Comparative Genetics of Nucleotide Binding Site-Leucine Rich Repeat Resistance Gene Homologues in the Genomes of Two Dicotyledons: Tomato and Arabidopsis

Corresponding author: Robert Fluhr, Weizmann Institute of Science, P.O. Box 26, Rehovot, Israel 76100., lpfluhr{at}weizmann.weizmann.ac.il (E-mail)

Communicating editor: C. S. GASSER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.

PLANTS require dominant or semidominant resistance (R) gene alleles to specifically recognize pathogen ingress. The protein products of such genes have been suggested to be receptors that specifically bind ligands encoded by the corresponding pathogen avirulence (avr) genes (gene-for-gene recognition; STASKAWICZ et al. 1995 ). The putative receptor-ligand complex initiates a series of signal transduction cascades leading to disease resistance (BAKER et al. 1997 ). Among the cellular events that characterize resistance are oxidative burst, cell wall strengthening, induction of defense gene expression, and rapid cell death at the site of infection.

Several R-genes have been isolated by map-based cloning or transposon-tagging strategies and were shown to restore pathogen-specific resistance (DE WIT 1997 ). R-genes can be divided into at least four broad structural classes. The first family belongs to the serine-threonine protein kinase. Pto is the only known member and confers resistance to bacterial speck disease in tomato (MARTIN et al. 1993 ). The second class of R-genes is represented by the Cf family of tomato resistance genes specific for leaf mold resistance and Hs1 for nematode resistance, which encode putative transmembrane receptors with extracellular LRR domains (JONES et al. 1994 ; DIXON et al. 1996 , DIXON et al. 1998 ; CAI et al. 1997 ; THOMAS et al. 1997 ). The third class encodes for a receptor-like kinase combining qualities of both classes above and is exemplified by Xa21 conferring resistance to rice bacterial blight (SONG et al. 1995 ).

The fourth class, representing by far the majority of functionally described R-genes, are the nucleotide binding site-leucine rich repeat (NBS-LRR) resistance genes. These genes contain at least three discernible domains: a variable N terminus, nucleotide-binding site, and leucine rich repeats. Two kinds of N termini are present in NBS-LRR. One type of NBS-LRR contains coiled coils (CC) that are thought to play a role in protein-protein interactions. CC motifs appear in the N terminus of both dicotyledons and cereals (PAN et al. 2000 ). The other type of N terminus has been described only in dicotyledons and shows homology to the Drosophila Toll or human interleukin receptor-like (TIR) regions that also contain LRR domains (WHITHAM et al. 1994 ; HAMMOND-KOSACK and JONES 1997 ). The C-terminal LRR region could participate in protein-protein interactions that underlie pathogen-specific gene-for-gene recognition (DE WIT et al. 1997; WARREN et al. 1998 ; ELLIS et al. 1999 ).

Distinct forms of NBS domains are found in many ATP- and GTP-binding proteins that serve as molecular switches, including the Ras superfamily and the recently described Ced-4 and Apaf-1 animal genes (TRAUT 1994 ; LI et al. 1997 ). The later genes regulate the activity of proteases that can initiate apoptotic cell death. As defense mechanisms in plants also include apoptotic-like hypersensitive responses, the common appearance of NBS domains in plants and animals is intriguing.

NBS-LRR homologues encode structurally related proteins, suggesting that they function in common signal transduction pathways, even though they confer resistance to a wide variety of pathogen types. The few functionally defined R-genes and the much larger repertoire of resistance gene homologues available in the databases provide a facile system to study the evolutionary biology and genetics of supergene families. Conserved motifs in the NBS domain have been used to isolate NBS sequences from soybean, potato, rice, barley, and Arabidopsis thaliana (KANAZIN et al. 1996 ; LEISTER et al. 1996 , LEISTER et al. 1998 ; YU et al. 1996 ; AARTS et al. 1998 ; SPEULMAN et al. 1998 ). A. thaliana expressed sequence tags containing LRR domains were mapped (BOTELLA et al. 1997 ). However, the identity of these sequences cannot be determined with certainty as LRR motifs are found in genes that do not act in disease resistance (BECRAFT 1998 ; MEYER et al. 1999 ; VAN DER KNAAP et al. 1999 ; YAO et al. 1999 ).

Recently, the results from comparative mapping in rice, barley, and foxtail millet have indicated a rapid reorganization of R-gene homologues in grasses (LEISTER et al. 1998 ). This raises the question of how R-gene homologues evolve in dicotyledon species. Tomato is a genetically well-characterized plant species with a relatively dense linkage map. Importantly, both potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum MILL.) are members of the Solanaceae, have the same basic chromosome number (x = 12), and show extensive conservation of the linear order of genetic markers (BONIERBALE et al. 1988 ). Here, we report the identification of 75 tomato clones and their division into phylogenetic clusters. Representative clones from all of the clusters were mapped. Comparison of orthologous sequence from the potato genome detected several conserved syntenic loci. However, the clusters were found to differ markedly from phylogenetic clusters in A. thaliana.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Amplification of genomic sequence:
Tomato L. esculentum cv. Motelle total genomic DNA was amplified in the presence of 1 µM of primers with 1 unit Taq DNA polymerase (Promega, Madison, WI) in 50 µl volume. DNA was denatured for 4 min at 94° followed by 35 amplification cycles as follows: 94° for 1 min, 40° for 1 min, and 72° for 1 min. PCR products were fractionated on 1% agarose gel and the fragments of expected NBS domain size (500–700 bp) were cloned in the pGEM-T vector (Promega). Primer sequences used are listed in Table 1.

Mapping by restriction fragment length polymorphism (RFLP) analysis of introgression lines:
Total genomic DNA was digested with appropriate restriction enzymes and fractionated on 1% agarose gels. The blotted DNA was hybridized to PCR-amplified NBS clones as indicated. The introgression lines showing L. pennellii polymorphism were scored by RFLP analysis as described (ESHED and ZAMIR 1995 ). The plant population used for mapping is a further refinement of a series of plant lines that contain the complete L. pennellii genome introgressed into the L. esculentum genome (ESHED and ZAMIR 1995 ; LIU and ZAMIR 1999 ). The current genetic map was constructed by Southern blot analysis of 75 nearly isogenic lines using standard tomato RFLP probes. The chromosomes are further divided into 107 bins or segments depending on the relative positions of the introgressed chromosomal intervals and overlaps between lines. Thus the average genetic size of a bin is ~12 cM (1274 cM-genome/107; TANKSLEY et al. 1992 ).

Sequence alignment and construction of phylogenetic tree:
Database searches were carried out by using both heuristic fast algorithms (ALTSCHUL et al. 1997 ) and the Smith-Waterman program for searches of more distantly related sequences (SMITH and WATERMAN 1981 ). Amino acid sequences were analyzed using the GCG9 (University of Wisconsin Genetics Computer Group, Madison) software package. Sequences were aligned using the PILEUP with the default settings of gap opening penalty at 3.0 and gap extension penalty at 0.1. No manual adjustment was found to be necessary. Phylogeny was examined by the neighbor-joining method (SAITOU and NEI 1987 ) using the neighbor-joining algorithm implemented in Clustal X software (THOMPSON et al. 1997 ; JEANMOUGIN et al. 1998 ). The substitution rates were corrected for multiple hits according to Dayhoff's PAM matrix with the pairwise gap removal option active.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Identification of novel tomato resistance gene homologues and pseudogenes:
NBS-containing genomic sequences were created by amplification of total tomato DNA using degenerate PCR primers described in Table 1. Fragments of an average 500–700-bp size were cloned and sequenced. Sequence analysis of the clones revealed that 75 of them are related to plant NBS-LRR genes because they contained additional confirmatory internal group-specific motifs (NBS II-NBS-VIII; Fig 1). Using this procedure the following types of amplified sequence will not be recovered: NBS-LRR that do not contain the conserved motifs used for priming and genes that contain large introns in this region. Inspection of A. thaliana NBS sequences in the genomic databases as well as other available monocotyledon and dicotyledon genomic NBS sequences suggests that all of the NBS-LRR-type genes contain the conserved motifs. In addition, analysis of the 13 functional NBS-LRR genes from dicotyledons and monocotyledons isolated to date show that only one (RPP8) contains an intron between the NBS motifs used here for amplification (MCDOWELL et al. 1998 ).

View larger version (125K): In this window In a new window Download PPT slide   Figure 1. Multiple sequence alignment of the deduced amino acid sequences of 37 representative tomato NBS clones with resistance genes from Solanaceae species. The disease resistance genes included are tobacco N gene (WHITHAM et al. 1994 ), tomato I2 (ORI et al. 1997 ), Mi (ROSSI et al. 1998 ), and Prf (SALMERON et al. 1996 ). Gaps (indicated with dashes) were introduced to improve the alignment. Conserved residues are shaded and their motifs are numbered as in PAN et al. 2000 . Stars indicate position of stop codons. Demarcation of the major NBS Groups I and II is indicated on the right.

The translated sequences were aligned in the GCG Pileup comparison program and representative NBS homologues (37/75), together with known Solanaceae resistance genes, are shown in Fig 1. Inspection of the sequence revealed the presence of stop codons in seven clones (note stars in Q136, Q97, and Q137) or ~10% of the NBS sequences recovered. No frameshift-type mutations were recovered. The stop codons unveiled are unlikely to be the result of random sequencing errors or PCR amplification vagaries because they appear in the same position in a series of closely related but not identical clones (data not shown). It is therefore possible that they reflect the duplication of genome mutational events that form small pseudogene families. We wished to establish whether a similar rate of pseudogene recovery could be anticipated from the A. thaliana genomic sequence. A BLAST search using different representative NBS motifs revealed 88 full-length NBS-like motifs of which 9 had introns. Direct inspection of their sequence revealed no intragenic stop codons.

Phylogenetic analysis of tomato NBS loci:
The plethora of tomato NBS sequences made available by genome amplification enable phylogeny construction and comparison. A phylogenetic tree based on the NBS sequence alignment was generated by the neighbor-joining method as described in MATERIALS AND METHODS. The reliability of the tree was then established by conducting 500 bootstrap resampling steps. Mammalian apoptosis-related protein Apaf1 (LI et al. 1997 ), which exhibits 20% sequence identity with plant resistance genes in the NBS region, was included as an outgroup for phylogenetic analysis. Two major branches designated as Groups I and II were obtained (Fig 2). This is consistent with previous results that were carried out on a smaller sequence set that contained a mixture of cereal and dicotyledon species (PAN et al. 2000 ). The internal stability of the two major branches is strongly supported by the bootstrapping experiment (500/500 for major group divisions). Members of the two groups are about equally represented in tomato. Sequences among the groups appear as clusters forming short segmented branches or as deep branches. The former represent gene families of common recent origin while the latter are either underrepresented due to PCR vagaries or represent diverged singular genes. To establish if the recent evolution of R-gene sequences predates speciation in the Solanaceae we added to the phylogeny analysis all the known NBS sequences from qualified Solanaceae NBS-type R-genes (N, Prf, Mi, and I2) as well as isolated potato NBS (St) sequence (Fig 2). The results show that all the Solanaceae R-genes and R-gene homologues are well distributed among the branches of the tomato phylogenetic tree, indicating that they arose from common ancestral genes before speciation.

View larger version (43K): In this window In a new window Download PPT slide   Figure 2. Phylogenetic tree of tomato and other Solanaceae R-genes and R-gene homologues. Amino acid sequences were aligned using the Clustal X program and a tree was generated using the neighbor-joining method. Sequences that appear in ovals are the representatives chosen from each cluster for mapping as shown in Fig 3 and Fig 4. The arrows, emanating from the ovals, point to the map locations as shown in Fig 4. Potato R-gene homologues (labeled St) were included and are indicated by boxes. The Solanaceae R-genes are N, Prf, I2C-1, and Mi. A candidate gene in potato for the resistance against S. endobioticum (Sen; HEHL et al. 1999 ) and its tomato homologues SenT is also included. A total of 500 bootstrapping runs were performed and only the significant branches (>80% reliability) are labeled. The scale indicates the average substitutions per site.

Mapping of tomato resistance gene homologues reveals null alleles and NBS sequence clustering:
Inspection of the amplified sequences suggested that many belong to multigene families. Therefore, conventional RFLP was chosen as the mapping method because it facilitates mapping of multiloci components. The introgression lines (ILs) used for mapping are described in MATERIALS AND METHODS. The phylogenetic analysis was used to select representatives of the clusters for physical mapping and 35 clones were chosen (ovals, Fig 2). For each clone a survey was conducted using seven different restriction enzymes as exemplified in Fig 3. In the case of Q118 the restriction enzyme Hae III yielded a distinct polymorphism between the tomato species and was then used to survey Southern blots of the introgression lines (Fig 3B). The results show polymorphism present in the introgressed L. pennellii lines 9-1 and 9-1-2, which indicates the precise mapping position of Q on the L. esculentum physical map (Fig 4; bin 9-A). In contrast, probe Q174 yielded a hybridization signal with L. esculentum DNA but no signal was detected with L. pennellii DNA, indicating that all its family members are absent in this genome (Fig 3C). Analysis of the complete IL set with Q174 shows lack of hybridization signal in lines 9-1, 9-1-2, and 9-1-3 positioning the null allele Q174 immediately below Q118 (Fig 4; bin 9B). In a similar fashion, 32 NBS sequences were found to map to 23 different bins on the genetic map of tomato (Fig 4). No additional null alleles were found.

View larger version (61K): In this window In a new window Download PPT slide   Figure 3. Hybridization of Q118 and Q174 to the L. pennellii series of introgression lines. (A) Genomic DNA isolated from L. pennellii and L. esculentum cultivar M82 were digested with seven different restriction enzymes and hybridized with Q118 as the probe. (B) HaeIII-digested genomic DNA from the introgression lines, M82, and L. pennellii were hybridized with PCR clone Q118. (C) Genomic DNA isolated from L. pennellii and M82 were digested with seven different restriction enzymes and hybridized with Q174 as the probe. (D) DraI-digested genomic DNA from the introgression lines, L. esculentum cultivar M82, and wild-type L. pennellii were hybridized with PCR clone Q174.

View larger version (109K): In this window In a new window Download PPT slide   Figure 4. Schematic summary of mapped NBS regions on the tomato IL map. The L. esculentum map is drawn as open bars and the L. pennellii introgressed segments appear as solid bars in which the boundary edge of each segment is indicated by inclusive (+) and exclusive (-) RFLP markers. All IL lines are homozygous for the indicated introgression except part of IL8-1. Bins are designated by the chromosome number followed by a capital letter and indicate unique area of IL overlap and singularity. Molecular and genetic markers are indicated to the right of each chromosome and the genetic distances (in centimorgans) and approximate centromere regions are indicated to the left. Hatched vertical bars represent bins to which loci have been mapped. The subscript indicates that the same probe detected more than one locus. Arrowheads point to known disease resistance gene loci that were mapped at high resolution whereas arrows pointing to bars indicate quantitative trait loci that were mapped to larger genomic intervals. Tomato loci for resistances are in boldface. Loci for resistance to viruses include Sw-5, resistance to tomato spotted wilt virus (STEVENS et al. 1995 ); Tm-1 and Tm-2, resistance to tobacco mosaic virus (YOUNG et al. 1988 ; LEVESQUE et al. 1990 ); and Ty-1, resistance to tomato yellow leaf curl virus (ZAMIR et al. 1994 ). Loci for resistance to bacteria are Pto, resistance to bacterial speck (MARTIN et al. 1994 ); Bw-1/2/3, resistance to bacterial wilt (DANESH et al. 1994 ); and rx-1, rx-2, and rx-3, resistance to Xanthomonas campestris (YU et al. 1995 ). Loci for resistance to fungi consist of Lv and Ol-1, resistance to powdery mildew (CHUNWONGSE et al. 1994 ; VAN DE BEEK et al. 1994 ); I1, I2, and I3, resistance to Fusarium wilt (BOURNIVAL et al. 1989 ; SARFATTI et al. 1989 , SARFATTI et al. 1991 ); I4, I5, and I6, resistance to Fusarium wilt (M. B. SELA-BUURLAGE and R. FLUHR, unpublished results), Frl, resistance to Fusarium crown rot (LATERROT and MORETTI 1995 ); Sm, resistance to gray leaf spot (BEHARE et al. 1991 ); Ve, resistance to Verticillium wilt (DIWAN et al. 1999 ); Cf-2, Cf-4, Cf-5, and Cf-9, resistance to tomato leaf mold (DICKINSON et al. 1993 ; BALINT-KURTI et al. 1994 ; DIXON et al. 1995 ); and Asc, resistance to Alternaria stem canker (VAN DER BIEZEN et al. 1995 ). The loci for nematode resistance are Mi (MESSEGUER et al. 1991 ), Mi-3 (YAGHOOBI et al. 1995 ), Gro 1 (BARONE et al. 1990 ), and Hero (GANAL et al. 1995 ). Potato loci for resistances are in outline letters. R3, R6, R7, resistance to P. infestens (LEISTER et al. 1996 ); Rysto, resistance to PVY (BRIGNETI et al. 1997 ); RMcl, resistance to nematodes (BROWN et al. 1996 ); and Gro1, resistance to nematodes (LEISTER et al. 1996 ).

In many cases, NBS sequences tend to map to multiple positions. The highly homologous probes Q4, Q66, and Q99 detected a multigene family, members of which mapped to eight loci (indicated by superscript a-h) on five chromosomes. Members of this gene family mapped to a locus on chromosome 2 at a position corresponding to that of resistance locus Tm-1 that encodes resistance to tomato mosaic virus. The seventh locus of this family was also identified on chromosome 12 centromeric region, where the resistance gene Lv is located. The presence of homologous NBS homologues at many independent loci indicates rapid radiation of these sequences throughout the genome.

The overall mapping data clearly shows that in the majority of map positions (18 bins/27 bins) NBS homologues of divergent sequence origin tend to cluster together as has been detected in other plant genomes (KANAZIN et al. 1996 ; MEYERS et al. 1998 ; SHEN et al. 1998 ). For example, 10 different PCR clones (Q1, Q3, Q4, Q7, Q8, Q66, Q99, Q144, Q147, and Q210) display amino acid sequence identities ranging from 24 to 99%. Based on sequence they can be divided into at least three distinct subgroups (Fig 2). However, they all mapped to an interval on chromosome 1 (bin 1-D, between genetic markers TG80 and TG71). This could result from sets of gene duplications arrayed in tandem. However, in at least two cases the mapping data show that distinct NBS domains from the same cluster are interpolated among each other. Thus, examination of chromosome 11 and 12 shows that the adjacent bins 11-D and 11-E and bins 12-D and 12-E each contain a similar Q sequence demonstrating their interspersion. The clustering of R-gene homologues may have biological significance in facilitating creation of novel resistance genes by intragenic or intergenic recombination.

Phylogenetic comparison between Solanaceae species:
Recently, map positions of six PCR-derived potato R-gene homologues were determined (LEISTER et al. 1996 ). To investigate the extent of conservation of synteny among the resistance loci, we first examined the phylogenetic relationships between tomato and potato R-gene homologues (Fig 2). Numbers in ovals and boxes in Fig 2 represent tomato and potato homologues, respectively. Inspection of the tree shows that the potato and tomato sequences form tight clusters. Thus, some tomato homologues are more closely related to potato than to other tomato-derived sequences and may either represent sequence orthologs or be members of a class that derived from common NBS homologues.

Phylogenetic comparison between Solanaceae species and A. thaliana:
The result of mutual clustering between related NBS sequences in Solanaceae prompted us to examine if conservation exists outside of this family. We extended the analysis to A. thaliana for which a comparable amount of NBS sequence information exists. A complete analysis of NBS homologues was carried out on the available A. thaliana genomic sequence (see MATERIALS AND METHODS) and more than 65 independent loci were recovered that contained cognate LRR regions. Most loci were found to contain on the average two to three NBS-LRR homologues. Multiple sequence alignment was then carried out with one representative member from each locus followed by neighbor-joining phylogeny analysis that included the representative Q sequence, qualified R-genes, and additional published NBS-LRR sequences as indicated (Fig 5). With the caveat in mind that different methods were used as a source for sequence (PCR vs. genomic sequence), the results show that the division into two main groups of R-gene homologues is maintained. However, it is apparent from the phylogenetic tree that all branches and their clusters contain sequences that originate from only one family type. Thus, the clusters are family specific and, in contrast to the results in Fig 2, the NBS sequences of the species shown have significantly diverged, suggesting that the major gene duplication events occurred during dicotyledon divergence into various taxa.

View larger version (50K): In this window In a new window Download PPT slide   Figure 5. Phylogenetic tree of NBS-LRR resistance genes and resistance gene homologues. The tree was constructed as in Fig 2 and includes mapped Q sequence, NBS homologues of Arabidopsis, and NBS of functional R-genes. The functionally characterized resistance genes include N gene from tobacco (WHITHAM et al. 1994 ), Prf, I2C1, and Mi from tomato (SALMERON et al. 1996 ; ORI et al. 1997 ; MILLIGAN et al. 1998 ), RPM1, RPS2, RPP5, RPS5, RPP1, and RPP8 from A. thaliana (BENT et al. 1994 ; MINDRINOS et al. 1994 ; GRANT et al. 1995 ; PARKER et al. 1996 ; BOTELLA et al. 1998 ; MCDOWELL et al. 1998 ; WARREN et al. 1998 ), M and L6 from flax (LAWRENCE et al. 1995 ; ANDERSON et al. 1997 ), RGC2 from lettuce (MEYERS et al. 1998 ), Xa1 from rice (YOSHIMURA et al. 1998 ), Cre3 from wheat (LAGUDAH et al. 1997 ), and Sen (gene candidate) from potato (HEHL et al. 1999 ). The letter Q indicates tomato R-gene homologues. The A. thaliana R-gene homologues are shown as gene accession number unless indicated otherwise. Subscripts indicate clusters that are within 0.5 substitution/site. The scale indicates the averaged substitutions per site.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Conservation of NBS homologues reveals the pace of evolution of R-gene homologues:
The dynamics of NBS-LRR homologue evolution has been described here by constructing gene phylogeny based on the conserved NBS sequences from species for which data are available. The use of NBS sequences for construction of R-gene homologue phylogeny can be justified for the following reasons. NBS regions in genomic databases are associated with cognate N-terminal TIR/CC and C-terminal LRR elements and therefore emulate parts of genuine R-gene homologues.

NBS have been divided into two major groups based on phylogenetic analysis. Inspection of clusters of NBS homologues portrayed by tomato and A. thaliana NBS shows no evidence for common sequence origin other than the conserved motif constraints that divide Group 1 and Group 2 sequences (Fig 2 and Fig 5). Very high bootstrap values can be obtained for division into multiple independent clusters of tomato, A. thaliana, and other species. When an arbitrary criterion of less than 0.5 substitution per site is applied to define a cluster, we note about 16 and 19 different phylogenetic clusters in tomato and A. thaliana, respectively (Fig 5). In all cases clusters are species or family specific. Seeming exceptions like the NBS sequences AB01687 and RPM1 that originate from Brassicaceae but appear to fall near families of Solanaceaea_a and Poaceae_b, respectively, have low bootstrap levels and their positioning is thus unreliable. In striking contrast to the lack of overlap between Brassicaceae and Solanaceae species, the phylogenetic comparison within the Solanaceae reveals a degree of conservation because all NBS sequences of the major genera examined, e.g., Nicotiana, Solanum, and Lycopersicon, fall into clusters containing mutual sequences. This observation is likely an indication of recent gene radiation from a common ancestral source of R-gene homologues. However, as shown in Fig 3, the continued rapid evolution of NBS homologues is exemplified by the existence of null alleles between L. esculentum and L. pennellii, a result that is consistent with the birth and death hypothesis for R-gene diversification (MICHELMORE and MEYERS 1998 ).

Multigene families in plants display diverse evolutionary patterns (CLEGG et al. 1997 ). For example, expansions and contractions in gene copy number were detected in rbcS (~10 members/genome) that may be the result of interlocus gene conversion (MEAGHER et al. 1989 ). Thus, duplication followed by gene divergence and frequent gene conversion events leads to a pattern of clustering within a family. This mode of evolution is evident in comparison of R-gene homologues. In contrast to this mode of evolution, members of the large Lhc gene family (~30 members/genome) show conservation among different gene types that transcends phyla (JANSSON 1999 ). In R-gene evolution the stochastic selection of genes during speciation followed by rapid differentiation led to the emergence of family-specific clusters that erased their mutual origin other than the major division into NBS Groups 1 and 2.

Genomic organization of R-genes:
Tomato NBS were found to be dispersed into many distinct bin locations. In many cases, the bins contain sequences of mixed origin, indicating clustering as has been found for soybean NBS sequence (KANAZIN et al. 1996 ). In at least two cases the sequences from clusters are physically interspersed on the tomato genetic map. This indicates that R-gene families and their homologues are kept distinct from general gene homogenization processes.

Local and long-distance gene duplications probably play a role in the expansion of gene families of R-gene homologues. In the case of tomato I2 at least seven gene members are found over a 1-Mb interval on chromosome 11 and other copies are on chromosomes 8 and 9 (ORI et al. 1997 ; SIMONS et al. 1998 ). In addition, many sequences have multiple nonlinked mapping locations. For example, Q4 maps to eight independent locations on six different chromosomes (Fig 4). The proposed mechanisms for these types of duplications are varied and could include slippage during replication, nonreciprocal recombination events, and gene duplication via reverse transcription.

While mechanisms for gene amplification abound, a related question is the driving force that fixes these events in the population and maintains the local and genome-wide copy number level. Presumably, the driving force that would fix gene duplications in the population would be positive selection of gene arrays that yielded beneficial resistances. Local copy number would be maintained by the rate of gene duplication as opposed to gene eradication due to unequal crossing over. The birth of "new R-genes" at more distant locations would enable a new round of gene duplication and permit increase in R-gene diversity and enable the creation of new specificities. In this respect, Q174 and its cluster group that are present in L. esculentum but absent in L. pennellii represent either "birth" of a new locus in L. esculentum by gene transfer and subsequent diversification or "death" of a locus in L. pennellii due to unequal crossing over.

What is the upper limit of R-genes in the genome? The 65 identified A. thaliana loci (57% complete) contain about two to three genes each and predict ~300 NBS-LRR/genome. Thus, a few hundred NBS-LRR genes together with the other less-abundant types of R-genes are sufficient to maintain integrity of the plant's defense stature. Amplification of tomato genomic sequences yielded 75 independent NBS sequences and by comparison to A. thaliana this would indicate that we are far from saturation of the tomato genome potential, although the similar number of phylogenetic clusters as defined in Fig 5 (15–19 clusters) indicates that the complexity of NBS types recovered is comparable in both cases.

Co-localization of NBS domains with resistance loci:
The tomato genetic map shown in Fig 4 indicates the position of 23 genetic resistances. We note that at the current resolution 28 (37%) of the NBS sequences comapped to bins that contain known tomato disease resistance loci. For example, a genetic linkage was uncovered on chromosome 7 (bin 7-F) between the Fusarium resistance locus I3 and the probes Q2, Q6, and Q112 (Fig 4). Further high-resolution linkage analysis at 2.0 cM resolution maintained the linkage only between the Q2 sequence and the I3 locus, but not Q6 (M. SELA, unpublished data). PCR product Q118 mapped to a position on top of chromosome 9, which is linked to the Ve locus, a dominant Verticillium wilt resistance gene in tomato. Six different clones (Q1, Q8, Q144, Q164, Q173, Q210) mapped in an interval on chromosome 11 where the Fusarium resistance locus I has been located. Although the fixed-type construction of the IL lines obviates their use for precise estimates of genetic distance, the linkage established with previously described resistance loci should be an important tool for future map-based cloning of these resistance loci.

Syntenic relationships in Solanaceae:
Comparing sequence similarity and chromosomal distribution of resistance gene homologues in related species will shed light on NBS-LRR homologue diversification during speciation. Potato and tomato were among the first plant species where genome colinearity was demonstrated by using common sets of RFLP markers (BONIERBALE et al. 1988 ). We can identify four loci between tomato and available potato sequence data that appear in syntenic genomic positions. The phylogenetic location of Q2 suggests its similarity with potato St3.3.2 (81% sequence identity; Fig 2). St3.3.2 is tightly linked to the potato nematode resistance Gro1 locus on chromosome 7 that appears to be syntenic with the resistance gene I3 and Q2 on tomato chromosome 7 (BALLVORA et al. 1995 ; Fig 4). Phylogeny test shows that four different tomato homologues Q1, Q8, Q173, and Q164, as well as the NBS domain from the tobacco N gene, are related to potato NBS locus St3.3.1.3. They all comapped to marker CP58A in a chromosomal interval (bin 11-B) where tomato locus I is located as well as resistance to the potato pathogen Synchytrium endobioticum (HEHL et al. 1999 ). Significantly, the gene candidate for Sen resitance locus exhibits high amino acid sequence identity with its tomato counterparts (Q1, 72.5%; Q8, 76.0%; Q164, 68.4%; Q173, 71.1%). Other candidate potato resistance loci are Ry_sto, and R_Mcl, which encode resistance to potato virus Y (PVY) and root knot nematodes, respectively, and are tightly linked to RFLP marker TG508 in this region (Fig 4; BROWN et al. 1996 ; BRIGNETI et al. 1997 ). An additional locus can be identified at I2 on chromosome 11. The I2 gene locus encodes for resistance to F.o.l. race 2 in tomato (ORI et al. 1997 ) and shows 77% identity with St1.2.1 that was found to be linked to the Phytophthora infestens resistance loci R3, R6, and R7 (LEISTER et al. 1996 ). All loci occupy similar positions in Lycopersicon and Solanum species, as indicated by the tightly linked reference marker TG105A (Fig 4). A fourth locus, identified by Q3, is most closely related to potato St1.2.4. In this case as well, the map location is syntenic with that of St1.2.4; however, no potato disease resistance locus has been mapped in this region (LEISTER et al. 1996 ).

The conservation of synteny in Solanaceae reported here is in contrast to the rapid reorganization of resistance gene loci that occurs between related cereal species (LEISTER et al. 1998 ). In that case, no synteny in R-gene homologue positioning could be detected among rice, barley, and foxtail millet, although these genomes generally exhibit tight colinearity (GALE and DEVOS 1998 ). This may be due to the relatively more ancient origin of members of the Poaceae relative to Solanaceae species (56 and 40 million years, respectively). Within the Solanaceae one can differentiate between clusters that have multiple mapping positions (Fig 2 and Fig 5). Presumably, the more independent locations in which any one cluster appears could be an indication of its earlier origin. In this context, we note that the syntenic relationships reported here all occur within multimapping loci, lending credence to the idea that these sequences have an earlier origin.

We conclude from this study and from the work of LEISTER et al. 1996 that there is a high degree of conservation of NBS homologues between tomato and potato facilitating syntenic positioning of R-gene homologues. Interestingly, all syntenic tomato/potato loci confer resistance to completely unrelated disease, suggesting that NBS-LRR resistance genes may have adopted different pathogen specialization. This result is expected because amino acid changes in the LRR region have been shown to alter specificity (ELLIS et al. 1999 ). Indeed, even a singular R-gene, Mi, has been shown to exhibit multiple specificities to such diverse pathogen types as nematodes and aphids (ROSSI et al. 1998 ). Alternatively, the same disease resistances exist in the locus and may reside in other family members of the cluster. Notwithstanding, the construction of a detailed genetic map of disease resistance gene homologues in these species will promote identification of functional resistance genes through a map-based cloning strategy.

Widely different orders of metazoan taxa from mammals to plants display common molecular components of innate resistance. The innate immunity homologues in mammals, insects, and plants are composed of similar TIR-LRR elements; however, in animals their number are few (ROCK et al. 1998 ; HOFFMANN et al. 1999 ). In plants, R-gene homologues have undergone massive recurrent schemes of amplification and selection that apparently enable them to carry out a more diversified biological function. The study of their evolution will lead to a better grasp of their biology.

	ACKNOWLEDGMENTS

We thank B. Baker for the N gene probe. We are also grateful to C. Gebhardt for sharing unpublished data. This work was supported by a grant from the German-Israeli Foundation for Scientific Research and Development.

Manuscript received September 15, 1999; Accepted for publication December 14, 1999.

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THOMPSON<