Intragenic Recombination and Diversifying Selection Contribute to the Evolution of Downy Mildew Resistance at the RPP8 Locus of Arabidopsis

Correspondence to: Jeffery L. Dangl, dangl{at}email.unc.edu (E-mail), 919-962-1625 (fax).

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Pathogen resistance (R) genes of the NBS-LRR class (for nucleotide binding site and leucine-rich repeat) are found in many plant species and confer resistance to a diverse spectrum of pathogens. Little is known about the mechanisms that drive NBS-LRR gene evolution in the host–pathogen arms race. We cloned the RPP8 gene (for resistance to Peronospora parasitica) and compared the structure of alleles at this locus in resistant Landsberg erecta (Ler-0) and susceptible Columbia (Col-0) accessions. RPP8-Ler encodes an NBS-LRR protein with a putative N-terminal leucine zipper and is more closely related to previously cloned R genes that confer resistance to bacterial pathogens than it is to other known RPP genes. The RPP8 haplotype in Ler-0 contains the functional RPP8-Ler gene and a nonfunctional homolog, RPH8A. In contrast, the rpp8 locus in Col-0 contains a single chimeric gene, which was likely derived from unequal crossing over between RPP8-Ler and RPH8A ancestors within a Ler-like haplotype. Sequence divergence among RPP8 family members has been accelerated by positive selection on the putative ligand binding region in the LRRs. These observations indicate that NBS-LRR molecular evolution is driven by the same mechanisms that promote rapid sequence diversification among other genes involved in non-self-recognition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

A broad range of microorganisms have evolved the ability to use plants as a nutritional resource, and plants in turn have evolved multiple lines of defense against pathogen invasion (Hammond-Kosack and Jones 1996a ). Inducible defenses are mediated through gene-for-gene systems in which the plant carrying a particular resistance (R) gene allele responds to pathogens carrying a matching avirulence (avr) gene (Flor 1971 ). Most plants contain large collections of highly specific R genes, which are thought to encode specialized receptors that recognize avr gene–dependent elicitors (Keen 1990 ). If the R gene or the corresponding avr gene is not functional, then recognition does not occur, defenses are not activated, and the plant is susceptible to infection. Thus, pathogens can circumvent gene-for-gene resistance by alteration or loss of avr genes. This places the host under selective pressure to evolve new recognition capabilities. avr gene mutations and deletions occur at high frequency in nature (van Kan et al. 1991 ; Rohe et al. 1995 ; Sweigard et al. 1995 ; Joosten et al. 1997 ), but the host's response in this evolutionary arms race is not well understood.

Two themes have emerged from recent molecular characterization of R genes. R genes are often members of tightly linked multigene families, which can be functionally diversified (Hammond-Kosack and Jones 1996b ). A second, somewhat unexpected generality is that all R genes characterized to date, with one exception (Martin et al. 1993 ), encode proteins with long stretches of leucine-rich repeats (LRRs) (Jones and Jones 1996 ). LRRs are present in a wide variety of proteins and participate in protein–protein interactions and ligand binding (Kobe and Deisenhofer 1995 ). Crystal structure analysis has demonstrated that the LRRs of a ribonuclease inhibitor form a solvent-exposed ß sheet structure that binds the ribonuclease (Kobe and Deisenhofer 1993 ). By analogy, LRRs in plant R proteins are thought to bind pathogen-derived signal molecules and thereby mediate recognitional specificity (Dixon et al. 1996 ), although direct biochemical evidence is currently lacking.

Two superfamilies of LRR-encoding pathogen R genes have been defined by putative functional motifs and predicted localization of the encoded proteins (Dangl 1995 ). One superfamily, represented by the tomato Cf genes (for resistance to the fungal pathogen Cladosporium fulvum) (Hammond-Kosack and Jones 1996b ) and the Xa21 gene family in rice (for resistance to the bacterial pathogen Xanthomonas campestris pv oryzae) (Song et al. 1995 ), encodes proteins that are predicted to be membrane bound and composed primarily of extracytoplasmic LRRs. The Cf R proteins do not contain any recognizable signaling domain, whereas Xa21 contains extracytoplasmic LRRs fused to a cytoplasmic kinase domain.

The second and larger R gene superfamily (referred to as NBS-LRR) encodes proteins with a predicted nucleotide binding site followed by a variable number of C-terminal LRRs (Bent 1996 ). NBS-LRR proteins do not contain a recognizable signal sequence and probably function inside the cell (Leister et al. 1996 ). Most NBS-LRR genes fall into one of two subclasses based on their N-terminal motifs (Bent 1996 ). The TIR-NBS-LRR subclass is defined by an N-terminal region that resembles the cytoplasmic signaling domain of the Toll and interleukin1 transmembrane receptors (Parker et al. 1997 ). This subclass includes genes that specify resistance to a virus (N in tobacco) (Whitham et al. 1994 ), fungi (L6 and M in flax) (Lawrence et al. 1995 ; Anderson et al. 1997 ), and oomycetes (RPP5, RPP1A, RPP1B, and RPP1C in Arabidopsis, where RPP signifies resistance to Peronospora parasitica) (Parker et al. 1997 ; Botella et al. 1998 ). The second subclass (LZ-NBS-LRR) contains a leucine zipper–like motif in place of the TIR domain and is represented by the genes RPM1 (Grant et al. 1995 ), RPS2 (Bent et al. 1994 ; Mindrinos et al. 1994 ), and Prf (Salmeron et al. 1996 ). These genes specify resistance to Pseudomonas syringae pathovars.

Recent comparative analyses of extracytoplasmic LRR gene clusters have provided insight into their evolution. The Cf-4/9 gene cluster contains related but functionally distinct genes that are subject to positive diversifying selection in the LRRs (Parniske et al. 1997 ). Sequence exchanges appear to occur between linked Cf-4/9 homologs; novel Cf-4/9 haplotypes, which differ in gene copy number, can be generated by unequal crossovers at homologous intergenic regions. Evidence for gene duplications, intragenic recombination, and diversifying selection also has been reported for the Xa21 gene cluster (Song et al. 1997 ; Wang et al. 1998 ). Thus, molecular evolution of gene clusters encoding extracytoplasmic LRR–containing R proteins is driven by the same mechanisms that generate diversity in other complex loci involved in non-self-recognition, such as the major histocompatibility complex (MHC) in animals (Dangl 1992 ; Parham and Ohta 1996 ; Hughes and Yeager 1997 ).

Although NBS-LRR genes are widespread in plants and recognize many types of pathogens, little is known about the mode of NBS-LRR gene evolution. The available NBS-LRR sequences are very divergent from each other and provide no evolutionary insight other than definition of the conserved motifs described above. The structural differences between putative extracytoplasmic LRR proteins and NBS-LRR proteins imply that these two R protein superfamilies are biochemically distinct, and it is therefore of interest to determine whether they have evolved by different mechanisms.

We have used the Arabidopsis–P. parasitica (downy mildew) pathosystem for comparative analysis of R gene evolution. P. parasitica is a biotrophic oomycete and a prominent natural pathogen of Arabidopsis in Europe (Koch and Slusarenko 1990 ; Holub and Beynon 1996 ). A large number of Arabidopsis RPP genes have been defined using P. parasitica isolates from natural Arabidopsis populations (Holub et al. 1994 ; Tor et al. 1994 ). These genes are functionally polymorphic among Arabidopsis accessions, suggesting that co-evolution of host and parasite has been rapid and dynamic. Thus, comparison of allelic variants will provide insight into R gene evolution. The Arabidopsis–P. parasitica pathosystem also provides the opportunity to examine R gene evolution in a naturally evolving interaction, thereby avoiding potential loss of genetic diversity from bottlenecks in selective breeding of crop species as well as phylogenetic artifacts caused by forced introgression of genes from wild species.

Four RPP genes recently have been shown to encode members of the TIR-NBS-LRR subclass (Parker et al. 1997 ; Botella et al. 1998 ). In contrast, we show in this study that the RPP8 gene is a member of the LZ-NBS-LRR subclass. Furthermore, sequence comparisons of resistant and susceptible RPP8 alleles provide evidence that intragenic recombination and positive selection interact to promote sequence diversification in NBS-LRR R gene evolution.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Genetic and Physical Definition of the RPP8 Locus
The RPP8 gene specifies resistance to the Emco5 isolate of P. parasitica in the Arabidopsis accession Landsberg erecta (Ler-0). Emco5 is compatible with accession Columbia-0 (Col-0). Therefore, we used the Dean and Lister Col-0 x Ler-0 recombinant inbred (RI) lines (Lister and Dean 1993 ) to map RPP8 genetically, as shown in Figure 1. When we used 100 RI lines, resistance to Emco5 segregated as a single locus (RPP8) on chromosome 5 in the interval between Dfr and Lfy. We identified 71 additional Dfr–Lfy recombinants from an additional 198 RI lines, and we used these recombinants to narrow the interval, defining Spl2 and Cra1 as closer markers on either side of RPP8. Spl2 identified one recombinant centromeric to RPP8, and Cra1 identified five recombinants telomeric to RPP8 (Figure 1A).

View larger version (26K): [in this window] [in a new window]   Figure 1. Genetic and Physical Map of the RPP8 Region. (A) Genetic map of the RPP8 interval. Molecular markers are shown above the line, and the number of recombinants that separate each marker from RPP8 are shown below the line. The minimum genetic interval of RPP8 is shown between the arrowheads at top. Dfr, spl2, and Cra1 are cleaved amplified polymorphic sequence markers. CK1 refers to the restriction fragment length polymorphism (RFLP) shown in (B). The remaining markers are RFLPs derived from yeast artificial chromosome (YAC) ends. YAC and bacterial artificial chromosome (BAC) clones are depicted below the genetic map, with approximate lengths shown at right. YAC clones CIC4E12, CIC6F12, and EW6E5, which also map in the same region, are not shown. Cen, centromeric; Tel, telomeric. (B) Gel blot of genomic DNA from Col-0 and Ler-0 that was digested with EcoRV and probed with the CK1 candidate gene fragment at moderate stringency. The RFLP cosegregating with RPP8 is shown by arrows. The Col-0 band that comigrates with the doublet in Ler-0 segregated independently of the doublet. DNA length standards are shown at right in kilobases. (C) Physical structure of the RPP8 locus in Ler-0 and Col-0. The Ler-0 segment represents 15 of the 23 kb that were sequenced from the 9L9 cosmid. Genomic subclones are depicted above the physical map. RPP8-Ler and RPH8A coding sequences are depicted by filled and stippled boxes, respectively, 5' and 3' untranslated regions are depicted by open boxes, and introns are represented by diagonal lines. The boxes labeled CycCH and NF22H represent regions of homology to rice cyclin C and a hypersensitive response–inducing gene (NF22) from tobacco, respectively. In Col-0, the region between RPP8-Ler and RPH8A has been deleted, as depicted by the dashed lines.

Yeast artificial chromosome (YAC) end probes and genetically anchored molecular markers were used to construct a physical map of the RPP8 interval (Figure 1A). The Spl2 and Cra1 markers both mapped within the YAC contig, demonstrating that the contig spanned the RPP8 locus. We genetically mapped four YAC ends as restriction fragment length polymorphisms (RFLPs) to refine further the RPP8 interval. 5F12LE and 13F5RE RFLPs both cosegregated with RPP8, whereas 8C12LE and 15C8RE detected one recombinant telomeric to RPP8. The Spl2–8C12LE interval thus defined the smallest possible genetic interval in our mapping population. This genetic distance corresponds to a maximum physical distance of ~100 to 300 kb (Figure 1A).

Identification and Mapping of an RPP8 Candidate Gene
A candidate for the RPP8 gene (CK1, described by M.G.M. Aarts et al. 1998 ) was amplified with degenerate polymerase chain reaction (PCR) primers from conserved R gene motifs. CK1 hypothetically encodes an LRR sequence with ~30% identity and 40% similarity to segments of the RPM1 gene and hybridized with a polymorphic multicopy family in both Col-0 and Ler-0 (Figure 1B). We genetically and physically mapped an EcoRV RFLP, which consists of a double band (~5.5 to 6 kb) in Ler-0 and a single ~4.5-kb band in Col-0 (Figure 1B). The Ler-0 doublet cosegregated with resistance to Emco5 in the subset of RI lines that contained recombinations between Dfr and Lfy. Conversely, the 4.5-kb Col-0 band was always present in Emco5-susceptible RI lines and hybridized with all of the Col-0 YACs and bacterial artificial chromosomes (BACs) spanning rpp8 (Figure 1A). The genetic and physical colocalization of the EcoRV RFLP with the RPP8 phenotype, in combination with its sequence similarity to known R genes and potential copy number polymorphism, implicated it as a candidate gene for RPP8.

Transgenic Complementation of RPP8 Function
We isolated genomic cosmid clones containing the EcoRV doublet from Ler-0 by using the CK1 probe. One cosmid (9L9) contains a 23-kb insert that includes both bands of the doublet. A second cosmid (25M19), which overlaps with 9L9 over ~17 kb, contains the upper band of the doublet and a fragment of the lower band (data not shown). Both cosmids were transformed into susceptible Col-0, and transgenic (T₁) seedlings were selected and allowed to set seed. T₂ progeny from multiple independent transformants were inoculated with Emco5 and assessed for resistance. Complementation experiments are summarized in Table 1. All 12 tested Col::9L9 transgenic lines segregated ~3:1 for resistant to susceptible in the T₂ generation, which is consistent with a single, dominant transgenic locus conferring Emco5 resistance. At least five of six tested Col::9L9 lines were independent transformants (data not shown). None of the seven tested Col::25M19 T₂ lines displayed resistance to Emco5 (Table 1), suggesting that the lower band of the doublet was necessary for Emco5 resistance. Neither 9L9 nor 25M19 provided resistance to the Madi1 or Noco2 isolates of P. parasitica (Table 1).

Only one CK1-hybridizing band was detectable in the Col-0 YACs and BACs spanning rpp8 (data not shown), suggesting that only one Col-0 CK1 family member is present in this >470-kb interval. Furthermore, mapping of other CK1-hybridizing bands demonstrated that no other CK1 family members are closely linked to RPP8 (described by M.G.M. Aarts et al. 1998 ). Cosmids containing additional CK1 family members conferred no resistance to any P. parasitica isolate in transgenic Col-0 (data not shown). These results suggest that resistance to Emco5 in Ler is conferred specifically by one member of the CK1 gene family.

Two Closely Related Genes Are Present at the RPP8 Locus in Ler-0
Sequencing of the 9L9 cosmid insert revealed two highly similar NBS-LRR genes (Figure 1C). We constructed subclones to separate these two genes (Figure 1C). All of the four lines transgenic for pRPP8 were completely resistant to Emco5, whereas all of the four lines transgenic for pRPH8A were as susceptible to Emco5 as is wild-type Col-0 (Figure 2A and Table 1). Thus, a single NBS-LRR gene, referred to hereafter as RPP8-Ler, is sufficient to provide Emco5 resistance in the Col-0 background. The second gene (named RPH8A for RPP8 homolog A) is insufficient for transgenic complementation of resistance to Emco5 in Col-0.

View larger version (91K): [in this window] [in a new window]   Figure 2. Interaction Phenotypes of Col-0::pRPP8 Transgenic Plants and Ler-0 rpp8 Mutants. (A) RPP8 from Ler-0 confers resistance to Emco5 in transgenic Col-0 plants. At 7 days after inoculation with Emco5, wild-type Col-0 cotyledons support heavy asexual sporulation (S, sporangiophores), whereas no sporulation is visible on wild-type Ler-0 or transgenic Col-0 seedlings containing the pRPP8 subclone from the 9L9 cosmid. (B) The rpp8-2 mutant in Ler-0 is susceptible to Emco5. The interaction phenotypes of cotyledons from F1 progeny of various crosses demonstrate that rpp8-2 is recessive to RPP8-Ler and allelic to rpp8-Col and rpp8-3. Cotyledons were stained at 7 days after inoculation with trypan blue, which is retained by parasite structures (H, hyphae; O, oospores) and dead host cells (HR).

RPP8-Ler and RPH8A are separated by a 3.7-kb segment containing a putative open reading frame with 75% amino acid similarity to cyclin C from rice (Figure 1C). A fourth open reading frame ~1 kb downstream of RPH8A resembles (~50% amino acid similarity) the tobacco gene NF22 (GenBank accession number U66266). NF22 was identified by its ability to induce a hypersenstive response–like reaction when overexpressed (Karrer et al. 1998 ). Subclones of the NF22 homolog or the cyclin C homolog conferred no resistance to Emco5 in transgenic Col-0 plants (Table 1).

The intron–exon structure of RPP8-Ler was deduced by comparison to RPP8 cDNAs and is diagrammed in Figure 1C. The RPP8 coding sequence contains two introns: intron 1 (129 bp) splits codon 292, and intron 2 (675 bp) splits codon 341. A third intron (123 bp) begins 4 bp downstream of the stop codon in the RPP8 cDNA. Sequence analysis of 11 independent RPP8-Ler clones revealed variable polyadenylation sites ~450 bp downstream of the stop codon. The gene structure of RPH8A could not be confirmed by cDNA comparison because no RPH8A cDNAs were isolated, but it is probably identical because conserved intron–exon border sequences were found at identical locations in the RPH8A coding sequence. Interestingly, the 3' ends of RPP8-Ler and RPH8A are identical over an 898-bp stretch, from codon 837 to 688 bp downstream of the stop codon (including the intron, 3' untranslated region, and downstream nontranscribed sequence). After this 898-bp stretch, similarity between the two genes is very low. The 5' flanking sequences of RPP8-Ler and RPH8A are almost completely dissimilar, except for a 90-bp stretch of 89% identity, which begins 473 and 692 bp upstream of the RPP8-Ler and RPH8A start codons, respectively.

RPP8 Encodes a Member of the LZ-NBS-LRR Subclass
Figure 3 and Figure 4 provide the primary structures of hypothetical proteins encoded by RPP8-Ler (906 amino acids), rpp8-Col (908 amino acids), and RPH8A (907 amino acids). The latter two genes encode full-length hypothetical proteins that share 92 and 91% amino acid identity, respectively, with RPP8-Ler (Figure 3 and Figure 4, and Table 3). Several putative functional motifs present in known R genes are apparent in the encoded proteins (Figure 3 and Figure 4). The C-terminal one-third of each gene is composed of 14 imperfect LRRs, which vary in length from 21 to 29 amino acids. A consensus nucleotide binding site and a hydrophobic domain conserved in all NBS-LRR genes are also apparent. Finally, a putative six-heptad leucine zipper is present near the N terminus. This motif clearly places RPP8 in a distinct structural subclass from the other RPP proteins that have been identified. RPP8-Ler is more closely related to the RPM1 bacterial resistance protein from Arabidopsis (26% identity and 39% amino acid similarity) than it is to any other known R protein. RPP8 has no significant similarity with RPP5, RPP1A, RPP1B, or RPP1C, except in the functional domains that define the putative nucleotide binding site.

View larger version (48K): [in this window] [in a new window]   Figure 3. Deduced Amino Acid Sequence of RPP8-Ler. Domains A to F are based on putative functional motifs. Domains B and D contain putative leucine zippers. Domains C, D, and E contain the NBS motifs and a conserved hydrophobic domain, shown in boldface. Domain F contains 14 imperfect LRRs defined by the conserved residues shown in boldface. The LRR subdomain XXLXLXXXX, which encompasses the putative ß strand/ß turn region identified from the porcine ribonuclease inhibitor crystal structure, is framed between the solid lines. Blue residues represent positions in which either RPH8A or rpp8-Col encodes a different amino acid from RPP8-Ler. Residues in red are different in all three proteins.

View larger version (36K): [in this window] [in a new window]   Figure 4. Amino Acid Sequence Alignment of RPP8-Ler, RPH8A, and rpp8-Col. Dashes represent identical amino acids, and dots represent deletions in RPP8-Ler and rpp8-Col compared with RPH8A. Amino acid substitutions are shown as lowercase letters. Amino acid changes in Ler rpp8 mutants are shown above the RPP8-Ler sequence in boldface. The XXLXLXXXX motifs are underlined. The corresponding nucleotide sequences have GenBank accession numbers AF089710 and AF089711 for RPP8-Ler and rpp8-Col, respectively.

The rpp8 Allele in Col-0 Is a Chimera of Progenitor Genes Related to RPP8-Ler and RPH8A
As shown in Figure 1C, the structure of the rpp8 locus in Col-0 is dramatically different from the RPP8 locus in Ler-0. Only one RPP8 homolog (named rpp8-Col) is present at the Col-0 locus. The 5' flanking sequence of rpp8-Col is almost identical to that of RPP8-Ler, whereas the 3' flanking sequence of rpp8-Col is almost identical to the segment extending from the end of RPH8A to the NF22 homolog (Figure 1C). The segment that separates RPP8 and RPH8A in Ler-0, including the cyclin C homolog, is deleted in Col-0. rpp8-Col thus appears to be derived from a precise in-frame unequal crossover within an ancestral Ler-like RPP8 haplotype.

Seven insertion/deletion sites, shown in Figure 5, were used as landmarks to localize the most likely recombination breakpoint. rpp8-Col shares with RPP8-Ler a 9-bp insertion (codons 147 to 149) and a 6-bp deletion (codons 484 to 485) relative to the RPH8A sequence (Figure 4 and Figure 5). rpp8-Col also shares four additional indels with RPP8-Ler in intron 2 (Figure 5). rpp8-Col shares a 6-bp insertion with RPH8A, relative to RPP8-Ler, at codons 560 to 561. The recombination breakpoint thus appears to be located between codons 486 and 559, which includes the region just upstream of the LRRs as well as part of the first LRR (Figure 3 and Figure 4). Interestingly, most of the indels encompass short direct repeats (Figure 5), which suggests that they could have been generated by transposon insertion and subsequent excision.

View larger version (35K): [in this window] [in a new window]   Figure 5. Location and Sequence of Insertion/Deletion Sites in RPP8-Ler, rpp8-Col, and RPH8A. Codon positions are numbered according to the multiple alignment in Figure 4. Intron positions are numbered from the first nucleotide of intron 2 in RPP8-Ler. Direct repeats are emphasized by italics. Nucleotide substitutions are indicated by lowercase letters. Dots indicate gaps. The intron 2 splice donor site is underlined.

The pattern of nucleotide polymorphisms between RPP8-Ler, RPH8A, and rpp8-Col is very complicated, as shown in Figure 6. We observed a lack of consistent sequence affiliation, based on shared nucleotide polymorphisms, between any pair of homologs. Instead, the three RPP8 homologs exhibit a patchwork pattern of affiliations in their coding sequences. For example, the majority of polymorphisms (23 of 39) in the first 1000 bp support an affiliation between RPP8-Ler and rpp8-Col, which is consistent with the hypothesis that the 5' end of rpp8-Col was derived from an RPP8-Ler–like ancestor. Similarly, the majority of 3' polymorphisms support an affiliation between rpp8-Col and RPH8A. However, there are segments of contiguous polymorphisms that support different affiliations. For example, nucleotides 130 to 301 contain seven polymorphisms that affiliate RPP8-Ler with RPH8A rather than rpp8-Col. This suggests that a recent exchange occurred between the two Ler-0 genes. Alternatively, this affiliation could reflect the accumulation of contiguous point mutations in the Col-0 allele. Comparisons with other RPP8 homologs are necessary to distinguish accurately between these possibilities.

View larger version (32K): [in this window] [in a new window]   Figure 6. Patchwork Distribution of Nucleotide Polymorphisms between RPP8-Ler, rpp8-Col, and RPH8A Coding Sequences. Polymorphic sites that distinguish the three genes are shown. Nucleotide positions, beginning from the start codon, are shown above the lines. Nucleotide positions for which all three genes are identical or for which each gene has a different nucleotide are omitted. A consensus (cons) sequence (two of three) is shown between the lines. Residues that conform to the consensus are represented by stars, and silent nucleotide substitutions are shown in lowercase letters. Gaps are represented by dashes. Colors indicate sequence affiliations based on shared polymorphisms. The 14 LRRs are separated by spaces, whereas nucleotides encoding amino acids in the XXLXLXXXX motif are shown by italics in the consensus sequence.

Several of the LRR-encoding segments are extremely divergent among the three genes (Figure 4 and Figure 6). The degree of divergence among the LRRs is variable, with LRRs 11 and 12 exhibiting the highest divergence and LRRs 3, 10, 13, and 14 exhibiting the highest degree of conservation. Perhaps the divergent LRRs are directly involved in recognition specificity, whereas the conserved LRRs play a structural role. Two highly variable regions also are apparent outside the LRRs (amino acid residues 432 to 442 and 480 to 489). They do not fall within any recognizable functional motif. We predict that these regions define a new set of functionally relevant residues.

Analysis of rpp8 Mutants in Ler-0
We isolated six independent rpp8 mutants from a screen of mutagenized Ler-0 plants. Disease ratings of all mutants are shown in Table 1, and the phenotype of the rpp8-2 mutant is presented in Figure 2B. The six mutants supported different levels of Emco5 sporulation, suggesting that they represent a series of alleles with mutations of differing severity. Although the levels of Emco5 growth varied somewhat between experiments, rpp8-2 was generally the most susceptible mutant, whereas rpp8-1 exhibited the weakest susceptibility phenotype. Typically, only 20 to 40% of rpp8-1 cotyledons produced sporangiophores. Each mutant retained wild-type levels of resistance to the Madi1 (resistance provided by RPP21) and Noco2 (RPP5) isolates. None of the mutants exhibited any obvious developmental phenotype.

Results from genetic analysis of the rpp8 mutants are summarized in Table 2. F₁ progeny from backcrosses of all six mutants to Ler-0 were resistant to Emco5 (Table 2 and Figure 2B), demonstrating that the mutations were recessive. Trypan blue staining of backcross F₁ plants revealed occasional trails of necrotic host cells in the cotyledons (Figure 2B), suggestive of a slightly delayed defense response. This phenomenon was also observed in F₁ progeny from a cross of wild-type Col-0 x Ler-0 (data not shown), suggesting that RPP8-Ler is not completely dominant with respect to rpp8-Col. F₂ progeny from the backcrosses of rpp8-2 and rpp8-3 to Ler-0 segregated ~3 resistant:1 susceptible, which is consistent with a single recessive mutation. F₂ progeny from the rpp8-1 x Ler-0 backcross did not segregate any individuals that supported sporulation. This most likely reflects the very weak effect of the rpp8-1 mutation, as suggested by the weak and inconsistent Emco5 growth in the rpp8-1 M3 seedlings described above.

Outcrosses of all of the six mutants to wild-type Col-0 as well as three intermutant crosses yielded susceptible F₁ progeny (Figure 2B and Table 2). Because RPP8 is the only locus for Emco5 resistance that segregates between Col-0 and Ler-0, the observed lack of complementation in F₁ progeny of these crosses strongly suggests that all seven mutations are in RPP8. F₂ segregation ratios from three tested outcrosses to Col-0 were consistent with this hypothesis. A significant proportion of F₂ progeny from the rpp8-1 x Col-0 cross did not support sporulation, most likely because of the weak effect of the rpp8-1 mutation. F₂ progeny from the intermutant crosses also segregated for disease-free individuals. This could reflect the additive effect of two partially functional mutations. Chi-square analysis (Table 2) strongly contradicts the hypothesis that the mutations are in unlinked second site loci (predicted 9 resistant:7 susceptible segregation in outcross and intermutant F₂ populations).

For further confirmation that these mutations are in the RPP8 gene, we compared the rpp8 coding sequence from four mutants with the wild-type RPP8-Ler sequence. In rpp8-1, a C-to-T mutation in codon 827 caused an S-to-L substitution in LRR12 (Figure 4). In rpp8-2, a G-to-A mutation in codon 553 caused an R-to-K substitution in LRR1. In rpp8-3, a G-to-A mutation in codon 418 caused a D-to-N substitution. In rpp8-4, a C-to-T mutation in codon 151 created a stop codon. These sequence alterations confirm that the R gene candidate is indeed RPP8.

Nucleotide Substitution Patterns Suggest That Positive Selection Has Been Acting on RPP8
We determined that RPP8 is under positive selection for amino acid diversification by comparing nonsynonymous (K_a) and synonymous (K_s) nucleotide substitutions in different segments of the rpp8-Col, RPP8-Ler, and RPH8A protein coding regions. In most cases in which evolution is conservative, the number of synonymous substitutions greatly exceeds that of nonsynonymous substitutions, leading to a K_a/K_s ratio <1. A K_a/K_s ratio >1 indicates selection for amino acid diversification (Kreitman and Akashi 1995 ).

Much of amino acid divergence among the three RPP8 family members was concentrated in a subdomain of the LRRs (XX[L]X[L]XXXX), where leucine, isoleucine, or valine residues are found at the conserved positions designated by an L (Figure 3 and Figure 4). This motif encompasses a predicted ß strand/ß turn region in which hydrophobic side chains at the conserved positions are buried in the core, and the nonconserved, interstitial residues (designated by X) are solvent exposed (Dixon et al. 1996 ; Jones and Jones 1996 ). Calculations of K_a and K_s (Table 3) support the hypothesis that positive selection is acting to diversify putative solvent-exposed residues. For example, K_a in the XX(L)X(L)XXXX codons was 15.8% between rpp8-Col and RPP8-Ler, whereas K_s was only 7.8% (K_a/K_s = 2.0). In the remainder of the coding sequence, excluding the XX(L)X(L)XXXX codons, K_a was fivefold lower, and the K_a/K_s ratio was 0.8, indicating a more conservative mode of evolution. A similar trend was apparent in the other two pairwise comparisons (Table 3).

	DISCUSSION

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Plants may have an inherent disadvantage in the gene-for-gene arms race, because loss-of-function mutations in pathogen avr genes are sufficient to disarm gene-for-gene resistance. In contrast, the host must respond with a corresponding gain of function (recognition), and accumulation of point mutations in preexisting R genes alone may not provide sufficient structural diversity for novel resistance specificities to evolve in a timely fashion. Below, we discuss the implications of our results that are relevant to this conundrum.

Structurally Distinct NBS-LRR Subclasses Can Function in P. parasitica Resistance
It seems likely that novel R genes are recruited from preexisting R genes. Genes at the L and M loci are highly related to each other, and the Cf genes in tomato have very similar structural features. Based on these precedents, one might predict that RPP8 is a member of the TIR-NBS-LRR subclass, like RPP5 and the RPP1 family members. However, RPP8 encodes an LZ-NBS-LRR protein and is most closely related to the RPM1 bacterial R gene, demonstrating that the TIR-NBS-LRR and the LZ-NBS-LRR subclasses can function in resistance to P. parasitica. Similarly, the Xa1 NBS-LRR gene and the Xa21 extracytoplasmic LRR gene specify resistance to different isolates of the same bacterial pathogen of rice (Yoshimura et al. 1998 ). These observations suggest that plants can recruit a wide range of R proteins to recognize structurally diverse elicitors from the same pathogen. This is likely to be a key adaptive mechanism, in view of the apparent ease with which pathogens can alter or discard certain avr genes (van Kan et al. 1991 ; Rohe et al. 1995 ; Sweigard et al. 1995 ; Joosten et al. 1997 ).

Recent genetic evidence suggests that RPP8-mediated resistance may operate through a different signaling pathway from RPP1 and RPP5. The Arabidopsis eds1 (for enhanced disease susceptibility) mutation abolishes the function of several RPP genes, including RPP5 and RPP1; however, eds1 has little or no effect on RPP8 function (N. Aarts et al. 1998 ). Similarly, the ndr1 mutation, which partially inactivates several RPP genes and completely inactivates the Arabidopsis LZ-NBS-LRR bacterial R genes, does not affect RPP8 (N. Aarts et al. 1998 ). RPP8 is the only cloned Arabidopsis R gene that does not require either NDR1 or EDS1 for function. RPP8 may therefore define a novel resistance pathway, or alternatively, NDR1 and EDS1 could be functionally redundant in RPP8-mediated resistance. We are currently constructing lines to test these possibilities.

A Novel rpp8 Haplotype Was Generated by an Unequal Crossover between Linked Genes
Genetic analyses of R gene clusters, such as Rp1 in maize and M in flax, have indicated that recombination between repeated sequences in R gene clusters is a critical mechanism in R gene evolution (reviewed in Ellis et al. 1997 ; Hulbert 1997 ), and the chimeric structure of the rpp8-Col allele adds to a growing body of molecular data that supports this proposal. Intra-allelic recombinants have been discovered in mutational screens at the M and RPP5 loci (Anderson et al. 1997 ; Parker et al. 1997 ). These recombinant alleles arose from ectopic recombination between LRR-encoding modules that caused expansions or contractions in LRR copy number, thereby inactivating the gene. Intragenic recombination also has been proposed to occur within a 5' region that is highly conserved between genes in the Xa21 cluster, resulting in "promoter swaps" with minimal alterations in the coding sequences (Song et al. 1997 ). Finally, expansions and contractions in gene copy number have been observed to occur in the Cf-4/9 complex by unequal crossing over between homologous intergenic regions (Parniske et al. 1997 ). The structure of rpp8-Col expands on these observations: rpp8-Col was generated by unequal crossing over between linked, nonallelic genes, it encodes a chimeric protein that differs dramatically from both progenitors, and it was present in at least one natural Arabidopsis population from which the Col-0 accession was derived. The observations that recombination can produce coding sequence chimeras, promoter swaps, and expansion or contraction in gene number and LRR copy number collectively underscore the role of recombination as a potent and versatile force in R gene evolution.

The functional roles of rpp8-Col and RPH8A are currently unknown. Neither gene is sufficient for resistance to Emco5 in Col-0, but both genes encode predicted full-length proteins. The nonrandom pattern of substitutions in ß strand/ß turn LRR-encoding motifs of both genes suggest that they are functional and remain under selection. We did not find RPH8A cDNAs among the 25 that were isolated, but rpp8-Col is expressed, as evidenced by complete identity to the Col-0 expressed sequence tag clone T14073. Therefore, it seems likely that these genes recognize currently undefined pathogens, and experiments are under way to define their functions genetically.

It is also possible that the rpp8-Col and RPH8A genes are obsolete or superfluous. A potential analogy may exist in the MHC, which contains functional class 1a antigen presentation genes as well as class 1b genes, which evolved from class 1a genes by duplication (Klein and O'hUigin 1994 ). Some class 1b genes are functional, whereas others are expressed at reduced levels and appear to be evolving into nonexpressed pseudogenes. Class 1 MHC genes are thought to undergo turnover through cycles of birth and death as inactive or obsolete genes are supplanted by more efficient copies arising from duplication and divergence (Nei and Hughes 1992 ). This process also may operate in plant disease resistance loci, which typically contain duplicated genes with unknown functions (Martin et al. 1994 ; Anderson et al. 1997 ; Wang et al. 1998 ). A significant fraction of these genes could be "molecular fossils" arising from gene turnover during the host–pathogen arms race. Nonfunctional R gene homologs may still play an important role, however, as repositories of sequence variation, as is seen among class 1 MHC genes (Hughes 1995 ). Indeed, close relatives of RPP8-Ler and RPH8A served as sequence donors when rpp8-Col was generated.

RPP8 Sequence Diversity Arises from Positive Selection
The divergence between the Col-0 and Ler-0 RPP8 alleles is much higher than is divergence among other Arabidopsis alleles (typically <0.01%) (Bergelson et al. 1998 ). Our analysis of nucleotide substitution patterns suggests that the divergence among RPP8 family members has been accelerated by positive diversifying selection. Clear evidence for positive selection in molecular evolution has rarely been observed (Kreitman and Akashi 1995 ). Interestingly, the majority of genes that appear to be under selection for protein diversification are involved in host–pathogen interactions (Endo et al. 1996 ). Members of the Cf-4/9 and Xa21 extracellular LRR gene families are under positive selection in the LRR subdomain that is predicted to form a ß strand/ß turn structure (reviewed in Jones and Jones 1996 ), and RPP8 appears to be evolving in an analogous fashion. The fact that two of four sequenced rpp8 mutations are missense substitutions in the XXLXLXXXX motif underscores the functional importance of this domain. It appears that both superfamilies of LRR disease resistance proteins are subject to diversifying selection, potentially for altered ligand binding capabilities in the LRRs. Interestingly, the divergence among the RPP8 family members is concentrated in a slightly longer motif than in the Cf-4/9 homologs (XXLXLXX) (Parniske et al. 1997 ). This possibly reflects adaptations for interactions with structurally dissimilar ligands.

What mechanisms generate the mutations upon which selection acts? Point substitutions are undoubtedly a primary source; however, we found it intriguing that most of the insertion/deletion sites among the three genes, including three indels that encompassed two or three codons, comprise direct repeats of varying degeneracies (Figure 5). This direct repeat structure suggests target site duplication and subsequent imprecise excision of a transposable element(s). Perhaps transposon insertions occurred in RPP8 immediately after the RPP8-Ler/RPH8A duplication, allowing one homolog to compensate for loss of the other until the transposon was excised. Periods of decreased pathogen pressure also could provide windows of opportunity for transposon insertions (or other sequence rearrangements) to accumulate at no cost to the plant. Regardless of whether the RPP8 indels were generated by transposons, their presence suggests alternative mutational mechanisms that augment diversification from point substitutions.

Recombination and gene conversion also may have generated sequence diversity at RPP8. Although these two mechanisms cannot create nucleotide substitutions, they can reassort existing mutations and cause amino acid substitutions by creating novel codons at recombination breakpoints, as seen in the Cf-4/9 complex (Parniske et al. 1997 ). The region of complete identity at the 3' end of RPP8-Ler and RPH8A is suggestive of a recent gene conversion or double crossover. The patchwork pattern of nucleotide polymorphisms among the three RPP8 family members also suggests that sequence exchanges have occurred during their evolution. Strong evidence for sequence exchanges among MHC genes exists, and theoretical simulations of MHC evolution have suggested that gene conversion is particularly important for the acquisition of polymorphism under conditions of weak selection (Parham and Ohta 1996 ). This may be particularly significant in interactions with biotrophic plant pathogens in which penalties to the host are subtle (Holub and Beynon 1996 ).

In combination with other recent comparative analyses of R gene structure, our results have established clear mechanistic parallels between the evolution of the two R gene superfamilies and other loci that determine the outcome of interactions. A growing body of data suggests that genes mediating coevolutionary self- and non-self-interactions are subject to a mode and tempo of evolution that differ dramatically from most other types of genes. Future studies expanding our understanding of the interplay between mutation, recombination, and selection in the generation of novel pathogen R genes should provide insights of broad academic and agricultural significance.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Emco5 Derivation and Pathogenicity Tests
The Peronospora parasitica isolate Emco5 was intentionally isolated for the purpose of cloning the RPP8 allele from Arabidopsis thaliana Landsberg erecta (Ler-0) (Holub and Beynon 1996 ). RPP8 was defined initially in Ler-0 by mapping a locus involved in recognition of the isolate Emoy2 by using recombinant inbred (RI) lines from a cross between Ler-W100 (Ler carrying nine phenotypic markers) and Wassilewskija (Ws-0) (Reiter et al. 1992 ). However, detailed mapping of RPP8 was difficult in this cross because segregation was complicated by the presence of two additional R genes: RPP1 from Ws-0 on chromosome 3 and RPP4 from Ler-W100 on chromosome 4. The Ler x Columbia (Col-0) RI mapping population could not be used to map RPP8 because Col-0 also carries a functional RPP4 allele. Consequently, a series of "baiting host lines" carrying a functional RPP4 allele from either Ler-0 or Col-0 were used to select natural recombinant variants that had lost the presumed ATR4 gene. This screen was initiated with the natural oospore (sexual inoculum) population from which Emoy2 was originally derived. Emco5 was eventually isolated using a Ler-0 x Col-0 RI line (LC175) carrying a Ler-0 RPP4 allele and a Col-0 RPP8 allele. The asexual inoculum from this isolate was used to confirm that it was compatible with both Col-0 and Ws-0 and was detected by a single RPP locus from Ler-0 in the two available RI mapping populations. This locus was closely linked to the phenotypic marker T T3 in the Ler-W100 x Ws-0 RI population that defined the RPP8 locus for Emoy2 resistance and cosegregated with agp6 in the Ler-0 x Col-0 RI population.

Pathogenicity tests and mutant screens were conducted by spraying 7-day-old seedlings with a suspension of asexual inoculum (5 x 10⁴ conidiosporangia mL^-1). Seedlings were then covered with a transparent dome to maintain high humidity and to contain the isolate throughout the experiment. Seedlings were grown for 7 days at 16 to 18°C with an 8-hr photoperiod in a Percival Scientific growth chamber (Boone, Iowa). P. parasitica growth was assessed visually at 7 days after inoculation by counting sporangiophores on both sides of the cotyledon and classifying plants as either N (no sporangiophores), L (1 to 10 sporangiophores), M (11 to 19 sporangiophores), or H (20 or more sporangiophores). To calculate the mean sporangiophore production shown in Table 1, we used actual numbers (0 to 10) for N and L cotyledons and assigned values of 15 (M) and 20 (H). Hyphal growth was assessed by staining inoculated seedlings with lactophenol–trypan blue (Koch and Slusarenko 1990 ).

Identification and Sequencing of rpp8 Mutants
We mutagenized Ler-0 seeds with 0.15% ethyl methanesulfonate for 8 hr. M₂ seed was collected from lots of ~50 M₁ plants. We inoculated 1500 to 2000 7-day-old M₂ seedlings from each lot with Emco5 and visually screened for asexual sporulation 7 days later. We screened 35 M₂ lots and identified Emco5-susceptible seedlings from nine lots. Mutants were rescued by treatment with a 1:400 dilution of Ridomil (0.1 g L^-1 metalaxyl; Novartis Ltd., Basel, Switzerland) and transferred to a 16-hr photoperiod at 23°C. Three mutants exhibited very inconsistent resistance phenotypes and are not described.

We used Ler-0 plants with the ttg marker in this screen to distinguish rogue seeds or outcross contaminants visually. In addition, we tested DNA from each mutant with a set of cleaved amplified polymorphic sequences and simple sequence length polymorphism markers from throughout the genome that distinguished polymorphisms between Ler-0 and two Emco5-compatible accessions, Col-0 and Ws-0. A Ler-0 pattern was observed for every marker tested in each mutant (data not shown), thereby demonstrating that the mutants were derived from the Ler-0 background.

We determined the sequence of the mutant Ler rpp8 alleles by polymerase chain reaction (PCR) amplification and direct sequencing of the entire PCR product. Multiple amplification products were sequenced to check for misincorporations during the amplification. We designed PCR primers based on the sequence variation that exists between RPP8-Ler and RPH8A to amplify specifically the RPP8-Ler gene. Gene specificity of the primer sets was confirmed using pRPP8 and pRPH8A as controls. Primer sequences will be provided upon request.

Yeast and Bacterial Artificial Chromosome Manipulation
Yeast artificial chromosome (YAC) clones that hybridized with markers Dfr and mi83 were kindly provided by R. Schmidt (Max Delbrück Laboratory, Cologne, Germany) and C. Dean (John Innes Centre, Norwich, UK) and assembled into a contig by hybridization with known nearby markers and YAC ends. The YAC ends were cloned by vectorette PCR (Matallana et al. 1992 ). TAMU bacterial artificial chromosome (BAC) clones (Choi et al. 1995 ) hybridizing with the 15C8RE and Spl2 markers were identified, and their integrity was confirmed by DNA gel blotting with several markers that span the contig.

cDNA and Genomic Clone Isolation
Two Ler-0 cDNA libraries (Parker et al. 1997 ) were kindly provided by M. Coleman (John Innes Centre). One library was size selected for inserts >1.8 kb. A total of ~1.8 million plaque-forming units were screened with the CK1 probe at 65°C in 2 x Denhardt's solution (1 x Denhardt's solution is 0.02% Ficoll, 0.02% PVP, and 0.02% BSA). Filters were washed at 65°C in 2 x SSC (1 x SSC is 0.15 M NaCl and 0.015% sodium citrate). Twenty-five clones that gave signals of varying intensity were purified. Sequence was obtained from both ends of each clone, and the clone that had the longest insert was completely sequenced. The 9L9 and 25M19 cosmids were obtained by screening a Ler-0 genomic DNA library in the pCLD04541 binary cosmid vector (kindly provided by M. Botella, John Innes Centre), as described above. Cosmid DNA was extracted by standard alkaline lysis procedures, and restriction digest patterns of each cosmid were compared with Ler-0 genomic DNA on gel blots to check for rearrangements in the insert.

RPP8 Subclones
Fragments of the 9L9 cosmid were subcloned directly into binary plasmid vectors by standard procedures. pRPP8 contains a 5488-bp EcoRI fragment that includes the entire RPP8-Ler coding sequence as well as 679 bp of the 5' flanking sequence and 1288 bp of the 3' flanking sequence. pRPH8A contains a 5672-bp EcoRI fragment that includes the entire RPH8A coding sequence, 1334 bp of the 5' sequence, and 815-bp of the 3' sequence. The cyclin C homolog was contained on a 4321-bp SacI subclone (2826 bp of the 5' sequence and 639 bp of the 3' sequence). The NF22 homolog was contained on a 6576-bp SacI fragment (>2 kb of both the 5' and 3' sequences). The CycH and NF22H subclones are in the pGPTV-Kan binary vector (Becker et al. 1992 ). pRPP8 and pRPH8A are in pBAR1, which was derived from pGPTV-Bar by replacing the ß-glucuronidase gene with the polylinker from pBluescript SK+ (Stratagene, La Jolla, CA) (B. Holt, D. Boyes, and J.L. Dangl, unpublished data).

Plant Transformation
Binary clones were transformed into Agrobacterium tumefaciens GV3101 by electroporation. Tetracycline at 12.5 mg mL^-1 was not a reliable selection for transformed Agrobacterium, but kanamycin at 50 mg mL^-1 worked consistently. We transformed plants using the vacuum infiltration method (Bechtold et al. 1993 ). We extracted cosmid DNA from an aliquot of each Agrobacterium culture used for transformation and checked for insert rearrangements by DNA gel blotting. Plants transformed with pGPTV-Kan were selected on Murashige and Skoog (Gibco BRL, Grand Island, NY) medium with 50 mg mL^-1 kanamycin. pBar1 transformants were selected by spraying seedlings at 5, 6, and 7 days after germination with a solution of 0.01% BASTA (200 g L^-1 glufosinate ammonium; AgrEvo USA, Wilmington, DE) and 0.01% Silwet L-77 (Lehle Seeds, Round Rock, TX). Plants that survived this selection were sprayed again at 14 and 15 days after germination.

DNA Sequencing
To obtain the Ler-0 sequence, we fragmented the 9L9 cosmid and shotgun subcloned ~1-kb fragments into the M13 vector. Recombinant M13 clones that contained Ler-0 DNA were identified by hybridization and sequenced with the M13 forward primer. These random sequences were assembled into contigs, and gaps were filled by primer walking. To determine the Col-0 sequence, we isolated two contiguous, RPP8-hybridizing, BglII subclones from the 2D9 BAC that spanned RPP8. We obtained most of the sequence from both inserts with a collection of primers derived from the Ler-0 sequence. Gaps were filled by primer walking.

Sequence similarity searches were conducted using the BLAST program with default settings (Altschul et al. 1990 ). Conceptual translations, pairwise comparisons, and multiple alignments were performed with default settings using the Translate, Gap, and Pileup programs of the software package, version 9.1, from the Genetics Computer Group (Madison, WI). Nonsynonymous (K_a) and synonymous (K_s) substitutions were calculated using the Genetics Computer Group Diverge program, which corrects for multiple hits and unequal rates of transitions versus transversions. Residues at the position designated by an L in the XX(L)X(L)XXXX motif were omitted from the calculations of K_a and K_s, based on the rationale that they are under selection for conservation of function (Parniske et al. 1997 ). Polymorphic sites were displayed with the Sequence Output program (B.G. Spratt, University of Sussex, Brighton, UK).

	FOOTNOTES

¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

We gratefully acknowledge the following contributions: Miguel Botella, Mark Coleman, and Jonathan Jones for providing Ler-0 genomic and cDNA libraries; Guillermo Cardon for providing the Spl2 sequence; Canan Can and Patricia Chimot for assistance with P. parasitica isolate characterization and confirmation of pathology data; Claire Lister, Caroline Dean, and Renate Schmidt for assistance with genetic and physical mapping; and Ian Crute, Ben Holt, Susanne Kjemtrup, and Rheinhard Kunze for providing helpful comments on the manuscript. This work was supported by grants from the U.S. Department of Agriculture National Research Initiative Competitive Grants Program (to J.L.D.), the U.K. Biotechnology and Biological Sciences Research Council (to E.B.H.), and The Netherlands Technology Foundation (to M.G.M.A.). J.M.M. was supported by a postdoctoral fellowship from the National Institutes of Health.

Received June 23, 1998; accepted September 11, 1998.

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