 ). None of the well-studied genes (such as NDR1, EDS1, PAD4, DND1, LSD1, and PBS2) map to the intervals on chromosome 4 identified in this study. Further experimental effort will be required to isolate and characterize the RPS2-interacting host factor(s) described in this study.

Direct protein associations among host factors known to be required for the R-avr signaling complex have yet to be demonstrated. In the closest example to date, Pto kinase has been shown to directly phosphorylate Pti1 (ZHOU et al. 1995 ). In a more immediate example, LEISTER and KATAGIRI 2000 used AvrRpt2 to coprecipitate RPS2 and another unidentified protein in antibody pull-down experiments. Interestingly, RPS2 could also be precipitated by AvrB despite the fact that RPS2 does not confer resistance to P. syringae that express avrB (LEISTER and KATAGIRI 2000 ). This result is consistent with genetic evidence for interference between RPS2 and RPM1 resistance signaling pathways when pathogens that express avrB or avrRpm1 and avrRpt2 are co-inoculated (REUBER and AUSUBEL 1996 ; RITTER and DANGL 1996 ).

Although the interacting loci found in this study are characterized as defense pathway loci, it is also possible that these loci are active in disease susceptibility. avrRpt2 has been shown to promote virulence in the absence of RPS2 (CHEN et al. 2000 ), and one or more of the loci identified in this study may encode a protein that is a target for the virulence activity of AvrRpt2.

Allele-specific interactions:
The strong resistance response of ecotype Col-0 to P. syringae that express avrRpt2 is known to be dependent on RPS2 (KUNKEL et al. 1993 ; YU et al. 1993 ). The lack of an effective response in Po-1 initially suggested that Po-1 does not carry a functional RPS2 allele. We discovered that Po-1 carries an allele of RPS2 that confers avrRpt2-specific resistance in other genetic backgrounds, implying that defects in other Po-1 loci cause loss of RPS2 function. Intriguingly, the Col-0 RPS2 allele under native RPS2 promoter complemented Po-1 for resistance when introduced by transformation, suggesting that Po-1 RPS2 is also partly responsible for the nonfunctional resistance in Po-1. As noted above, we found that the LRR is the domain responsible for the RPS2 component of these allele-specific interactions.

Allele-specific interactions were not confined to the Po-1 allele of RPS2. The discovery of Po-1 x Col-0 F₂ individuals and F₃ families that were homozygous for Col-0 RPS2 but disease susceptible indicated that, in certain mixed Po-1/Col-0 genetic backgrounds, allele-specific interactions among resistance-modulating loci could also prevent resistance signaling through the otherwise functional Col-0 RPS2. The fully resistant phenotype of the Po-1 x Col-0 F₁ indicated that the nonproductive interaction between alleles that prevent Col-0 RPS2 function is recessive.

In contrast to the above, nonproductive interactions were dominant when we monitored interaction between Po-1 RPS2 and the RPS2-interacting loci. The F₁ of Po-1 x Col-0 rps2/rps2 mutants were HR^- (Table 2). Po-1 RPS2 could function in concert with the Col-0 alleles at these other loci (Fig 4A), but could not function in the heterozygous background of these F₁.

As a separate matter, we were intrigued that complementation experiments involving all or part of Po-1 RPS2 often produced a weak or intermediate HR (Fig 3). Our interpretation of this result is that Po-1 RPS2 (including the Po-1 amino terminus/Col-0 LRR fusion), even when functional, cannot interact with other host factors as effectively as Col-0 RPS2. It may also be the case that Po-1 RPS2 does not recognize the avrRpt2 ligand as effectively. Although some quantitative reduction in responsiveness to the avrRpt2 ligand cannot be excluded, the constructs containing domains from Po-1 RPS2 could clearly mediate responses to P. syringae that express avrRpt2. In contrast, the host genotype at loci other than RPS2 had a pronounced effect, correlating with the presence or near-complete absence of a response to pathogen (Fig 4).

A separate example of allele-specific interactions that affect expression of resistance was recently provided by the demonstration of monogenic and novel digenic resistance mediated by three RXC loci in the Arabidopsis ecotypes Col-0 and Landsberg erecta (Ler) in response to the bacterial pathogen Xanthomonas campestris (BUELL and SOMERVILLE 1997 ). In the RXC defense system, monogenic resistance is determined by the presence of the Col-0 allele of RXC2 while in its absence, digenic resistance is specified by the presence of the Col-0 allele of RXC4 in conjunction with the Ler allele of RXC3. Numerous combinations of the six RXC alleles were shown to confer intermediate levels of resistance (BUELL and SOMERVILLE 1997 ). The lack of resistance in Po-1 carrying Po-1 RPS2, and in some mixed Col-0/Po-1 backgrounds carrying Col-0 RPS2, may or may not have a similar molecular basis as the allele-specific interactions observed for RXC loci.

In studies on the lesion mimic Arabidopsis mutant cep, mapping crosses between genetically heterogeneous ecotypes showed that expression of the mutant phenotype was conditioned not only by the cep locus but also by two other loci that were designated CPR20 and CPR21 (SILVA et al. 1999 ). CPR20 mapped to the lower arm of chromosome 4 and was required for the cep phenotype, while CPR21 of chromosome 1 was often but not always required for the cep phenotype (SILVA et al. 1999 ). The genetic interval encompassing CPR20 does not overlap with the genetic intervals on chromosome 4 that were found to contribute to the avrRpt2-resistance phenotype.

Sequence differences among R gene alleles have been shown to cause quantitative variation in the defense response in many systems (reviewed in ELLIS et al. 2000 ). The general finding of quantitative variation in defense responses has been observed in many additional disease resistance systems (MICHELMORE 1995 ; CRUTE and PINK 1996 ). Our study highlights the fact that this variation can be due as much to altered interaction among host factors as to altered interaction between R gene product and pathogen-derived elicitors. Responsiveness to P. syringae that express avrRpt2 could be observed with all natural and synthetic alleles of RPS2 that were studied. Allele-specific interaction between other host factors and the LRR domain of RPS2 played the primary role in determining whether or not gene-for-gene defense responses were triggered.

In the future, it should be particularly informative to isolate and characterize the RPS2/avrRpt2-pathway gene(s) implicated by this study, and to determine the precise structural determinants that control effective interaction between the RPS2 protein and its interacting host factors.

	ACKNOWLEDGMENTS

We thank Josef Herzog, Roger Innes, Brian Staskawicz, and Gracia Zabala for assistance with experiments; Torbert Rocheford, Bernie Kaufman, and Aldi Kraja for assistance with early stages of the quantitative trait analysis; and Christine Pfund for comments on the manuscript. This work was funded by the National Institutes of Health (GM53595) and the U.S. Department of Agriculture (NRICGP 9500945).

Manuscript received December 5, 2000; Accepted for publication February 14, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AUSUBEL, F. M., R. BRENT, R. E. KINGSTON, D. D. MOORE, J. G. SEIDMAN et al., 1997 Current Protocols in Molecular Biology. John Wiley & Sons, New York.

BANCROFT, I., K. LOVE, E. BENT, S. SHERSON, and C. LISTER et al., 1997  A strategy involving the use of high redundancy YAC subclone libraries facilitates the contiguous representation in cosmid and BAC clones of 1.7 Mb of the genome of the plant Arabidopsis thaliana.. Weeds World 4:1-9.

BECHTOLD, N., J. ELLIS, and G. PELLETIER, 1993  In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris Life Sci. 316:1194-1199.

BELL, C. J. and J. R. ECKER, 1994  Assignment of 30 microsatellite loci to the linkage map of Arabidopsis.. Genomics 19:137-144[Medline].

BENDAHMANE, A., K. KANYUKA, and D. C. BAULCOMBE, 1999  The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 11:781-791[Abstract/Free Full Text].

BENT, A. F., B. N. KUNKEL, D. DAHLBECK, K. L. BROWN, and R. L. SCHMIDT et al., 1994  RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes. Science 265:1856-1860[Abstract/Free Full Text].

BITTNER-EDDY, P. D., I. R. CRUTE, E. B. HOLUB, and J. L. BEYNON, 2000  RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica.. Plant J. 21:177-188[Medline].

BRAUN, T., P. R. SCHOFIELD, and R. SPRENGEL, 1991  Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity. EMBO J. 10:1885-1890[Medline].

BUELL, C. R. and S. C. SOMERVILLE, 1997  Use of Arabidopsis recombinant inbred lines reveals a monogenic and a novel digenic resistance mechanism to Xanthomonas campestris pv. campestris. Plant J. 12:21-29[Medline].

CHEN, Z., A. P. KLOEK, J. BOCH, F. KATAGIRI, and B. N. KUNKEL, 2000  The Pseodomonas syringae avrRpt2 gene product promotes pathogen virulence from inside plant cells. Mol. Plant Microbe Interact. 13:1312-1321[Medline].

CLOUGH, S. J. and A. F. BENT, 1998  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.. Plant J. 16:735-743[Medline].

CRUTE, I. R. and D. A. C. PINK, 1996  The genetics and utilization of pathogen resistance in plants. Plant Cell 8:1747-1755[Medline].

ELLIS, J. G., G. J. LAWRENCE, J. E. LUCK, and P. N. DODDS, 1999  Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity. Plant Cell 11:495-506[Abstract/Free Full Text].

ELLIS, J., P. DODDS, and T. PRYOR, 2000  Structure, function and evolution of plant disease resistance genes. Curr. Opin. Plant Biol. 3:278-284[Medline].

FALCONER, D. S., and T. F. C. MACKAY, 1996 Introduction to Quantitative Genetics. Longman, Essex, England.

FLETCHER, J. C., U. BRAND, M. P. RUNNING, R. SIMON, and E. M. MEYEROWITZ, 1999  Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems (see comments). Science 283:1911-1914[Abstract/Free Full Text].

GLAZEBROOK, J., 1999  Genes controlling expression of defense responses in Arabidopsis.. Curr. Opin. Plant Biol. 2:280-286[Medline].

GOODMAN, R. N., and A. J. NOVACKY, 1994 The Hypersensitive Reaction in Plants to Pathogens: A Resistance Phenomenon. APS Press, St. Paul.

GRANT, M. and J. MANSFIELD, 1999  Early events in host-pathogen interactions. Curr. Opin. Plant Biol. 2:312-319[Medline].

GREENBERG, J. T., 1997  Programmed cell death in plant-pathogen interactions. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:525-545.

HAMMOND-KOSACK, K. E. and J. D. G. JONES, 1997  Plant disease resistance genes. Annu. Rev. Plant Mol. Biol. 48:575-607.

HAMMOND-KOSACK, K. E., D. A. JONES, and J. D. G. JONES, 1994  Identification of two genes required in tomato for full Cf-9 dependent resistance to Cladosporium fulvum.. Plant Cell 6:361-374[Abstract].

HERBERS, K., J. CONRADS-STRAUCH, and U. BONAS, 1992  Race-specificity of plant resistance to bacterial spot disease is determined by repetitive motifs in a bacterial avirulence protein. Nature 356:172-174.

HINSCH, M. and B. STASKAWICZ, 1996  Identification of a new Arabidopsis disease resistance locus, RPS4, and cloning of the corresponding avirulence gene, avrRps4, from Pseudomonas syringae pv. pisi. Mol. Plant Microbe Interact. 9:55-61[Medline].

INNES, R. W., 1998  Genetic dissection of R gene signal transduction pathways. Curr. Opin. Plant Biol. 1:299-304[Medline].

JIA, Y., S. A. MCADAMS, G. T. BRYAN, H. P. HERSHEY, and B. VALENT, 2000  Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. EMBO J. 19:4004-4014[Medline].

KLEMENT, Z., G. I. FARKAS, and L. LOVREKOVICH, 1964  Hypersensitive reaction induced by phytopathogenic bacteria in the tobacco leaf. Phytopathology 54:474-477.

KOBE, B. and J. DEISENHOFER, 1994  The leucine-rich repeat: a versatile binding motif. Trends Biochem. Sci. 19:415-421[Medline].

KONCZ, C. and J. SCHELL, 1986  The promoter of the T_L-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.

KONIECZNY, A. and F. M. AUSUBEL, 1993  A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J. 4:403-410[Medline].

KOOMAN-GERSMANN, M., G. HONEE, G. BONNEMA, and P. J. G. M. DE WIT, 1996  A high-affinity binding site for the AVR9 peptide elicitor of Cladosporium fulvum is present on plasma membranes of tomato and other solanaceous plants. Plant Cell 8:929-938[Abstract].

KUNKEL, B. N., A. F. BENT, D. DAHLBECK, R. W. INNES, and B. J. STASKAWICZ, 1993  RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2.. Plant Cell 5:865-875[Abstract/Free Full Text].

LEISTER, R. T. and F. KATAGIRI, 2000  A resistance gene product of the nucleotide binding site—leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo.. Plant J. 22:345-354[Medline].

LUCK, J. E., G. J. LAWRENCE, P. N. DODDS, K. W. SHEPHERD, and J. G. ELLIS, 2000  Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination. Plant Cell 12:1367-1378[Abstract/Free Full Text].

MARINO, M., L. BRAUN, P. COSSART, and P. GHOSH, 2000  A framework for interpreting the leucine-rich repeats of the Listeria internalins. Proc. Natl. Acad. Sci. USA 97:8784-8788[Abstract/Free Full Text].

MARTIN, G. B., S. H. BROMMONSCHENKEL, J. CHUNWONGSE, A. FRARY, and M. W. GANAL et al., 1993  Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262:1432-1436[Abstract/Free Full Text].

MCDOWELL, J. M., M. DHANDAYDHAM, T. A. LONG, M. G. AARTS, and S. GOFF et al., 1998  Intragenic recombination and diversifying selection contribute to the evolution of downy mildew resistance at the RPP8 locus of Arabidopsis. Plant Cell 10:1861-1874[Abstract/Free Full Text].

MEYERS, B. C., K. A. SHEN, P. ROHANI, B. S. GAUT, and R. W. MICHELMORE, 1998  Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection. Plant Cell 10:1833-1846[Abstract/Free Full Text].

MICHELMORE, R., 1995  Molecular approaches to manipulation of disease resistance genes. Annu. Rev. Phytopathol. 33:393-427.

MINDRINOS, M., F. KATAGIRI, G.-L. YU, and F. M. AUSUBEL, 1994  The A. thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats. Cell 78:1089-1099[Medline].

NAM, H.-G., J. GIRAUDAT, B. DEN BOER, F. MOONAN, and W. D. B. LOOS et al., 1989  Restriction fragment length polymorphism linkage map of Arabidopsis thaliana.. Plant Cell 1:699-705[Abstract/Free Full Text].

NELSON, J. C., 1997  QGene: software for marker-based genomic analysis and breeding. Mol. Breeding 3:239-245.

NOEL, L., T. L. MOORES, E. A. VAN DER BIEZEN, M. PARNISKE, and M. J. DANIELS et al., 1999  Pronounced intraspecific haplotype divergence at the RPP5 complex disease resistance locus of Arabidopsis.. Plant Cell 11:2099-2112[Abstract/Free Full Text].

PARNISKE, M., K. E. HAMMOND-KOSACK, C. GOLSTEIN, C. M. THOMAS, and D. A. JONES et al., 1997  Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato. Cell 91:821-832[Medline].

REUBER, L. and F. M. AUSUBEL, 1996  Isolation of Arabidopsis genes that differentiate between resistance responses mediated by the RPS2 and RPM1 disease resistance genes. Plant Cell 8:241-249[Abstract].

RHEE, S. Y., S. WENG, D. FLANDERS, J. M. CHERRY, and C. DEAN et al., 1998  Genome maps 9: Arabidopsis thaliana, wall chart. Science 282:663-667[Free Full Text].

RITTER, C. and J. L. DANGL, 1996  Interference between two specific pathogen recognition events mediated by distinct plant disease resistance genes. Plant Cell 8:251-257[Abstract].

ROGERS, S. O., and A. J. BENDICH, 1988 Extraction of DNA from plant tissues, pp. 1–10 in Plant Molecular Biology Manual. Kluwer, Dordrecht, The Netherlands.

SALMERON, J. M., S. J. BARKER, F. M. CARLAND, A. Y. MEHTA, and B. J. STASKAWICZ, 1994  Tomato mutants altered in bacterial disease resistance provide evidence for a new locus controlling pathogen recognition. Plant Cell 6:511-520[Abstract].

SALMERON, J. M., G. E. D. OLDROYD, C. M. T. ROMMENS, S. R. SCOFIELD, and H. S. KIM et al., 1996  Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster. Cell 86:123-133[Medline].

SCOFIELD, S. R., C. M. TOBIAS, J. P. RATHJEN, J. H. CHANG, and D. T. LAVELLE et al., 1996  Molecular basis for gene-for-gene specificity in bacterial speck disease of tomato. Science 274:2063-2065[Abstract/Free Full Text].

SHIRASU, K., T. LAHAYE, M. W. TAN, F. ZHOU, and C. AZEVEDO et al., 1999  A novel class of eukaryotic zinc-binding proteins is required for disease resistance signaling in barley and development in C. elegans. Cell 99:355-366[Medline].

SILVA, H., K. YOSHIOKA, H. K. DOONER, and D. F. KLESSIG, 1999  Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance. Mol. Plant-Microbe Interact 12:1053-1063[Medline].

SUZUKI, N., H.-R. CHOE, Y. NISHIDA, Y. YAMAWAKI-KATAOKA, and S. OHNISHI et al., 1990  Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins. Proc. Natl. Acad. Sci. USA 87:8711-8715[Abstract/Free Full Text].

TANG, X., R. D. FREDERICK, J. ZHOU, D. A. HALTERMAN, and Y. JIA et al., 1996  Initiation of plant disease resistance by physical interaction of AvrPto and Pto kinase. Science 274:2060-2063[Abstract/Free Full Text].

THOMAS, C. M., D. A. JONES, M. PARNISKE, K. HARRISON, and P. J. BALINT-KURTI et al., 1997  Characterization of the tomato Cf-4 gene for resistance to Cladosporium fulvum identifies sequences that determine recognitional specificity in Cf-4 and Cf-9.. Plant Cell 9:2209-2224[Abstract].

WARREN, R. F., A. HENK, P. MOWERY, E. HOLUB, and R. W. INNES, 1998  A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes. Plant Cell 10:1439-1452[Abstract/Free Full Text].

WHALEN, M., R. INNES, A. BENT, and B. STASKAWICZ, 1991  Identification of Pseudomonas syringae pathogens of Arabidopsis thaliana and a bacterial gene determining avirulence on both Arabidopsis and soybean. Plant Cell 3:49-59[Abstract/Free Full Text].

WHITE, F. F., B. YANG, and L. B. JOHNSON, 2000  Prospects for understanding avirulence gene function. Curr. Opin. Plant Biol. 3:291-298[Medline].

YOUNG, N. D., 2000  The genetic architecture of resistance. Curr. Opin. Plant Biol. 3:285-290[Medline].

YU, G.-L., F. KATAGIRI, and F. M. AUSUBEL, 1993  Arabidopsis mutations at the RPS2 locus result in loss of resistance to Pseudomonas syringae strains expressing the avirulence gene avrRpt2.. Mol. Plant-Microbe Interact. 6:434-443[Medline].

YU, I.-C., J. PARKER, and A. F. BENT, 1998  Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proc. Natl. Acad. Sci. USA 95:7819-7824[Abstract/Free Full Text].

ZHOU, J., Y.-T. LOH, R. A. BRESSAN, and G. B. MARTIN, 1995  The tomato gene Pti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the hypersensitive response. Cell 83:925-935[Medline].

This article has been cited by other articles:

				R. M.P. Van Poecke, M. Sato, L. Lenarz-Wyatt, S. Weisberg, and F. Katagiri Natural Variation in RPS2-Mediated Resistance among Arabidopsis Accessions: Correlation between Gene Expression Profiles and Phenotypic Responses PLANT CELL, December 1, 2007; 19(12): 4046 - 4060. [Abstract] [Full Text] [PDF]

				Y. Cao, X. Ding, M. Cai, J. Zhao, Y. Lin, X. Li, C. Xu, and S. Wang The Expression Pattern of a Rice Disease Resistance Gene Xa3/Xa26 Is Differentially Regulated by the Genetic Backgrounds and Developmental Stages That Influence Its Function Genetics, September 1, 2007; 177(1): 523 - 533. [Abstract] [Full Text] [PDF]

				U. Orgil, H. Araki, S. Tangchaiburana, R. Berkey, and S. Xiao Intraspecific Genetic Variations, Fitness Cost and Benefit of RPW8, A Disease Resistance Locus in Arabidopsis thaliana Genetics, August 1, 2007; 176(4): 2317 - 2333. [Abstract] [Full Text] [PDF]

				A. Al-Daoude, M. de Torres Zabala, J.-H. Ko, and M. Grant RIN13 Is a Positive Regulator of the Plant Disease Resistance Protein RPM1 PLANT CELL, March 1, 2005; 17(3): 1016 - 1028. [Abstract] [Full Text] [PDF]

				C. A. Wright and G. A. Beattie Pseudomonas syringae pv. tomato cells encounter inhibitory levels of water stress during the hypersensitive response of Arabidopsis thaliana PNAS, March 2, 2004; 101(9): 3269 - 3274. [Abstract] [Full Text] [PDF]

				L. E. Rose, P. D. Bittner-Eddy, C. H. Langley, E. B. Holub, R. W. Michelmore, and J. L. Beynon The Maintenance of Extreme Amino Acid Diversity at the Disease Resistance Gene, RPP13, in Arabidopsis thaliana Genetics, March 1, 2004; 166(3): 1517 - 1527. [Abstract] [Full Text] [PDF]

				A. J. Hayes, S. C. Jeong, M. A. Gore, Y. G. Yu, G. R. Buss, S. A. Tolin, and M. A. S. Maroof Recombination Within a Nucleotide-Binding-Site/Leucine-Rich-Repeat Gene Cluster Produces New Variants Conditioning Resistance to Soybean Mosaic Virus in Soybeans Genetics, January 1, 2004; 166(1): 493 - 503. [Abstract] [Full Text] [PDF]

				L. Deslandes, J. Olivier, N. Peeters, D. X. Feng, M. Khounlotham, C. Boucher, I. Somssich, S. Genin, and Y. Marco Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus PNAS, June 24, 2003; 100(13): 8024 - 8029. [Abstract] [Full Text] [PDF]

				K. A. Shepard and M. D. Purugganan Molecular Population Genetics of the Arabidopsis CLAVATA2 Region: The Genomic Scale of Variation and Selection in a Selfing Species Genetics, March 1, 2003; 163(3): 1083 - 1095. [Abstract] [Full Text] [PDF]

				R. Mauricio, E. A. Stahl, T. Korves, D. Tian, M. Kreitman, and J. Bergelson Natural Selection for Polymorphism in the Disease Resistance Gene Rps2 of Arabidopsis thaliana Genetics, February 1, 2003; 163(2): 735 - 746. [Abstract] [Full Text] [PDF]

				J. A. Harton, M. W. Linhoff, J. Zhang, and J. P.-Y. Ting Cutting Edge: CATERPILLER: A Large Family of Mammalian Genes Containing CARD, Pyrin, Nucleotide-Binding, and Leucine-Rich Repeat Domains J. Immunol., October 15, 2002; 169(8): 4088 - 4093. [Abstract] [Full Text] [PDF]

				N. A. Eckardt, T. Araki, C. Benning, P. Cubas, J. Goodrich, S. E. Jacobsen, P. Masson, E. Nambara, R. Simon, S. Somerville, et al. Arabidopsis Research 2001 PLANT CELL, September 1, 2001; 13(9): 1973 - 1982. [Full Text] [PDF]

				J. Bergelson, M. Kreitman, E. A. Stahl, and D. Tian Evolutionary Dynamics of Plant R-Genes Science, June 22, 2001; 292(5525): 2281 - 2285. [Abstract] [Full Text] [PDF]