Content uploaded by Laurent D Noël
Author content
All content in this area was uploaded by Laurent D Noël
Content may be subject to copyright.
The Plant Cell, Vol. 11, 2099–2111, November 1999, www.plantcell.org © 1999 American Society of Plant Physiologists
Pronounced Intraspecific Haplotype Divergence at the
RPP5
Complex Disease Resistance Locus of Arabidopsis
Laurent Noël,
1,2
Tracey L. Moores,
1
Erik A. van der Biezen,
1
Martin Parniske, Michael J. Daniels,
Jane E. Parker, and Jonathan D. G. Jones
3
Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom
In Arabidopsis ecotype Landsberg
erecta
(L
er
),
RPP5
confers resistance to the pathogen
Peronospora parasitica.
RPP5
is part of a clustered multigene family encoding nucleotide binding–leucine-rich repeat (LRR) proteins. We compared 95
kb of DNA sequence carrying the L
er
RPP5
haplotype with the corresponding 90 kb of Arabidopsis ecotype Columbia
(Col-0). Relative to the remainder of the genome, the L
er
and Col-0
RPP5
haplotypes exhibit remarkable intraspecific
polymorphism. The
RPP5
gene family probably evolved by extensive recombination between LRRs from an
RPP5
-like
progenitor that carried only eight LRRs. Most members have variable LRR configurations and encode different num-
bers of LRRs. Although many members carry retroelement insertions or frameshift mutations, codon usage analysis
suggests that regions of the genes have been subject to purifying or diversifying selection, indicating that these genes
were, or are, functional. The
RPP5
haplotypes thus carry dynamic gene clusters with the potential to adapt rapidly to
novel pathogen variants by gene duplication and modification of recognition capacity. We propose that the extremely
high level of polymorphism at this complex resistance locus is maintained by frequency-dependent selection.
INTRODUCTION
Plant resistance to animal, fungal, bacterial, and viral pathogens
often is governed by gene-for-gene interactions between
host resistance (
R
) genes and corresponding pathogen avir-
ulence (
Avr
) genes (Crute, 1994). In the absence of matching
R
genes,
Avr
gene products presumably enhance pathogen
virulence (Collmer, 1998). Different pathogen strains carry
different arrays of
Avr
genes (e.g., Holub and Beynon, 1996),
which may indicate some functional redundancy. In crop
monocultures,
R
genes impose strong selection on patho-
gen
Avr
genes for mutations to virulence; as a consequence,
plant breeders must continuously recruit new
R
genes from
wild relatives. In natural populations that are genetically and
spatially more diverse, the evolutionary forces at work are
less clear. However, the population dynamics of all plant–
pathogen systems are strongly influenced by genetic varia-
tion in resistance and virulence determinants (Crute, 1994;
Simms, 1996). Parallels have been proposed between the
evolution of plant
R
genes and of genes in the major histo-
compatibility complex (MHC) of vertebrates (Dangl, 1992;
Michelmore and Meyers, 1998). It is of major interest, there-
fore, to understand the molecular basis for the evolution of
host–pathogen specificity and to characterize intraspecific
natural variation at
R
gene haplotypes. Interspecific compar-
isons have been reported elsewhere (Parniske et al., 1997;
Thomas et al., 1998).
The capacity to adapt rapidly to a broad spectrum of
novel pathogen variants imposes a need for a mechanism
that generates new
R
gene alleles. Many
R
genes are mem-
bers of gene families residing at complex loci that can carry
several distinct pathogen recognition specificities (Hammond-
Kosack and Jones, 1997).
R
genes, therefore, probably
evolve novel Avr recognition capacities through gene dupli-
cation, diversification, and subsequent selection. Most
cloned
R
genes encode a leucine-rich repeat (LRR) domain
(Staskawicz et al., 1995; Hammond-Kosack and Jones,
1997), a motif that has been associated with protein–protein
interactions (Kobe and Deisenhofer, 1994). The LRRs in
R
gene products, therefore, are envisaged to specify pathogen
recognition by direct or indirect interaction with Avr mole-
cules (Jones and Jones, 1996). Indeed, in the porcine ribo-
nuclease inhibitor protein, the solvent-exposed residues of a
parallel
b
sheet form a surface for ligand interactions (Kobe
and Deisenhofer, 1995). In several
R
gene products, the pre-
dicted solvent-exposed residues in the
b
-sheet regions are
hypervariable and correlate with differential pathogen recog-
nition (Parniske et al., 1997; Thomas et al., 1997; Botella et
al., 1998; Dixon et al., 1998; McDowell et al., 1998; Meyers
et al., 1998b; Ellis et al., 1999).
Variability in the solvent-exposed LRR residues probably
1
These authors contributed equally to this work.
2
Current address: Institute of Genetics, Martin-Luther-Universität,
Weinbergweg 22, D-06120 Halle, Germany.
3
To whom correspondence should be addressed. E-mail jonathan.
jones@bbsrc.ac.uk; fax 1603-250024.
2100 The Plant Cell
is accomplished through accumulation of and selection for
point mutations. The excess of nonsynonymous substitu-
tions over synonymous substitutions within codons encod-
ing the predicted ligand-interacting LRR residues indicates
that diversifying selection acts on these amino acids
(Michelmore and Meyers, 1998). The same criterion was
used to infer diversifying selection in the peptide binding re-
gion of the MHC class I genes (Hughes and Yeager, 1998).
In contrast, purifying selection appears to act on regions en-
coding the presumed signal effector portion of the
R
gene
product (Parniske et al., 1997; Wang et al., 1998; Botella et
al., 1998; McDowell et al., 1998; Meyers et al., 1998b). The
meiotic instability and the generation of novel
R
gene speci-
ficities at some
R
gene loci suggested that unequal recom-
bination and gene conversion could also contribute to
diversity (Ellis et al., 1997; Hulbert, 1997). This conclusion
was supported recently by comparative sequence analysis
of several
R
genes (Parniske et al., 1997; Botella et al., 1998;
McDowell et al., 1998; Meyers et al., 1998b; Ellis et al., 1999).
The small crucifer Arabidopsis is a model for plant molec-
ular genetics, and its genome sequence is being deter-
mined. The biotrophic oomycete pathogen
Peronospora
parasitica
naturally infects and completes its life cycle in
Arabidopsis (Holub and Beynon, 1996). In Arabidopsis
ecotype Landsberg
erecta
(L
er
), a single gene,
RPP5
, on
chromosome 4 (Parker et al., 1997) confers resistance to
strain Noco2 of
P. parasitica.
In contrast, resistance to the
same strain in ecotype Wassilewskija is conferred by the
RPP1
locus, which is located on chromosome 3.
RPP1
con-
sists of at least three very similar genes, each probably rec-
ognizing a different
Avr
gene product (Botella et al., 1998).
The
RPP5
and
RPP1
genes are members of the largest class
of plant
R
genes that encode nucleotide binding (NB) sites
and LRR domains (NB-LRR proteins; Staskawicz et al.,
1995; Hammond-Kosack and Jones, 1997). Both
RPP5
and
RPP1
are further grouped into a TIR-NB-LRR subclass
based on their N termini, which possess similarity to the cy-
toplasmic effector domains of the Drosophila and human
Toll and interleukin-1 receptors (TIR domain, for Toll, inter-
leukin-1 receptor, and R protein; Parker et al., 1997; Botella
et al., 1998). The central part of all NB-LRR proteins com-
prises three motifs predicted to constitute an NB pocket and
several short motifs without known function (Hammond-
Kosack and Jones, 1997). This region is shared with the ani-
mal apoptosis regulatory proteins CED-4 and Apaf-1 and
has been designated the NB-ARC domain (ARC, for Apaf-1,
R protein, and CED-4; Van der Biezen and Jones, 1998a),
and also the Ap-ATPase domain (Aravind et al., 1999). NB-
LRR proteins have a C-terminal LRR domain that includes
21 LRRs in RPP5 (Parker et al., 1997) and either nine or 10
LRRs in the RPP1 proteins (Botella et al., 1998).
DNA gel blot analysis showed that all analyzed Arabidop-
sis ecotypes contain multiple, highly polymorphic,
RPP5
-
and
RPP1
-related sequences (Parker et al., 1997; Botella et
al., 1998). Therefore, it is of interest to study the evolutionary
forces that generate this striking level of intraspecific poly-
morphism between complex disease resistance haplotypes.
We determined the DNA sequence of the entire L
er
RPP5
haplotype containing 10 homologs and compared it with
that of the ecotype Columbia (Col-0), which contains eight
homologs (Bevan et al., 1998). This analysis showed that the
two
RPP5
gene clusters have diverged to an extraordinary
degree compared with most other L
er
and Col-0 loci. The
RPP5
family members have different numbers of LRRs rang-
ing from 13 to 23, and they could have evolved from a pro-
genitor gene with only eight LRRs. In addition, the predicted
ligand-interacting LRR residues are hypervariable and ap-
pear to be subject to diversifying selection, indicating that
the
RPP5
family members function or functioned in recogni-
tion of different pathogen Avr products. Gene conversion,
point mutation, retrotransposition, and unequal recombina-
tion all contributed to the high mutation rate responsible for
the divergence of the
RPP5
haplotypes. This intraspecific
DNA sequence comparison of two complex disease resis-
tance haplotypes reveals that pathogens exert strong selec-
tive pressure for enhanced polymorphism at
R
gene loci.
Possible mechanisms for this selection are discussed.
RESULTS
Structural Divergence of the L
er
and Col-0
RPP5
Haplotypes
Analysis of the 95 kb of DNA sequence of the L
er
RPP5
hap-
lotype revealed the presence of 10
RPP5
homologs:
La-A
(
RPP5
) to
La-J
(Figure 1). Apart from a truncated member at
the telomeric end (
La-J
), all other homologs are oriented in
the same direction. In addition, sequence analysis identified
three
Ty1
/
Copia
–like retroelements (Bennetzen, 1996). Fur-
thermore, one complete copy and one truncated copy of a
mitochondrial 5S rRNA gene and eight truncated copies of a
serine/threonine protein kinase gene are present (Figure 1).
By contrast, the 90-kb sequence of the
RPP5
haplotype in
Col-0, which is susceptible to infection by
P. parasitica
Noco2, consists of eight homologs (
Col-A
to
Col-H
); most of
these also are oriented in the same direction (Bevan et al.,
1998; Figure 1). As in L
er
, the Col-0
RPP5
haplotype con-
tains eight truncated copies of the same protein kinase
pseudogene. It also contains a
Ty1
/
Copia
–like and a
Ty3
/
Gypsy
–like retroelement inserted in two
RPP5
homologs.
The
Ty1
/
Copia
–like retroelements inserted in
La-D
and
La-G
are identical; the retroelements in
La-H
and
Col-D
differ from
those in
La-D
and
La-G
, and from each other.
Structural comparison of the L
er
and Col-0 haplotypes
shows that the regions flanking the gene clusters are almost
identical, including
La-J
and
Col-H
, which are two truncated
homologs (Figure 1). However, colinearity is almost com-
pletely absent within these boundaries. First, the relative lo-
cation and number of homologs within each haplotype differ
markedly, accounting for the high level of polymorphism re-
Evolution of the
RPP5
Resistance Gene Family 2101
vealed by DNA gel blot analysis (Parker et al., 1997). Sec-
ond, the position in the cluster of a particular L
er
homolog
does not correspond to the position of the most closely re-
lated member in the Col-0 haplotype (and vice versa). For
example,
La-B
is most similar to
Col-C
(96% identity). Third,
aside from the presence of truncated protein kinase se-
quences, limited sequence similarity exists in the intergenic
regions. For example, no mitochondrial 5S rRNA gene is
present in the Col-0 region. The extensive sequence differ-
ences between the L
er
and Col-0
RPP5
gene clusters are in
remarkable contrast with the generally observed low level of
polymorphisms between these ecotypes (e.g., Bergelson
et al., 1998). Therefore, we conclude that since the evolu-
tionary separation of the ecotypes, the L
er
and Col-0
RPP5
haplotypes have diversified extensively, whereas little diver-
gence has occurred at the vast majority of other loci.
RPP5
Family Members Are Expressed but Have
Disrupted Reading Frames
All homologs, including promoters, introns, and 3
9
regions,
have a similar overall structure. Three members have an
eighth exon, including
RPP5
,
Col-A
, and
Col-F.
However,
deletion of this last short exon does not compromise
RPP5
function (Parker et al., 1997) and therefore it may not be re-
quired for the other members. The most variable region en-
codes the LRRs, and this region often differs by multiple
duplications and deletions (see next three sections). The nu-
cleotide sequence identity between pairs of genes ranges
from 74% for
Col-B
/
Col-G
to 99% for
La-J
/
Col-H. Distance
analysis does not preferentially group family members from
one haplotype (data not shown). Thus, family members
within one haplotype (paralogs) are not more similar to each
other than they are to those from the other haplotype
(orthologs). Phylogenetic analysis of the RPP5 family con-
ducted with full-length gene sequences, however, provides
limited information, because considerable sequence ex-
change has occurred between homologs (see RPP5 Family
Members Are Mosaics of Shared Gene Segments, below).
Apart from RPP5 itself, none of the other nine Ler ho-
mologs is predicted to encode full-length open reading
frames (ORFs). At the Col-0 haplotype, only two homologs
(Col-B and Col-F) encode intact ORFs (Figure 1). Five mem-
bers are severely truncated (La-E, La-F, La-H, La-J, and Col-H).
One member (La-D) contains duplicated exon 2 and exon 3
sequences, possibly resulting from the insertion of the retro-
element present at this location. However, most other ho-
mologs appear to be full length and carry only one ORF-
disrupting mutation (Figure 1): two members carry a retro-
element insertion (La-G and Col-A), and five members con-
tain a single point mutation (La-C, La-I, Col-D, Col-E, and
Col-G). The single mutations in Col-A, Col-D, and Col-G re-
side close to the 39 end of the gene, beyond the region en-
coding the LRRs. Because a 39 truncated RPP5 transgene is
still functional (Parker et al., 1997), it is possible that these
three homologs also encode functional proteins. La-B and
Col-C carry three point mutations, and the latter also carries
a retroelement insertion.
RNA gel blot analysis of the expression of the RPP5 ho-
mologs in healthy Ler and Col-0 leaves revealed multiple
mRNA transcripts (Figure 2). The RPP5 transcript is 4.2 kb in
length and is not abundantly expressed. A smaller (1.9 kb)
and more abundant transcript in Ler is encoded by La-G,
and its corresponding cDNA was previously designated SFH
(for sequence from homolog; Parker et al., 1997). The trun-
cated transcript’s presence and size show remarkable re-
semblance to transcripts resulting from alternative splicing
of the structurally related R genes N from tobacco and L6
from flax (Whitham et al., 1994; Ellis et al., 1997; Parker et
al., 1997). The 39 end of the corresponding cDNA consists of
sequences belonging to the retroelement inserted in La-G.
Several other rare RPP5-related transcripts are present in
Ler, but their corresponding genes have not been defined. In
Col-0, transcripts of z4.5 kb, slightly longer than that from
RPP5, are present. Of the six Col-0 expressed sequence
Figure 1. Representation of the DNA Sequence of the Ler and Col-0 RPP5 Haplotypes.
The RPP5 homologs in Ler and Col-0 are named from A to J and are shown as boxes, with the direction of transcription indicated by arrow-
heads. The hatched areas at the centromeric (left) and telomeric (right) ends indicate almost completely (96%) identical sequences. Asterisks in-
dicate the positions of the open reading frame (ORF)–disrupting point mutations. Retroelement insertions are shown as black arrows (Ty1/Copia
class) and as a hatched arrow (Ty3/Gypsy class). Similarity to a serine/threonine protein kinase pseudogene is shown as filled triangles; the two
open triangles indicate sequences with similarity to a mitochondrial 5S rRNA gene.
2102 The Plant Cell
tags present in the Arabidopsis AtEST database (September
1999), five correspond to Col-F (GenBank accession num-
bers AA86077, T41662, T03992, Z46716, and Z46715), and
one corresponds to Col-C (N96078), indicating that at least
these members are expressed.
RPP5 Family Members Are Mosaics of Shared
Gene Segments
For determination of sequence relationships, the RPP5 fam-
ily members were fingerprinted by analysis of informative
polymorphic sites (IPSs). IPSs are characteristic nucleotide
substitutions shared between homologous sequences and
distinguish these from other homologous sequences
(Parniske et al., 1997). Sequence affiliations were identified
when three or more consecutive IPSs are contained within
homologous segments. For example, in exon 1 of Col-F, all
IPSs are shared with those of RPP5 (Figure 3), except be-
tween base pairs 234 and 243 in which four consecutive
IPSs are shared with exon 1 of several other homologs (La-D,
La-I, Col-A, Col-E, and Col-G). Overall, this analysis showed
an extensive patchwork distribution of nucleotide polymor-
phisms between most family members (Figure 3), as also
was inferred in a previous interspecific haplotype compari-
son at the Cladosporium fulvum Cf-4/Cf-9 locus (Parniske et
al., 1997). The IPS analysis clearly revealed traces of un-
equal recombination and/or gene conversion events against
a background of homolog-specific polymorphisms. Three
pairs of family members are almost identical in sequence:
Col-E/Col-G (91%), La-B/Col-C (96%), and La-J/Col-H
(99%).
From the number of distinct gene segments shared be-
tween the RPP5 haplotypes, the minimum number of family
members present before the separation of Ler and Col-0
can be deduced. For example, five different exon 1 gene
segments are shared between the Ler and Col-0 haplo-
types, suggesting that in the ancestral haplotype, the RPP5
gene family consisted of at least five members. After the
separation of the ecotypes, subsequent independent se-
quence exchanges between paralogs or with other RPP5
haplotypes presumably contributed to further divergence of
the gene clusters.
Figure 2. Transcript Analysis of RPP5 Family Members in Ler and
Col-0.
Poly(A)1 RNA gel blot analysis was conducted. Transcripts encoded
by RPP5 and La-G are indicated, with approximate transcript sizes
indicated in kilobases.
Figure 3. Sequence Exchange among the RPP5 Multigene Family.
Gene structures are represented with exons as wide rectangles; pro-
moters and introns are shown as narrow rectangles. Sequence affili-
ations were inferred from runs of at least three consecutive IPSs,
with each shown in a different color. Asterisks indicate the position
of the ORF-disrupting mutations, and the black arrowheads indicate
the locations of the retroelement insertions. Deletions within the
genes are indicated by open triangles. The spaces in some ho-
mologs are created because other homologs carry duplications in
that region. The numbers of LRRs in the homologs are shown at the
right.
Evolution of the RPP5 Resistance Gene Family 2103
RPP5 Family Members Evolve through Shuffling of LRRs
RPP5 encodes a predicted protein with 21 LRRs. These
LRRs can be classified into eight groups (A to H) and consist
of duplicated LRR-encoding segments (Parker et al., 1997).
These duplications most likely result from successive un-
equal intragenic recombination events; those involving type
B LRRs generated additional introns, which are contained in
these gene segments. Experimental evidence for intragenic
duplication within the LRR region was provided by plant FL-
387 (Parker et al., 1997). This plant was identified in a fast
neutron–mutagenized Ler population and carries two un-
linked mutations, one in PAD4 (for PHYTOALEXIN DEFI-
CIENT 4; necessary for camalexin synthesis; Zhou et al.,
1998; J.E. Parker, unpublished data) and one in RPP5. This
RPP5-1 allele contains a 270-bp duplication encoding four
LRRs, resulting in a predicted protein with 25 LRRs (Figure
4). Genetic separation from the pad4 mutation showed that
the RPP5-1 allele fully retained its function in conferring re-
sistance to P. parasitica Noco2.
The sequence analysis of the RPP5 family members in
Col-0 and Ler suggests a model for RPP5 homolog evolu-
tion. All homologs carry similar LRR duplications (or rem-
nants thereof), as described for RPP5. However, Col-D and
Col-F are the only members with 21 LRRs arranged identi-
cally to RPP5. Most members lack the duplication of four
LRRs that generated exon 4 in RPP5 (e.g., Col-B and La-C),
and some members also lack the duplication of four LRRs
that gave rise to exon 6 (e.g., Col-C and La-B) in RPP5 and
other members (Figure 3). Thus, the RPP5 family can be in-
terpreted as having evolved from ancestors with 13, 17, or
21 LRRs, which themselves probably were derived from a
single RPP5-like gene that lacked the internal duplications
and had only eight LRRs (Figure 4). Furthermore, many
members carry additional and mostly unique LRR duplica-
tions and deletions. For example, Col-B and Col-E probably
were derived from an RPP5 ancestor with 17 LRRs, but they
have both undergone subsequent independent rearrange-
ments generating 23 and 14 LRRs, respectively (Figure 4).
The different number of introns in the LRR regions correlates
with the number of type B LRRs undergoing duplications or
deletions.
Distance Analysis of Putative Structural and Signaling
Domains within the RPP5 Family
Comparison of the predicted proteins of the 12 nontruncated
members (RPP5, La-B, La-C, La-G, La-I, and Col-A to Col-G)
revealed considerable sequence conservation (Figures 5A
Figure 4. Model for the Evolutionary History of the RPP5 Multigene Family.
The RPP5 homologs are shown as rectangles, with the TIR domains (left, N-terminal) and LRR domains (right, C-terminal) as open rectangles,
and the central NB-ARC domains as black rectangles. Open arrowheads indicate intron positions. All individual LRRs among the entire RPP5
family can be classified into eight different groups (A to H). These LRRs are thought to be derived from an RPP5 ancestor (top center) with eight
progenitor LRRs (shown in red). A series of intragenic duplications may have led to an increase of these ancestral LRRs from eight to 13 LRRs
(curved arrow and duplication in blue), to 17 LRRs (curved arrow and duplication in black), to 21 LRRs (curved arrow and duplication in green).
The Ler RPP5 gene and the Col-0 homologs Col-F and Col-D have an identical LRR configuration with 21 LRRs. Most family members (most not
shown), however, lack some of the ancestral duplications but carry other duplications and deletions in the LRR-encoded region; for example,
Col-B carries two duplications (gray and yellow), and Col-A has undergone a deletion event (D). In Ler, the functional RPP5-1 allele with 25 LRRs
was recovered (curved arrow and duplication in pink).
2104 The Plant Cell
and 6). Most homologs have highly related exon 1–encoded
TIR domains (Figure 5B), which is consistent with the TIR
domain’s predicted role as part of the effector portion of the
protein (Whitham et al., 1994; Staskawicz et al., 1995). The
exon 2–encoded NB-ARC (or Ap-ATPase) domain is homol-
ogous to the animal apoptosis proteins Apaf-1 and CED-4
and includes an NB site and several short conserved motifs
with unknown function (Van der Biezen and Jones, 1998a;
Aravind et al., 1999). Distance analysis shows extreme con-
servation of the predicted NB site (Figure 5C), which is con-
sistent with the presence of a preserved ATP or GTP binding
pocket in all NB-LRR proteins. The two similar members,
La-B and Col-C, diverge significantly from the NB site con-
sensus (Figure 5C). There appear to be three distinct domain
classes within the second half of exon 2, designated the
ARC domain (Figure 5D). One conserved ARC class is repre-
sented by RPP5 and five other members. A second ARC
class is represented by the only two Col-0 members, Col-B
and Col-F, that have intact ORFs; this class also includes
the diverged pair La-B and Col-C. A third ARC type is
present in the two highly similar and diverged homologs,
Col-E and Col-G (Figure 5D). The three ARC domain classes
within the RPP5 gene possibly reflect functional differences,
such as interaction with different effector proteins.
Solvent-Exposed Residues of LRRs Are Hypervariable
In RPP5, as in many NB-LRR proteins, the canonical LRRs
consist of 23 or 24 residues, of which z19 either are part of
a predicted a-helical region connecting adjacent LRRs or
are hydrophobic residues (mostly leucine residues) buried in
the hydrophobic core (Jones and Jones, 1996). Each LRR
also carries a short b-strand/b-turn region with the xxLxLxx
motif in which the five interstitial residues (x) are predicted to
be solvent exposed. Collectively, the b-strand/b-turn regions
are predicted to form a parallel b sheet, which could create
a surface for ligand interactions (Kobe and Deisenhofer,
1995). Comparisons between RPP5 family members showed
high conservation of most of the LRR residues, which con-
forms to the prediction that they serve a structural role.
However, extensive amino acid variation occurs in the pre-
dicted solvent-exposed residues in the xxLxLxx motif. For
example, comparison of RPP5 with Col-F (both of which
possess 21 LRRs) showed that 49% (50 of 103) of these
LRR residues are different, whereas only 11% (41 of 377) of
different residues are present in the remainder of the LRRs.
We then determined which residues within the entire family
are hypervariable (greater than or equal to four different resi-
dues among the compared members; Figure 6). This analysis
revealed that within the RPP5 family, 23% (24 of 103) of the
predicted solvent-exposed LRR residues are hypervariable
as opposed to 0.8% (3 of 377) in the remaining structural
LRR region. In several R proteins, hypervariability of solvent-
exposed residues correlates with differential pathogen rec-
ognition (Parniske et al., 1997; Thomas et al., 1997; Botella
et al., 1998; Dixon et al., 1998; Meyers et al., 1998b).
Positive Selection of Ligand-Interacting Residues in
the LRRs
To examine further the idea that RPP5 family members func-
tion or functioned in differential pathogen recognition, we
Figure 5. Dendrograms Showing Distance Relationships between
Conceptual Protein Sequences of RPP5 Family Members from the
Ler and Col-0 Haplotypes.
Sequence distance trees were calculated using the neighbor-joining
algorithm. Relative branch lengths (0.05) are indicated by bars belo
w
the trees. La, Ler; Lu, Linum usitatissimum; Nt, Nicotiana tabacum;
Ws, Arabidopsis thaliana land race Ws-0.
(A) Distance tree of entire gene products of the RPP5 family and
other R proteins containing N-terminal TIR domains.
(B) Distance tree of TIR domains (residues 1 to 160 in RPP5) of the
RPP5 family members and other R proteins.
(C) Distance tree of NB sites (residues 202 to 333 in RPP5) of the
RPP5 family members.
(D) Distance tree of ARC domains (residues 334 to 518 in RPP5) of
the RPP5 family members.
Evolution of the RPP5 Resistance Gene Family 2105
subjected predicted functional domains to pairwise analysis
for substitutions at synonymous (Ks) and nonsynonymous
(Ka; Table 1) sites. Synonymous substitutions have no ap-
parent selective advantage or disadvantage; therefore, the
Ks values give an estimate for the background (random) sub-
stitution rate. However, if in a certain region more nonsynon-
ymous than synonymous substitutions are found (i.e., Ka/Ks .
1), it provides solid evidence that these changes have been
positively selected for (Parniske et al., 1997; Hughes and
Yeager, 1998; Meyers et al., 1998b).
The exon 1–encoded TIR domains show low Ka/Ks ratios
(on average Ka/Ks 5 0.6), which is consistent with conserva-
tion of a proposed effector function. Low Ka/Ks ratios also
were calculated for the regions encoding the NB site (on av-
erage, Ka/Ks 5 0.6). The only significant deviation from this
average is observed in the two members (La-B and Col-C)
that possess more than one ORF-disrupting mutation.
These homologs have a Ka/Ks ratio of 1.1, indicating that no
selection pressure is acting on these members and classify-
ing La-B and Col-C as pseudogenes. Consistent with this
finding is the observation that these two highly similar genes
diverge significantly from the rest of the family (see Distance
Analysis of Putative Structural and Signaling Domains within
the RPP5 Family; Figure 5). Therefore, these pseudogenes
serve as an internal control for the Ka/Ks analysis and rein-
force the suggestion that the other members have been
functional in the recent evolutionary past. Low Ka/Ks ratios
also are observed for the LRR residues predicted to serve a
structural role (on average Ka/Ks 5 0.6; Table 1). The low Ka/Ks
values calculated for the TIR domain, the NB site, and the
structural LRR residues are consistent with the predicted
structural and functional constraints of these domains and
indicate that purifying selection operated such that the ma-
jority of amino acid substitutions are not tolerated.
In the region encoding the hypervariable solvent-exposed
LRR residues, the Ka rate substantially exceeds the Ks rate,
resulting in Ka/Ks ratios that average 2.8 (omitting the
pseudogenes La-B and Col-C; Table 1). The generally high
Ka/Ks ratios in this region suggest that diversifying selection
acted on the predicted ligand-interacting LRR residues.
Similar conclusions were drawn from Ka/Ks analyses of other
R genes (Parniske et al., 1997; Wang et al., 1998; Botella et
al., 1998; McDowell et al., 1998; Meyers et al., 1998b).
Stability of the Ler RPP5 Haplotype
To investigate the stability of the Ler RPP5 haplotype and
the basis for the mutations in the Ler homologs, we ana-
lyzed the La-0 ecotype, the progenitor of Ler used in this
Figure 6. Conserved and Hypervariable Amino Acids among Mem-
bers of the RPP5 Multigene Family.
The RPP5 protein sequence and its predicted TIR, NB-ARC, and
LRR structures are shown as given in Parker et al. (1997). Intron–
exon boundaries are indicated by diamonds, and exon numbers are
indicated at the right. Predicted proteins of 12 full-length family
members are aligned, including those of RPP5, La-B, La-C, La-G,
La-I, and Col-A to Col-G. Residues shown in lowercase letters either
are part of highly variable regions and cannot be aligned or, like the
residues that constitute exon 8, are absent from most members.
Conserved amino acids (black) are defined as at most one different
residue among the compared members. Hypervariable amino acids
(red) are defined as four or more different types of residues among
the compared members. Other amino acids are moderately variable
(two or three different types of residues among the compared mem-
bers) and are shown in blue. Because LRR-encoded regions were
missing from some of the members, limited sequence comparison of
LRRs 5 to 8 (five members) and LRRs 16 to 19 (four members) was
possible. As a consequence, the hypervariable residues in these
LRRs are possibly underestimated. Conserved kinase motifs that
constitute an NB pocket and conserved hydrophobic residues within the LRRs are shown in boldface letters. Predicted solvent-exposed
residues (x in the xxLxLxx motif) are shown between vertical lines.
2106 The Plant Cell
study. DNA gel blots showed identical RPP5-hybridizing
fragments in La-0 as is in Ler (data not shown). All poly-
merase chain reaction (PCR) primers designed to detect the
retroelement insertions unique to the Ler haplotype ampli-
fied an identical product from the wild-type La-0 progenitor.
In addition, the sequences of PCR-amplified fragments from
the Ler homologs show point mutations in La-0 identical to
those in Ler (data not shown). This analysis indicates that
the loss of function of the Ler members is not an artifact of
recent domestication through propagation in greenhouses in
the absence of selection for P. parasitica resistance.
To analyze further the genetic stability of the RPP5 haplo-
type, we made an outcross of male sterile Ler to Col-0 and
tested the progeny for susceptibility to P. parasitica Noco2.
Among z7500 heterozygous progeny, no susceptible indi-
viduals were recovered, again suggesting that this locus is
not highly meiotically unstable when homozygous. This ex-
periment would have detected high instability (as at the
maize Rp1-G disease resistance haplotype) but not low in-
stability (as at the maize Rp1-D haplotype; Hulbert, 1997).
DISCUSSION
Intraspecific Haplotype Divergence at a Complex
Disease Resistance Locus
Many different R genes have been isolated (Hammond-
Kosack and Jones, 1997), and several R gene haplotypes
have been compared. Interspecific comparisons of complex
R gene haplotypes have been reported (Parniske et al.,
1997; Thomas et al., 1997, 1998; Dixon et al., 1998), and in-
traspecific comparisons have been conducted with simple
loci that contain only one R gene homolog (Grant et al.,
1998; McDowell et al., 1998; Caicedo et al., 1999; Ellis et al.,
1999; Henk et al., 1999). The unique availability of the com-
plete DNA sequence of the Ler and Col-0 RPP5 haplotypes
permits analysis of intraspecific variation at a complex R
gene locus and a comparison of R gene locus polymor-
phism to the level of polymorphism at other loci.
The clearest conclusion from the Ler and Col-0 RPP5
haplotype comparisons is that this locus exhibits an extraor-
dinary degree of intraspecific polymorphism. Variation was
observed in the number and relative location of homologs, in
the presence and location of retroelements, and in other
DNA sequences (such as the 5S rDNA sequences) as well as
in numerous point mutations. This pronounced lack of syn-
teny over z75 kb of sequence may have been the source of
the significant recombination suppression noted in this re-
gion during the map-based cloning of RPP5 (Parker et al.,
1997). The meiotic stability of the homozygous Ler RPP5
haplotype is consistent with the results of crossing plants
expressing Cf9 to those expressing Cf0 to create 20,000
testcross progeny in which no spontaneous disease-sensi-
tive recombinants were recovered (Parniske et al., 1997).
However, it is in contrast with the results of crossing several
Cf gene transheterozygotes to disease-sensitive lines; in
these experiments, recombination events were detected
within the locus (Parniske et al., 1997) and between ho-
mologs (Dixon et al., 1998). These data suggest that when a
complex haplotype is homozygous, the different homologs
rarely mutate or exchange sequence information. This ap-
pears paradoxical, because R gene loci have been antici-
pated to be sites of frequent recombination that could give
rise to new recognition specificities. However, the rate of re-
combination between R haplotypes can depend on the spe-
cific haplotype combination (Hulbert, 1997). The idea that
some haplotypes pair better than others would have consid-
erable consequences for the rate of sequence exchange
Table 1. Pairwise Ka/Ks Ratios in the Predicted Solvent-Exposed LRR Residues and Structural LRR Residues among the RPP5 Familya
RPP5 La-B La-C La-G La-I Col-A Col-B Col-C Col-D Col-EbCol-F Col-Gb
RPP5 30.9 1.3 2.8 3.0 1.5 2.5 1.8 2.0 — 2.5 —
La-B 0.8 30.7 0.9 0.6 1.0 1.0 0.5 0.8 — 0.5 —
La-C 0.7 0.6 31.4 1.3 2.8 1.1 1.0 0.9 — 1.0 —
La-G 0.6 0.8 0.4 31.7 2.5 3.0 1.3 5.7 — 2.5 —
La-I 0.9 0.6 0.6 0.4 34.2 4.4 0.9 5.8 — 2.2 —
Col-A 0.6 0.6 0.6 0.3 0.8 34.7 1.8 6.6 — 4.1 —
Col-B 0.7 0.6 0.6 0.4 0.6 0.6 31.4 3.2 — 1.8 —
Col-C 1.1 0.5 0.7 0.7 0.8 0.8 0.8 31.1 — 1.2 —
Col-D 0.9 0.6 0.6 0.4 0.5 0.7 0.4 0.7 3— 1.9 —
Col-Eb— ————————3— 1.7
Col-F 0.9 0.5 0.7 0.4 0.6 0.8 0.4 0.6 1.4 — 3—
Col-Gb— ————————0.5—3
aSolvent-exposed residues are shown above the diagonal, and structural residues are shown below the diagonal.
bCol-E and Col-G are highly similar but significantly diverge in their LRR-encoded DNA sequences from the rest of the family; as a consequence,
the Ka/Ks analysis did not produce meaningful results (denoted by dashes).
Evolution of the RPP5 Resistance Gene Family 2107
within and between R loci. In Arabidopsis, which reproduces
predominantly by self-fertilization, haplotype mispairing is
likely to be rare. Occasional outcrosses with genotypes car-
rying distinct haplotypes would create greater opportunity
for mispairing to create evolutionary novelty (Hulbert, 1997;
Parniske et al., 1997).
Mechanisms of DNA Sequence Divergence in the Col-0
and Ler RPP5 Haplotypes
We propose that the progenitor of the RPP5 gene family
contained only eight LRRs (Figure 4) and was flanked by a
protein kinase gene. Gene duplication generated at least five
RPP5 homologs before the separation of the Ler and Col-0
ecotypes. A series of intragenic duplications within the re-
gion encoding the LRRs presumably produced RPP5 pro-
genitors with 13, 17, and 21 LRRs. This progenitor RPP5
multigene family diverged into dissimilar gene clusters with
eight members in Col-0 and 10 in Ler, which were inter-
spersed with fragments from the ancestral protein kinase
gene. In both haplotypes, members with 21 LRRs are
present. However, most members lack one or more ances-
tral duplications. Furthermore, some members carry addi-
tional and mostly unique deletions and duplications within
the region encoding the LRRs.
Both haplotypes carry an identical truncated copy, which
is in inverse orientation relative to the other homologs. This
curious structure could be a relic of the original duplication
event, perhaps involving aberrant behavior of the DNA repli-
cation fork. In contrast to most of the Arabidopsis genome,
the RPP5 haplotypes have undergone gross structural rear-
rangements and accumulated numerous point mutations.
This probably indicates that many of the RPP5 homologs
are under reduced selection for conservation or retention of
function. An interesting corollary of this interpretation is that
DNA sequence changes at the RPP5 locus might reflect the
mutagenic mechanisms that are constantly at work in the re-
mainder of the genome, where their consequences are more
rapidly purged by selection.
The RPP5 multigene family is composed of a limited num-
ber of homologous but distinct sequence segments distin-
guished by runs of IPSs. The shared and exchanged gene
segments most likely result from unequal recombination
and/or gene conversion. Meiotic misalignment involving dif-
ferent homologs followed by intergenic or intragenic cross-
ing-over would result in loss of one or more members on
one chromosome and duplication of these members on the
other. Intragenic crossing-over, in addition, would result in
exchanges of gene segments. Unequal recombination also
could involve mispairing of LRR-encoding regions followed
by an intragenic crossing-over. Homologs with more than
eight LRRs carry several duplications, which increase the
probability of intragenic misalignments. Such events would
generate additional duplications or deletions and probably
account for the variable number of LRRs. Unequal intragenic
recombination presumably gave rise to a novel RPP5-1 al-
lele with an internal duplication of four LRRs (Parker et al.,
1997).
The predicted solvent-exposed LRR residues are hyper-
variable in the RPP5 family (and other R proteins). Such
changes are more likely to have arisen from point mutations
than from sequence exchanges, and based on Ka/Ks ratios,
this hypervariability is due to diversifying selection. Thus, al-
though sequence exchange and LRR shuffling greatly con-
tributed to diversification of the gene family members, point
mutations in codons for the predicted solvent-exposed resi-
dues also must play a crucial role in generating novel recog-
nition specificities. DNA shuffling enhances the potential of a
point mutation to generate a useful recognition capacity, be-
cause it can be combined with other variants to create an al-
lelic series of distinct, parallel b-sheet recognition surfaces.
“Birth and Death” of RPP5 and Other Plant R Genes
It has been suggested that R genes at complex haplotypes
evolve by mechanisms similar to those at work in the verte-
brate MHC (Michelmore and Meyers, 1998). These multi-
gene families are proposed to evolve through a birth and
death process rather than through concerted evolution (Nei
et al., 1997), explaining why, when different MHC haplo-
types are compared, orthologs are usually more similar than
paralogs.
There are more complete data available for haplotype
comparisons at the MHC than for R gene loci. Comparisons
of haplotypes at two complex Cf loci suggested significant
sequence exchange between paralogs (Parniske et al.,
1997; Thomas et al., 1997, 1998; Dixon et al., 1998). Many
other R genes are also members of clustered multigene
families (Whitham et al., 1994; Anderson et al., 1997; Song
et al., 1997; Botella et al., 1998; Meyers et al., 1998a, 1998b;
Simons et al., 1998), but complete comparisons of corre-
sponding haplotypes in related species or ecotypes have
not been reported.
At the Col-0 and Ler RPP5 haplotypes, some family mem-
bers are more closely related to orthologs in the other
ecotype than to paralogs. These include La-B/Col-C, La-J/
Col-H, and possibly also RPP5/Col-A (Figures 1 and 3). The
latter two pairs of orthologs (RPP5/Col-A and La-J/Col-H)
reside at the proximal and distal borders of the cluster, re-
spectively. However, orthologous relationships are difficult
to discern between members located in the middle of the
cluster. As noted at other loci (Michelmore and Meyers,
1998), if sequence exchange between paralogs were fre-
quent, it would not be possible to detect orthologs when
two haplotypes are compared. At the RPP5 haplotypes, se-
quence exchange has been sufficiently frequent to obscure
orthology, especially in the middle of the cluster, but has not
lead to homogenization of the gene clusters. However,
orthologous relationships are easier to discern when subdo-
mains are compared (Figure 3; Meyers et al., 1998a). Given
2108 The Plant Cell
the pronounced polymorphism at R gene loci in natural pop-
ulations and the enhanced frequency of mispairing when
haplotypes are combined in trans, it is probably unhelpful to
maintain too rigid a distinction between paralogs and
orthologs, because unequal crossovers could turn one into
the other.
Selective Pressures on RPP5 Haplotypes and Gene
Family Members
The TIR domains, NB sites, and structural LRR residues
within the RPP5 family are highly conserved and show evi-
dence for purifying selection. In contrast, the predicted
ligand-interacting LRR residues are hypervariable and,
based on their Ka/Ks ratios (Table 1), are subject to diversify-
ing selection. The evidence for diversifying and purifying se-
lection strongly suggests that even the apparently mutant
RPP5 homologs are and/or were functional.
Three distinct classes of ARC domains are present within
the RPP5 family. Interestingly, a similar divergence in the
ARC domain has been observed in the Ler RPP8 gene and
its homolog in Col-0 (McDowell et al., 1998). This domain
may play a role in intramolecular signaling (Van der Biezen
and Jones, 1998b) and therefore may coevolve with the di-
verging LRR domains. In the apoptosis proteins Apaf-1 and
CED-4, the NB-ARC (or Ap-ATPase) domain functions as a
conformation-dependent protein–protein interaction module
(Srinivasula et al., 1998; Yang et al., 1998). The diverged
ARC domains in R proteins, therefore, may indicate interac-
tions with distinct proteins.
In addition to RPP5 in Ler, two family members in Col-0
have intact ORFs, and three have nearly full-length ORFs.
Most of the other 12 family members either are truncated
(five homologs) or encode ORFs that are disrupted at the 59
end (seven homologs). However, eight members have ORFs
that are only disrupted by a single mutational event (point
mutation or retroelement insertion) and have not accumu-
lated additional apparent deleterious mutations, suggesting
that these genes have been functional in the recent evolu-
tionary past. The five Col-0 homologs with intact or nearly
full-length ORFs also may be functional R genes. If so, one
of these Col-0 homologs could be the Col-0 RPP4 gene that
recognizes the P. parasitica strain Emoy2 (Tör et al., 1994).
Apparently, nonfunctional R gene homologs also might
serve a repository function and allow mutation, unequal re-
combination, and gene conversion to generate novel R gene
variants upon which diversifying selection can operate.
Disease resistance has been associated with a fitness
“cost” (Crute, 1994; Bergelson and Purrington, 1996; Simms,
1996). In the (temporary) absence of pathogen pressure, in-
dividual plants with mutated R genes therefore may have se-
lective advantages. The high proportion of mutant RPP5
family members supports the idea that some R genes can
impose an evolutionary cost. Such costs might arise if the R
gene confers some constitutive activity or if it weakly recog-
nizes an endogenous ligand. Single R genes also can have a
high proportion of mutant alleles (Grant et al., 1998; Caicedo
et al., 1999; Henk et al., 1999; Stahl et al., 1999).
Maintenance of Extreme Polymorphism at R Gene Loci
Because pathogens such as P. parasitica can readily over-
come a specific R gene by loss or mutation of the corre-
sponding Avr gene (Holub and Beynon, 1996), it is likely that
any pathogenicity function conferred by such Avr genes is
redundant. Pathogens can escape recognition through loss
of function of Avr genes, whereas novel R genes in plants
evolve through presumably rarer gain-of-function mutations.
In natural populations, if evolution to virulence exceeds evo-
lution to resistance, how do plants tolerate pathogen pres-
sure?
At least two theories have been proposed to explain the
extreme polymorphism at the MHC, which, like complex R
gene haplotypes, encodes multiple proteins that have the
capacity to detect multiple nonhost recognition determi-
nants (Hughes and Yeager, 1998). One theory maintains that
heterozygote advantage (or overdominance) is key and that
high levels of polymorphism are essential to ensure that most
individuals are heterozygous. The alternative interpretation
is frequency-dependent selection in which any advanta-
geous MHC allele could be overcome by pathogens if it
were at too high a frequency in the population; if an MHC al-
lele is rare, there is little selective pressure on the pathogen
to overcome it. In Arabidopsis, an inbreeder, the heterozy-
gote advantage or overdominance theory is ruled out be-
cause most individuals are homozygous. It is interesting to
speculate, however, that in agricultural systems, in which
there is potential for one pathogen strain to cause an epi-
demic in a genetically uniform crop, overdominance at R
gene loci (which are distributed throughout the genome)
might contribute to F1 hybrid vigor. Frequency-dependent
selection due to minority advantage provides a simple ex-
planation for the high level of natural intraspecific polymor-
phism between haplotypes at the RPP5 locus and perhaps
at other complex R gene loci. Recently, Stahl et al. (1999)
also inferred frequency-dependent selection to explain poly-
morphism at the Arabidopsis RPM1 locus.
METHODS
Construction of a Physical Cosmid Contig of the Entire
Landsberg erecta RPP5 Haplotype
Screening of an Arabidopsis thaliana ecotype Landsberg erecta (Ler)
genomic library with the C18 probe resulted in the isolation of seven
cosmids, including pCLD29L17 containing the RPP5 gene (Parker et
al., 1997). To obtain additional cosmids, we screened the abi3 yeast
artificial chromosome (YAC) library by hybridization with the RPP5
gene. This identified the YAC clone abi3-8D3 (180 kb) containing all
Evolution of the RPP5 Resistance Gene Family 2109
RPP5-hybridizing fragments observed in gel blots with genomic Ler
DNA (Parker et al., 1997). DNA of the abi3-8D3 clone was partially di-
gested with Sau3A and ligated in the SLJ755I5 cosmid vector
(www.uea.ac.uk/nrp/jic/s3d_plas.htm). Four thousand clones were
screened by hybridization with RPP5, and 30 different cosmid clones
were identified. These were assembled into physical contigs by using
inverse polymerase chain reaction (PCR) products and direct end-
sequencing, restriction digest, DNA fingerprinting, and DNA gel blot
hybridizations with RPP5-derived sequences. Finally, a contig of
eight cosmids covering z110 kb was established.
DNA Sequencing
Sequencing templates were prepared from random-sheared DNA gen-
erated by sonication, repaired with T4 DNA polymerase (Pharmacia),
and cloned into dephosphorylated SmaI-digested pUC18 plasmids
(Pharmacia). The double-stranded plasmid DNA was sequenced us-
ing the PRISM Ready Reaction Terminator Cycle Sequencing Sys-
tem (Perkin-Elmer). The reaction products were separated on an
Applied Biosystems 377 DNA sequencer (Foster City, CA), and the
data were assembled using UNIX versions of the Staden package (R.
Staden, Cambridge, UK). Single-stranded gaps were completed us-
ing specifically designed primers. Deletions in clones were generated
to establish or confirm putative contigs. Double-stranded sequenc-
ing with an average of fourfold redundancy was achieved.
Sequence Analysis
Sequences of six cosmids were assembled into a contig of 92 kb
covering the entire Ler RPP5 haplotype. The RPP5 family member
La-J was amplified by PCR from the Ler cosmid abi3-8D3-21 by us-
ing primers based on the corresponding ecotype Columbia (Col-0)
homolog Col-H. The Col-0 haplotype sequence was obtained from a
90.5-kb bacterial artificial chromosome clone IGF3D5 (Bevan et al.,
1998) and the two cosmids cc33M3 and cc16N19. Analysis and
alignments of assembled sequences were performed using Genetics
Computer Group (Madison, WI) programs. Alignments were optimized
manually. Putative splice sites were based on the sequence of RPP5 and
Col-F cDNA and reverse transcription–PCR products and confirmed
by the NetPlantGene program (www.cbs.dtu.dk/NetPlantGene.html).
Informative polymorphic sites (IPSs) were displayed using the Se-
quence Output program (B.G. Spratt, University of Sussex, Brighton,
UK). Distance trees were calculated using the neighbor-joining algo-
rithm from the Clustal X package (Thompson et al., 1997). Nonsynon-
ymous (Ka) and synonymous (Ks) substitution ratios were calculated
with NewDiverge (Genetics Computer Group) for each pair of se-
quences.
Nucleic Acid Manipulations
All DNA and RNA manipulations, including YAC and PCR analyses,
were performed essentially as described by Parker et al. (1997). The
autoradiogram of the RNA gel blot shown in Figure 2 was made from
z2 mg of poly(A)1 RNA, which was size-fractionated through a verti-
cal 1.4% agarose gel and blotted onto a Hybond N filter (Amersham),
hybridized with a radiolabeled sequence encoding exons 1 and 2 of
Col-F (NcoI-NsiI fragment, base pairs 1 to 1712) for 16 hr, stringently
washed with 0.1 3 SSC (1 3 SSC is 0.15 M NaCl and 0.015 M so-
dium citrate), 0.1% SDS at 658C, and exposed to Kodak X-Omat AR
film for 2 days with intensifying screens at 2708C.
Plant and Pathogen Handling
Arabidopsis growth and Peronospora parasitica infections were as
described previously (Parker et al., 1997).
Accession Numbers
The EMBL nucleotide sequence database accession numbers of the
Ler RPP5 gene cluster (La-A to La-I) and the La-J homolog are
AF180942 and AF180943, respectively.
ACKNOWLEDGMENTS
We thank David Baker and Patrick Bovill (Sainsbury Laboratory) for
operating the sequencing equipment and Alan Cavill (Sainsbury
Laboratory) for supportive management. Roger Innes (Indiana Uni-
versity, Bloomington) is thanked for critically reading the manuscript.
We are grateful to Ian Bancroft and Mike Bevan (John Innes Centre)
for supplying Col-0 cosmid libraries and bacterial artificial chromo-
some clones, and to Clare Lister and Caroline Dean (John Innes
Centre) for providing the Ler YAC library. Maarten Koornneef (Agri-
cultural University, Wageningen, The Netherlands) is thanked for the
gift of La-0 seeds. The determination and analysis of the sequence of
the Col-0 RPP5 haplotype were conducted under the European
Scientists Sequencing Arabidopsis program sponsored by the Euro-
pean Community. E.A.v.d.B. and M.P. were supported by fellowships
from the European Community. The Sainsbury Laboratory is sup-
ported by the Gatsby Charitable Foundation.
Received July 16, 1999; accepted September 13, 1999.
REFERENCES
Anderson, P.A., Lawrence, G.J., Morrish, B.C., Ayliffe, M.A.,
Finnegan, E.J., and Ellis, J.G. (1997). Inactivation of the flax
resistance gene M associated with loss of a repeat unit within the
leucine-rich repeat coding region. Plant Cell 9, 641–651.
Aravind, L., Dixit, V.M., and Koonin, E.V. (1999). The domains of
death: Evolution of the apoptosis machinery. Trends Biochem.
Sci. 24, 47–53.
Bennetzen, J.L. (1996). The contributions of retroelements to plant
genome organization, function and evolution. Trends Microbiol. 4,
347–353.
Bergelson, J., and Purrington, C.B. (1996). Surveying patterns in
the cost of resistance in plants. Am. Nat. 148, 536–558.
Bergelson, J., Stahl, E., Dudek, S., and Kreitman, M. (1998).
Genetic variation within and among populations of Arabidopsis
thaliana. Genetics 148, 1311–1323.
Bevan, M., et al. (1998). Analysis of 1.9 Mb of contiguous sequence
from chromosome 4 of Arabidopsis thaliana. Nature 391, 485–488.
2110 The Plant Cell
Botella, M.A., Parker, J.E., Frost, L.N., Bittner-Eddy, P.D., Beynon,
J.L., Daniels, M.J., Holub, E.B., and Jones, J.D.G. (1998). Three
genes of the Arabidopsis RPP1 complex resistance locus recog-
nize distinct Peronospora parasitica avirulence determinants.
Plant Cell 10, 1847–1860.
Caicedo, A.L., Schaal, B.A., and Kunkel, B.N. (1999). Diversity and
molecular evolution of the RPS2 resistance gene in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. USA 96, 302–306.
Collmer, A. (1998). Determinants of pathogenicity and avirulence in
plant pathogenic bacteria. Curr. Opin. Plant Biol. 1, 329–335.
Crute, I.R. (1994). Gene-for-gene recognition in plant–pathogen
interactions. Philos. Trans. R. Soc. Lond. Biol. Sci. 346, 345–349.
Dangl, J.L. (1992). The major histocompatibility complex à la carte:
Are there analogies to plant disease resistance genes on the
menu? Plant J. 2, 3–11.
Dixon, M.S., Hatzixanthis, K., Jones, D.A., Harrison, K., and
Jones, J.D.G. (1998). The tomato Cf-5 disease resistance gene
and six homologs show pronounced allelic variation in leucine-
rich repeat copy number. Plant Cell 10, 1915–1925.
Ellis, J., Lawrence, G., Ayliffe, M., Anderson, P., Collins, N.,
Finnegan, J., Frost, D., Luck, J., and Pryor, T. (1997). Advances
in the molecular genetic analysis of the flax–flax rust interaction.
Annu. Rev. Phytopathol. 35, 271–291.
Ellis, J.G., Lawrence, G.J., Luck, J.E., and Dodds, P.N. (1999).
The 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.
Grant, M.R., McDowell, J.M., Sharpes, A.G., De Torres Zabala,
M., Lydiate, D.J., and Dangl, J.L. (1998). Independent deletions
of a pathogen-resistance gene in Brassica and Arabidopsis. Proc.
Natl. Acad. Sci. USA 95, 15843–15848.
Hammond-Kosack, K.E., and Jones, J.D.G. (1997). Plant disease
resistance genes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48,
575–607.
Henk, A.D., Warren, R.F., and Innes, R.W. (1999). A new Ac-like
transposon of Arabidopsis is associated with a deletion of the
RPS5 disease resistance gene. Genetics 151, 1581–1589.
Holub, E.B., and Beynon, J.L. (1996). Symbiology of mouse ear
cress (Arabidopsis thaliana) and oomycetes. Adv. Bot. Res. 24,
227–273.
Hughes, A.L., and Yeager, M. (1998). Natural selection at major
histocompatibility complex loci of vertebrates. Annu. Rev. Genet.
32, 415–435.
Hulbert, S.H. (1997). Structure and evolution of the Rp1 complex
conferring rust resistance in maize. Annu. Rev. Phytopathol. 35,
293–310.
Jones, D.A., and Jones, J.D.G. (1996). The role of leucine-rich
repeat proteins in plant defences. Adv. Bot. Res. 24, 89–167.
Kobe, B., and Deisenhofer, J. (1994). The leucine-rich repeat: A
versatile binding motif. Trends Biochem. Sci. 19, 415–421.
Kobe, B., and Deisenhofer, J. (1995). A structural basis of the inter-
actions between leucine-rich repeats and protein ligands. Nature
374, 384–386.
McDowell, J.M., Dhandaydham, M., Long, T.A., Aarts, M.G.M.,
Goff, S., Holub, E.B., and Dangl, J.L. (1998). Intragenic recombi-
nation and diversifying selection contribute to the evolution of
downy mildew resistance at the RPP8 locus of Arabidopsis. Plant
Cell 10, 1861–1887.
Meyers, B.C., Chin, D.B., Shen, K.A., Sivaramakrishnan, S.,
Lavelle, D.O., Zhang, Z., and Michelmore, R.W. (1998a). The
major resistance gene cluster in lettuce is highly duplicated and
spans several megabases. Plant Cell 10, 1817–1832.
Meyers, B.C., Shen, K.A., Rohani, P., Gaut, B.S., and Michelmore,
R.W. (1998b). Receptor-like genes in the major resistance locus of
lettuce are subject to divergent selection. Plant Cell 10, 1833–
1846.
Michelmore, R.W., and Meyers, B.C. (1998). Clusters of resistance
genes in plants evolve by divergent selection and a birth-and-
death process. Genome Res. 8, 1113–1130.
Nei, M., Gu, X., and Sitnikova, T. (1997). Evolution by the birth-and-
death process in multigene families of the vertebrate immune sys-
tem. Proc. Natl. Acad. Sci. USA 94, 7799–7806.
Parker, J.E., Coleman, M.J., Szabò, V., Frost, L.N., Schmidt, R.,
Van der Biezen, E.A., Moores, T., Dean, C., Daniels, M.J., and
Jones, J.D.G. (1997). The Arabidopsis downy mildew resistance
gene RPP5 shares similarity to the Toll and interleukin-1 receptors
with N and L6. Plant Cell 9, 879–894.
Parniske, M., Hammond-Kosack, K.E., Golstein, C., Thomas,
C.M., Jones, D.A., Harrison, K., Wulff, B.B.H., and Jones,
J.D.G. (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.
Simms, E.L. (1996). The evolutionary genetics of plant–pathogen
systems. Bioscience 46, 136–145.
Simons, G., et al. (1998). Dissection of the Fusarium I2 gene cluster
in tomato reveals six homologs and one active gene copy. Plant
Cell 10, 1055–1068.
Song, W.-Y., Pi, L.-Y., Wang, G.-L., Gardner, J., Holsten, T., and
Ronald, P.C. (1997). Evolution of the rice Xa21 disease resistance
gene family. Plant Cell 9, 1279–1287.
Srinivasula, S.M., Ahmad, M., Fernandes-Alnemri, T., and
Alnemri, E.S. (1998). Autoactivation of procaspase-9 by Apaf-1–
mediated oligomerization. Mol. Cell 1, 949–957.
Stahl, E.A., Dwyer, G., Maurico, R., Kreitman, M., and Bergelson,
J. (1999). Dynamics of disease resistance polymorphism at the
Rpm1 locus of Arabidopsis. Nature 400, 667–671.
Staskawicz, B.J., Ausubel, F.M., Baker, B.J., Ellis, J.G., and
Jones, J.D.G. (1995). Molecular genetics of plant disease resis-
tance. Science 268, 661–667.
Thomas, C.M., Jones, D.A., Parniske, M., Harrison, K., Balint-
Kurti, P.J., Hatzixanthis, K., and Jones, J.D.G. (1997). Charac-
terization of the tomato Cf-4 gene for resistance to Cladosporium
fulvum identifies sequences that determine recognitional specific-
ity in Cf-4 and Cf-9. Plant Cell 9, 2209–2224.
Thomas, C.M., Dixon, M.S., Parniske, M., Golstein, C., and
Jones, J.D.G. (1998). Genetic and molecular analysis of tomato
Cf genes for resistance to Cladosporium fulvum. Philos. Trans. R.
Soc. Lond. Biol. Sci. 353, 1413–1424.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and
Higgens, D.G. (1997). The CLUSTAL_X windows interface: Flexi-
ble strategies for multiple sequence alignment aided by quality
analysis tools. Nucleic Acids Res. 25, 4876–4882.
Evolution of the RPP5 Resistance Gene Family 2111
Tör, M., Holub, E.B., Brose, E., Musker, R., Gunn, N., Can, C.,
Crute, I.R., and Beynon, J.L. (1994). Map positions of three loci
in Arabidopsis thaliana associated with isolate-specific recogni-
tion of Peronospora parasitica (downy mildew). Mol. Plant-
Microbe Interact. 7, 214–222.
Van der Biezen, E.A., and Jones, J.D.G. (1998a). The NB-ARC
domain: A novel signaling motif shared by plant resistance gene
products and regulators of cell death in animals. Curr. Biol. 8,
R226–R227.
Van der Biezen, E.A., and Jones, J.D.G. (1998b). Plant disease
resistance proteins and the gene-for-gene concept. Trends Bio-
chem. Sci. 23, 454–456.
Wang, G.L., Ruan, D.-L., Song, W.-Y., Sideris, S., Chen, L., Pi,
L.-Y., Zhang, S., Zhang, Z., Fauquet, C., Gaut, B.S., Whalen,
M.C., and Ronald, P.C. (1998). Xa21D encodes a receptor-like
molecule with a leucine-rich repeat domain that determines race-
specific recognition and is subject to adaptive evolution. Plant
Cell 10, 765–779.
Whitham, S., Dinesh-Kumar, S.P., Choi, D., Hehl, R., Corr, C.,
and Baker, B. (1994). The product of the tobacco mosaic virus
resistance gene N—Similarity to Toll and the interleukin-1 recep-
tor. Cell 78, 1101–1115.
Yang, X., Chang, H.Y., and Baltimore, D. (1998). Essential role of
CED-4 oligomerization in CED-3 activation and apoptosis. Sci-
ence 281, 1355–1357.
Zhou, N., Tootle, T.L., Tsui, F., Klessig, D.F., and Glazebrook, J.
(1998). PAD4 functions upstream from salicylic acid to control
defense responses in Arabidopsis. Plant Cell 10, 1021–1030.
