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Comparative Genetics of Nucleotide Binding Site-Leucine Rich Repeat
Resistance Gene Homologues in the Genomes of Two Dicotyledons:
Tomato and Arabidopsis
Qilin Pan,* Yong-Sheng Liu,† Ofra Budai-Hadrian,* Marianne Sela,* Lea Carmel-Goren,‡
Dani Zamir‡ and Robert Fluhr*
*Department of Plant Science, Weizmann Institute of Science, Rehovot 76100, Israel, †Institute of Botany, Chinese Academy of Sciences,
Beijing 100093, People’s Republic of China and ‡Department of Field and Vegetable Crops, Faculty of Agriculture, Environment and
Food Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Manuscript received September 15, 1999
Accepted for publication December 14, 1999
ABSTRACT
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 racespecific 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.
P
LANTS 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
Corresponding author: Robert Fluhr, Weizmann Institute of Science,
P.O. Box 26, Rehovot, Israel 76100.
E-mail: lpfluhr@weizmann.weizmann.ac.il
Genetics 155: 309–322 (May 2000)
resistance, which encode putative transmembrane receptors with extracellular LRR domains ( Jones et al.
1994; Dixon et al. 1996, 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
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Q. Pan et al.
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 apoptoticlike 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, 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
Amplification of genomic sequence: Tomato L. esculentum
cv. Motelle total genomic DNA was amplified in the presence
of 1 ␮m of primers with 1 unit Taq DNA polymerase (Promega,
Madison, WI) in 50 ␮l 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 ⵑ12 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
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; Figure 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).
The translated sequences were aligned in the GCG
Pileup comparison program and representative NBS ho-
Resistance Gene Homologues in Tomato
311
TABLE 1
Primer sequences used for amplification of genomic DNA
Primer
Motifsa
Groupb
Motifs
Sequencec
15912
15913
15914
15915
16403
16409
16410
17696
17697
28106
28107
28108
29407
29408
29409
30392
30393
NBS-I
NBS-VI
NBS-I
NBS-VI
NBS-I
NBS-I
NBS-I
NBS-IX
NBS-IX
NBS-IX
NBS-IX
NBS-IX
NBS-IX
NBS-IX
NBS-IX
NBS-VI
NBS-VI
—
—
—
—
—
—
—
I
II
II
I
II
I
I
I
I
I
GGVGKTT
GLPLAL
GGVGKTT
GLPLAL
GGMGKTT
GGSGKTT
GGLGKTT
FLDIACF
LKRCFLY
FAYCSLF
FLHIACF
YCALFPE
FRHIACI
FLHIACF
FKCIACF
LCGNLPL
PLGLRVMG
GGT GGG GTT GGG AAG ACA ACG
CAA CGC TAG TGG CAA TCC
GGI GGI GTI GGI AAI ACI AC
AGI GCI AGI GGI AGI CC
GGI GGI ATI GGI AAA ACI AC
GGI GGI WSI GGI AAR ACI AC
GGI GGI YTI GGI AAR ACI AC
RAA RCA IGC SAT RTC IAR RAA
RTA IAG RAA RCA ISK YAG
RAA IAR ISW RCA RTA IGC RAA
RAA RCA IGC DAT RTG IAR RAA
YTC IGG RAA IAR IGC RCA RTA
ATR CAN GCN ATR TGN CKR AA
RAA NAC NGC NAT RTG NAR RAA
RAA RCA NCG NAT RCA YTT RTT
ARI GGI ARR TTI CCR CAI AR
CCC ATI ACI CKI ARI CCI AR
a
Primers are based on conserved motifs as shown in Figure 1.
Primers are Group I or Group II specific or general (—).
c
Codes for degenerate positions are I, Inosine; R, A/G; Y, C/T; K, G/T; N, A/G/C/T; D, A/G/T; W,
A/C; S, G/C.
b
mologues (37/75), together with known Solanaceae resistance genes, are shown in Figure 1. Inspection of the
sequence revealed the presence of stop codons in seven
clones (note stars in Q136, Q97, and Q137) or ⵑ10%
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 (Figure 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 (Figure 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.
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,
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Q. Pan et al.
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.
Resistance Gene Homologues in Tomato
313
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 Figures 3 and 4. The arrows, emanating from
the ovals, point to the map locations as shown in Figure 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.
Figure 2). For each clone a survey was conducted using
seven different restriction enzymes as exemplified in
Figure 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 (Figure 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 (Figure 4; bin 9-A). In contrast, probe Q174 yielded
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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.
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 (Figure
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 (Figure 4; bin 9B). In a similar fashion, 32 NBS
sequences were found to map to 23 different bins on
the genetic map of tomato (Figure 4). No additional
null alleles were found.
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 pres-
Resistance Gene Homologues in Tomato
ence 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 (Figure 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 (Figure 2). Numbers in ovals and
boxes in Figure 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 (Figure 5). With the
315
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 Figure 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.
DISCUSSION
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 (Figures 2 and 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 (Figure 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 Solanaceaeaa and Poaceaeb, 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 Figure 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).
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Q. Pan et al.
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 (ⵑ10 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 (ⵑ30 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 (Figure 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 ⵑ300
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 Figure 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:
䉴
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, 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).
Resistance Gene Homologues in Tomato
317
318
Q. Pan et al.
The tomato genetic map shown in Figure 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 (Figure 4). Further high-resolution linkage analy-
Figure 4.—Continued.
sis 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
Resistance Gene Homologues in Tomato
319
Figure 5.—Phylogenetic tree of NBS-LRR resistance genes and resistance gene homologues. The tree was constructed as in
Figure 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.
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; Figure 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;
Figure 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
320
Q. Pan et al.
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 Rysto, and RMcl, which encode resistance
to potato virus Y (PVY) and root knot nematodes, respectively, and are tightly linked to RFLP marker TG508 in
this region (Figure 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
(Figure 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
(Figures 2 and 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.
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.
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Communicating editor: C. S. Gasser