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Copyright 2005 by the Genetics Society of AmericaDOI: 10.1534/genetics.104.039339

Natural Variation in the Pto Pathogen Resistance Gene Within Species ofWild Tomato (Lycopersicon). I. Functional Analysis of Pto Alleles

Laura E. Rose,*,1 Charles H. Langley,* Adriana J. Bernal and Richard W. Michelmore

*Center for Population Biology, Department of Plant Pathology and The Genome Center and the Department of Plant Sciences,University of California, Davis, California 95616

Manuscript received December 3, 2004Accepted for publication May 30, 2005

ABS TRACT

Disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) in the cultivatedtomato, Lycopersicon esculentum, and the closely related L. pimpinellifolium is triggered by the physicalinteraction between plant disease resistance protein, Pto, and the pathogen avirulence protein, AvrPto.To investigate the extent to which variation in the Ptogene is responsible for naturally occurring variationin resistance to Pst, we determined the resistance phenotype of 51 accessions from seven species ofLycopersicon to isogenic strains of Pst differing in the presence of avrPto. One-third of the plants dis-played resistance specifically when the pathogen expressed AvrPto, consistent with a gene-for-gene in-teraction. To test whether this resistance in these species was conferred specifically by the Ptogene, allelesof Pto were amplified and sequenced from 49 individuals and a subset (16) of these alleles was tested in

plantausing Agrobacterium-mediated transient assays. Eleven alleles conferred a hypersensitive resistanceresponse (HR) in the presence of AvrPto, while 5 did not. Ten amino acid substitutions associated with theabsence of AvrPto recognition and HR were identified, none of which had been identified in previousstructure-function studies. Additionally, 3 alleles encoding putative pseudogenes of Ptowere isolated fromtwo species of Lycopersicon. Therefore, a large proportion, but not all, of the natural variation in thereaction to strains of Pst expressing AvrPto can be attributed to sequence variation in the Pto gene.

THE specificity of interactions between plants andpathogens is often determined by single genes inboth host and parasite. The genetic basis for diseaseresistance in plants and virulence in pathogens was firstdetermined in detail by Flor(1956). Studies of flax andflax rust showed that resistance occurs only when thedominant resistance allele (R-gene) in the plant ismatched by a dominant allele for avirulence in thepathogen (Flor1971; Islam and Mayo 1990; Islam andShepherd 1991). This gene-for-gene model led to thehypothesis that pathogens express ligands that aredetected by receptors in resistant plants (Pryor 1987).Specific recognition of a pathogen by these R-gene re-ceptors initiates a signal transduction cascade leadingto resistance (reviewed in Dangl and Jones 2001;Hammond-Kosack and Parker 2003; Innes 2004).In the absence of matching avirulence and resistance

genes, a compatible interaction ensues and the path-ogen successfully colonizes the host.

The highlyspecific gene-for-geneinteraction betweenhost and pathogen creates complex and dynamicselection pressures on both host and pathogen. Recip-

rocal selection on plant and pathogen populationsresults in the coevolution of genes determining speci-ficity in both genomes. Negative frequency-dependentselection acting on loci determining resistance was rec-ognized over 50 years ago as potentially important inmaintaining polymorphism (Haldane 1949). Modelingof frequency-dependent selection in a two-allele systemin host and parasite confirmed this potential (Barrett1988; Seger 1988; Leonard 1994). However, elevatedlevels of polymorphism are not predicted in all scenariosof host-parasite coevolution. Under certain circum-stances a novel R-gene may spread to fixation. Direc-tional selection for resistance and virulence, rather thannegative frequency-dependent selection, could result inan arms race between host and pathogen and thesequential evolution and fixation ofR-genes (May and

Anderson 1983; Frank 1992).

Extensive intraspecific variation in phenotypic dis-ease resistance has been documented, often as the resultof germplasm screens. However, little is known aboutthe evolutionary dynamics of disease resistance in natu-ral plant populations. Population genetic analyses ofallelic variation in Arabidopsis (Rps2, Rpm1, and Rps5)and Lycopersicon spp. (Cf-9) revealed that pathogenrecognition specificity corresponded to sequence vari-ation at these loci (Caicedo et al. 1999; Stahl et al.1999; van der Hoorn et al. 2001; Tian et al. 2002;Mauricio etal. 2003). Additional sequence-based analyses

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. DQ019170DQ019221.

1Corresponding author:Department Biologie II, Grosshadernerstrasse 2,Planegg-Martinsried 82152, Germany.E-mail: [email protected]

Genetics 171: 345357 (September 2005)

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combined with functional tests are needed to explorethe evolutionary consequences of interactions betweenhosts and pathogens.

The interaction between species of Lycopersicon andthe bacterial pathogen, Pseudomonas syringae pv. tomato(Pst), provides an excellent opportunity for evolution-ary studies because both the pathogen ligand and re-sistance protein have been extensively characterized at

the molecular level (reviewed in Sessa and Martin2000; Bogdanove 2002; Wu et al. 2004). Since the phys-ical interaction of these molecules elicits the resistanceresponse (Scofield et al. 1996; Tang et al. 1996), theyprovide the possibility to study the origin and recogni-tion specificity of these molecules and the functionalconsequences of amino acid variation on an importantaspect of plant fitness. Furthermore, Lycopersicon hasmany useful attributes for population genetic studies.

All nine species are diploids and there are extensive,well-documented collections from natural populationsof all species in the group (http://tgrc.ucdavis.edu);also Lycopersicon esculentum, the cultivated tomato, is a

model species for plant genetics and detailed geneticmaps exist for several others (e.g., Tanksley et al. 1992;van Ooijen et al. 1994; Monforte and Tanksley 2000).

The Pto gene confers resistance to strains of Pst ex-pressing avrPtoand was introgressed into the cultivatedspecies, L. esculentum, from the sister species L. pimpi-nellifolium (Pilowsky and Zutra 1982; Martin et al.1993). Pto is a small gene; the open reading frame(ORF) consists of 963 nucleotides, has no introns, andencodes a functional serine-threonine kinase (Martinet al. 1993; Loh and Martin 1995). Specific residues

within the Pto resistance protein that are requiredfor ligand binding, autophosphorylation, and down-stream signaling have been identified through site-directed mutagenesis and domain swaps between Ptoand an allele of a related paralog, Fen, that encodesa protein lacking AvrPto recognition (Scofield et al.1996; Tang et al. 1996; Frederick et al. 1998; Rathjenet al. 1999; Sessa et al. 2000; Wu et al. 2004). Thecurrent model for Pto activation involves Pto bindingto AvrPto within the plant cell and then becomingactivated, possibly by a change in protein conforma-tion induced through ligand binding. The activatedPto protein then transduces the AvrPto signal andis dependent upon Prf to elicit the resistance re-

sponse (Rathjen et al. 1999; Sessa and Martin 2000;Bogdanove 2002).

The Ptoresistance gene belongs to a small multigenefamily of five to six members in Lycopersicon (Martinet al. 1993). The genomic region containing the Ptogene family has been sequenced from a susceptibleL. esculentum cultivar and a resistant cultivar contain-ing the introgressed Pto locus from L. pimpinellifolium(D. Lavelle and R. Michelmore, unpublished results;GenBank accession nos. AF220602 and AF220603).Orthologous relationships of the Ptogene family mem-

bers between the resistant and susceptible cultivars weredetermined on the basis of positional information andsequence identity. The two haplotypes share five orthol-ogous, clustered genes (Pth2, Pth3, Pth4, Pth5, and Fen).Paralogs ofPtorange in nucleotide sequence identity toPto from 79 to 91%. One of the paralogs, Fen, conferssensitivity to the insecticide fenthion (Martin et al.1994). Functions have not been ascribed to the other

paralogs, although most are expressed and several arecapable of downstream signaling (Chang et al. 2002).Members of the Pto gene family have also been se-quenced in another species of this genus, L. hirsutum(Riely and Martin 2001).

The pathogen, Pst, invades the intercellular space ofits host and secretes proteins into the host cells via atype III secretion system (Jin et al. 2003). One of thesesecreted proteins is AvrPto. The specific recognition of

AvrPto by the host Pto protein results in the induction ofthe hypersensitive response (HR), which is character-ized by localized cell death (Ronald et al. 1992; Martinet al. 1993). A second avirulence gene, avrPtoB, whose

product is also recognized by Pto, has been isolatedfrom Pst (Kim et al. 2002). This gene has little overallsequence similarity to avrPto. However, some of the sameresidues that are required for interaction of AvrPto

with Pto are present in AvrPtoB (Bernal et al. 2005).Avirulence conferred byavrPtoBis Pto dependent, indi-cating that Pto has dual recognition specificity for the

AvrPto and AvrPtoB ligands (Kim et al. 2002).In this article, we describe variation in disease resis-

tance within and among seven species of Lycopersicon.We determined the nucleotide sequences of 52 allelesof Pto and tested 16 alleles for specific recognition ofthe AvrPto ligand and activation of HR in planta. Thisanalysis allowed the functional characterization of 41

variant amino acid positions among these alleles.Additionally we identified three putative pseudogenesofPtosegregating within natural populations of speciesof Lycopersicon. The implications of the naturally oc-curring protein variation found at Pto and variation inhost resistance are discussed in the context of host-parasite coevolution.

MATERIALS AND METHODS

Plant materials: Populations of each species of Lycopersi-con were sampled across their ranges (Figure 1). Plants weregrown from seed collected from natural populations in Peruand Chile (Table 1). More information on these collections isavailable from the Tomato Genetics Resource Center (TGRC;http://tgrc.ucdavis.edu). Seed from additional populations

was obtained from the U.S. Department of Agriculture,Agricultural Research Service Plant Genetic Resources Unitin Geneva, New York. For the outbreeding species (L. chilense,L. hirsutum, L. pennellii, and L. peruvianum), this study utilizedthe primary seed collected from native populations. Forthe inbreeding species (L. chmielewskii, L. parviflorum, andL. pimpinellifolium), selfed seed was used. Seeds were soaked in

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a 50% bleach solution for 30 min and incubated on germina-tion paper (Anchor, St. Paul) at 22 with 24-hr fluorescentlight. Seedlings were transferred to soil 2 weeks after germi-nation and grown under greenhouse conditions at Davis,California. Cuttings to provide replicated materials were made

when the plants were 3 months old.Assays for resistance to P. syringae pv. tomato: A total of 51

individuals from seven species of Lycopersicon were testedwith isogenic strains of Pst (strain T1) expressing a vector-borne copy of AvrPto or containing the empty vector to deter-mine if the plants were resistant to this pathogen and whetherthe resistance was AvrPto dependent. Separate inoculations

with isogenic strains of Pst-T1 containing pDSK519:AvrPtoor pDSK519 (empty vector) were conducted for each plantgenotype. Bacteria were grown overnight in Kings B media[20 g/liter of Difco (Detroit) protease peptone no. 3, 1.5 g/liter of K2HPO4, 1.6 g/liter of MgSO43 7H20, and 10 ml/literof glycerol] containing 50 mg/ml rifampicin and 20 mg/ml ofkanamycin at 28 with shaking. Cells were washed in 10 mmMgCl2 and resuspended in 10 mm MgCl2 to an OD600 of 1.00.

The concentrated Pst solution was then diluted in 10 mmMgCl2 to 13 105 colony-forming units (CFU)/ml. Two or more

individuals from each population of the self-incompatiblespecies, excluding L. pennellii, were tested, while only a singleindividual per population of each self-compatible species wastested. Previous studies had demonstrated that populations ofthe self-compatible species tended to be monomorphic atmultiple loci and the greatest diversity could be detected bysampling between populations of self-compatible Lycopersi-con species (Miller and Tanksley 1990). Inoculations witheach bacterial genotype were replicated three times on eachhost. In results presented here, all independent replicatesshowed the same level of resistance to the corresponding

bacterial strain. As described above, multiple replicates of hostmaterial were made by propagating cuttings of each plant.Cuttings were madebefore theplants were inoculated with Pst.The near-isogenic L. esculentumgenotypes, cv. Rio Grande 76Rthat contains Pto, and cv. Rio Grande 76S that doesnot contain

Pto (Seminis Seeds, Woodland, CA), were used as controls.Vacuum infiltration was used to separately inoculate plants ofeach genotype with each pathogen strain. Following inocula-tion, plants were placed in a growth chamber at 80% humidity,25, and 16 hr of light. Three and 7 days after inoculation,plants were scored for visible symptoms of the disease (i.e.,

water-soaked lesions, black specks surrounded by chlorotichalos). Three leaf discs per plant (28 mm2 in area) were alsocollected at 3 and 7 days after inoculations to estimate thenumber of bacteria within these plants. These leaf discs wereground in 1 ml of 10% glycerol solution and serial dilutions

were plated on to Kings B medium (see above) to estimate thenumber of colony-forming units. A plant was classified asresistant if at 3 and 7 days after inoculation the ground leafdiscs showed the same or fewer CFUs as the Rio Grande 76R

resistant control plant that had been inoculated with Pstexpressing AvrPto during the same experiment. This corre-sponded to significantly,105 CFUs at 3 days after inoculation.

A plant was classified as susceptible if at 3 and 7 days afterinoculation it showed the same or more CFUs as compared tothe respective susceptible controls (i.e., Rio Grande 76R in-oculated with Pstexpressing empty vector and Rio Grande 76Sinoculated with either strain ofPst). This corresponded to.107

CFUs at 3 days after inoculation.Analyses of genomic DNA: DNA was isolated using a CTAB

method (Doyle and Doyle 1987) from 2 g of leaf tissuecollected from each plant. The DNA was resuspended in3001000 ml TE, depending on yield. Alleles ofPtofrom each

Figure 1.Map of western coast of SouthAmerica indicating locations of populationsused in this study. The populations are labeledby an abbreviated form of the species nameand an accession number corresponding tothe accession numbers in Table 1.

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TABLE 1

Origin and resistance phenotype of individuals in this study

Population(individual identifier)a Locality

Pst1 emptyvectorb Pst1 AvrPto Pto allelec Trans. assaye

L. pimpinellifoliumLA114 Pacasmayo, Peru S S pim114cLA373 Culebras, Peru S R pimPto2

LA400 Haciendo Buenos Aires, Peru S R pimPto HR LA411 Pichilingue, Ecuador S R pimPto2

L. parviflorumLA247 Chavinillo, Peru S S NDd

LA1322 Limatambo, Peru R R parv80 HR LA2200 Choipiaco, Peru R R parv94 HR LA2133 Ona, Ecuador R R ND

L. chmielewskiiLA1306 Tambo, Peru R R chm106 HR LA2695 Chihuanpampa, Peru R R chm115 HR LA3653 Matara, Peru S S chmPto HR

L. hirsutumLA 407 (c1) Mirador, Ecuador S R hir540

LA 407 (c2) Mirador, Ecuador S R LA1775 (7216) Rio Casma, Peru R R NDLA1775 (7219) Rio Casma, Peru hir46 No HR LA1777 (7220) Rio Casma, Peru R R NDPI 129157 (c1) Banos, Ecuador R R hir132 HR PI 129157 (c2) Banos, Ecuador S R PI 134417 (c1) Ferrocarril a la Costa, Ecuador R R hir183 HR PI 134417 (c2) Ferrocarril a la Costa, Ecuador S R PI 134418 (c1) Ferrocarril a la Costa, Ecuador R R hir186 HR PI 134418 (c2) Ferrocarril a la Costa, Ecuador R R PI 251305 (c1) Sibambe, Ecuador R R hir137 HR PI 251305 (c2) Sibambe, Ecuador R R

L. pennelliiLA716 Atico, Peru S S ND

LA1912 Cerro Locari, Peru S S NDLA3791 Caraveli, Peru S S ND

L. chilenseLA460 (c1) Palca, Peru R R LA460 (c2) Palca, Peru R R LA2884 (7177) Ayaviri, Chile chi487 No HR LA2884 (7179) Ayaviri, Chile chi489 HR LA2884 (7180) Ayaviri, Chile chi493LA2884 (7183) Ayaviri, Chile chi497LA2884 (7184) Ayaviri, Chile chi502LA2884 (7185) Ayaviri, Chile S R chi580 HR LA2750 (1) Mina La Despreciada, Chile S S chi558 No HR LA2750 (2) Mina La Despreciada, Chile S S chi591LA3355 (7186) Cacique de Ara, Peru R R chi151 chi551 HR

LA3355 (7189) Cacique de Ara, Peru S R chi151 chi620 HR LA3355 (7190) Cacique de Ara, Peru R R chi566 chi565

L. peruvianumLA2744 (1) Sobraya, Chile S S peru554 HR LA2744 (2) Sobraya, Chile S S peru554 HR LA2744 (7232) Sobraya, Chile peru505c peru602cLA2744 (7233) Sobraya, Chile peru603LA2744 (7234) Sobraya, Chile S S peru515 peru605LA2744 (7235) Sobraya, Chile peru517 peru518 HR LA2744 (7236) Sobraya, Chile peru608 peru521

(continued)


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species were PCR amplified using Pfupolymerase (Stratagene,La Jolla, CA). Reaction times and annealing temperatures

varied depending on the primer used (supplementary TableS1 at http://www.genetics.org/supplemental/). However, thestandard protocol was 94 for 5 min, 253 (94 for 30 sec,5060 for 30 sec, 72 for 90 sec), followed by 72 for 10 min.Products were gel purified using QIAGEN (Valencia, CA)Gene Clean or Prep-A-Gene (Bio-Rad, Hercules, CA) kits.

These products were cloned into the pCR-Blunt, pCR2.1-TOPO, or pDONR vectors (Invitrogen, Carlsbad, CA). Mul-tiple (101) clones were sequenced from 49 individualsrepresenting seven species of Lycopersicon and one speciesof Solanum. Sequencing was performed using an ABI 377automated DNA sequencer. A total of 552 clones yielded 163unique sequences of the Pto gene family. Nearly one-thirdof these unique sequences (51) encoded Pto alleles, whiletwo-thirds (112) encoded paralogs of Pto including Fen, Pth2,Pth3, Pth4, and Pth5. Sequences were aligned by ClustalX(Thompson et al. 1997) and these alignments were refined vi-sually. Phylogenetic analyses, including the Kishino-Hasegawatest, were completed using PAUP (Swofford 1999).

Genomic Southern blots were conducted to determine thecomplexity and level of haplotype variation within and be-

tween species of Lycopersicon. Genomic DNA of 25 individ-uals was digested with DraI, which digests each of the paralogsin L. pimpinellifolium and L. esculentum into individual frag-ments. The digested DNA was fractionated electrophoreticallyand transferred to a Hybond N1 membrane (Amersham Bio-sciences, San Francisco). The Pto ORF was used as the DNAtemplate for probe synthesis using PCR. The blot was hy-bridized with the 32P-labeled probe at 65 and washed at 0.53SSPE, 0.1% SDS. The membrane was exposed to film at80.

Functional in planta analysis of Pto alleles from multipleLycopersicon species: The function of 16 Pto alleles from

wild Lycopersicon species was tested using Agrobacterium-mediated transient expression in a transgenic line ofNicotiana

benthamiana that had been previously engineered to containthe avrPtogene under a dexamethasone (DEX)-inducible pro-moter (Chang etal. 2002).These16 Ptoalleles were selected torepresent the sequence diversity found among the differentspecies of Lycopersicon. The Ptogenes that had been clonedinpCR2.1-TOPO were excised using XbaI and EcoRI, ligated intothe T-DNA vector pCB302-3 (Xiang et al. 1999), and trans-formed into strain GV2260 of Agrobacterium tumefaciens by

the freeze-thaw method. A 2-ml Luria broth (LB) culture wasgrown for 2 days at 28. A 7.5-ml culture was inoculated with0.2 ml of the 2-day-old culture and this culture was grownovernight in LB with 100 mm acetosyringone at 28. Thebacteria were washed in infiltration buffer (10 mm MES pH5.6,10 mm MgCl2,150mm acetosyringone)and diluted to an OD600of 1.00. Leaves of 7-week-old transgenic N. benthamianaplantscarrying the avrPtogene under the control of a DEX-induciblepromoter (Chang et al. 2002) were pressure infiltrated with A.tumefaciens carrying alleles of Pto from the wild Lycopersiconspecies. Up to eight alleles were tested per leaf. Two days afterinfiltration, expression ofavrPtowas induced by swabbing theleaves with 30 mm DEX. One leaf per plant was induced withDEX and one was not. Leaves of nontransgenic control plants

were also treated with DEX. Leaves were scored for macro-

scopic HR 24 hr after induction with DEX. Transient assays ofeach allele were replicated on at least two avrPto-expressingplants, one plant transformed with the empty vector and onenontransgenic N. benthamianaplant.

Site-directed mutagenesis and yeast two-hybrid analysis: Toinvestigate the functional effects of particular amino acidsubstitutions, additional functional analyses involving a subsetofPtoalleles were carried out. Specifically site-directed muta-genesis was used to replace the alanine residue at position 313in the protein sequence of Pto from L. pimpinellifoliumand theL205D-Pto (constitutively active mutant) by a glutamic acidto investigate how a negatively charged amino acid at thisposition affected Pto function. Site-directed mutagenesis was

TABLE 1

(Continued)

Population(individual identifier)a Locality

Pst1 emptyvectorb Pst1 AvrPto Pto allelec Trans. assaye

L. peruvianumLA3636 (7252) Coayllo, Peru S R peru592 No HR LA3636 (7258) Coayllo, Peru S R peru570 No HR

LA3636 (7259) Coayllo, Peru S R peru594LA3636 (7260) Coayllo, Peru S R peru567 HR PI126444 (1) Rio Canta, Peru S S peru245 peru246PI126444 (2) Rio Canta, Peru S R PI128654 (1) Azapa Valley, Chile S R peru234 peru236PI128654 (2) Azapa Valley, Chile S SPI128659 (1) Tacna, Peru S S peru238 peru239PI128659 (2) Tacna, Peru S R peru243LA2151 (1) Morochupa, Peru R R LA2151 (2) Morochupa, Peru R R LA3218 (1) Arequipa, Peru R R LA3666 (1) Ica, Peru R S

a Identifier of specific individuals of these populations.b Plants resistant (R) or susceptible (S) when inoculated with isogenic strains ofPstexpressing empty vector or avrPtobased on

pathogen growth.c Name of Pto allele isolated from this individual.d Pto allele not detected (ND) even though multiple independent PCR reactions were attempted. In all individuals, alleles of

other paralogs were amplified and sequenced, indicating that the gene family does exist in these individuals.e Results of transient expression of these Pto alleles in N. benthamiana (HR, hypersensitive response).


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carried out by recombinant PCR using a mixture of Klentaq(DNA PolymeraseTechnology, St. Louis) and Pfupolymerases.Oligonucleotides that introduced the desired mutations wereused. Products were cloned in pDONR207 using the Gatewaycloning system (Invitrogen).The pCB302-3 (Xiang etal. 1999)and pAS2.1 vectors were modified to be compatible with theGateway system as described by themanufacturer. Clonesfrom

which the expected sequence was confirmed were transferredto the pCB302-3 and pAS2.1 vectors.

Yeast two-hybrid analysis of two alleles from L. hirsutumand

one allele from L. pimpinellifolium was conducted using theMATCHMAKER GAL4 system following the protocols of themanufacturer (CLONTECH, Palo Alto, CA). These sequences

were cloned in pAS2.1 and were cotransformed with avrPtoinpACT2 (Scofield et al. 1996) into yeast strain Y190. Cotrans-formed yeast cells were selected by growth on selective mediaand replicated on filters (Whatman no. 1) prior to assaying foractivation of b-galactosidase using the substrate 5-bromo-4-chloro-3-indolyl b-d-galactoside (X-gal). The b-galactosidaseassays were performed as described by CLONTECH.

RESULTS

Variation in resistance within and between species:

Levels of disease resistance to two pathogen isolates dif-fered between individuals of both self-compatible andself-incompatible species (Table 1). Resistance varied notonly within species, but also within populations of thefollowing species:L. hirsutum, L. chilense,and L. peruvianum.One-third of the plants tested were resistant specifically

when the pathogen was expressing the AvrPto ligandand susceptible otherwise, consistent with the conser-

vation of AvrPto recognition conferred by the Ptogenein these different species. One-quarter of the plants weresusceptible whether or not the pathogen was expressingthe avrPto gene.

Approximately 37% of the accessions were resistant toPstregardless of the expression of AvrPto. These plantsmay recognize other avirulence proteins expressed bythe T1 strain of Pst. Resistance to Pst independent ofavrPtoexpression by the pathogen reflects the evolutionof recognition involving other determinants of aviru-lence. An unexpected phenotype was observed in indi-

viduals from the LA3636 population of L. peruvianum.These plants were susceptible specifically to Pst withavrPto, but not to Pst expressing empty vector. One ex-planation for this unexpected phenotype is that theseindividuals may be capable of recognizing an as yet un-characterized avirulence factor from Pst strain T1;

however, this recognition may be specifically nullifiedby the action of AvrPto.

Functional analyses of Pto alleles: Alleles from bothsusceptible and resistant individuals were expressedtransiently in plants known to have an intact down-stream Pto signaling pathway to determine whetherthese alleles encoded proteins capable of recognizing

AvrPto and initiating downstream signaling. These al-leles were derived from a total of 22 individuals from thedifferent Lycopersicon species, except L. pennelliifrom

which no Ptoalleles could be amplified. Alleles from 11

individuals conferred an AvrPto-dependent HR (Table 1;Figure 2). These alleles conferring AvrPto recognitionin the transient assay were designated A1 alleles. Themajority of the A1 alleles (6/11) originated fromindividuals that were resistant to Pst, regardless of theexpression of AvrPto by the pathogen. Since these in-dividuals are also resistant when AvrPto is not expressedby the pathogen, they must be capable of detecting mul-tiple avirulence determinants. Three of the A1 allelesoriginated from individuals that exhibited an AvrPto-dependent resistance when inoculated with Pst. Two A1alleles (peru554 and chmPto) were isolated from plantsthat were susceptible to Pst whether or not AvrPto wasexpressed by the pathogen. The lack of resistance inthese plants may be dueto downstreamgenes being non-functional or the lack of expression of these Ptoalleles.

Five alleles did not confer an HR when transientlyexpressed in the presence of AvrPto. These alleles were

designated the A

alleles. One of these A

alleles wasisolated from a plant that was susceptible to both strainsofPst, consistent with this plant lacking a version of Ptothat could recognize the AvrPto ligand. Two additionalA alleles were isolated from plants that showed AvrPtorecognition in the inoculation assays with the pathogen;i.e., these plants possess A alleles although they wouldhave been expected to possess A1 alleles. Both of theseplants were from the LA3636 population ofL. peruvianum.Given the high levels of polymorphism observed inthis species, it is possible that these individuals were

Figure 2.Variable amino acids in the alleles transientlyexpressed in the susceptible N. benthamiana line containingavrPto. The numbers at the top indicate the corresponding co-don position in the predicted amino acid sequence of the Ptoallele (LA400) from L. pimpinellifolium. Positions invariantamong these 16 alleles are removed. A dot indicates an aminoacid residue matching the LA400 sequence, while a dash in-dicates a gap. (A) The first 11 alleles conferred an avrPto--dependent HR (hypersensitive response). These alleles weredesignated A1 alleles. (B) The last five alleles did not conferan HR. These alleles were designated A alleles. Amino acidsubstitutions limited to the alleles that did not confer an HRare underlined. The reactionof the genotypesfrom whicheachallelewas derived when inoculated with Pstexpressing avrPtoisindicatedto theright of theallele sequence (Res,resistant; Sus,susceptible; and n.t., not tested).


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heterozygous atPto and only one Ptosequence was am-

plified from these individuals. Thus, these individualscould have possessed a second allele conferring an A1phenotype. Alternatively, the product of another genemay recognize AvrPto in these plants. The remainingtwo A alleles were isolated from plants that were nottested for resistance to Pst.

In summary, for nearly three-quarters (74%) of theplants for which both assays were conducted, a 1:1 cor-respondence was observed between recognition of Pstexpressing avrPtoand activation of HR in the presenceof AvrPto in the transformation assay (Figure 3). In gen-eral, plants that were resistant to Pst expressing AvrPto

were also likely to possess A1 alleles and vice versa. Theexceptional cases, in which the outcomes of the twoassays differed from each other (i.e., the plant wasresistant but its Pto allele was A or the plant wassusceptible but its allele was A1), may reflect equallyinteresting biological phenomena, such as convergentevolution for resistance and/or the role of additional locithat are functionally relevant for resistance (Figure 3,shaded cells).

Origin of A1 and A alleles: The nucleotidesequences of an additional set of 36 alleles were de-termined from these seven species of Lycopersicon. Aphylogenetic analysis of the nucleotide sequences of all

Pto alleles suggested that the A

alleles were indepen-dently derived, because the A sequences are distrib-uted across the tree rather than clustered together(Figure 4). To test for the independent origin of the Aalleles, maximum parsimony trees of the A1 and Aalleles were generated. A tree in which the A alleles

were constrained to be monophyletic was comparedto the tree in which the A1 and A alleles were notconstrained. The constrained tree was significantlylonger (i.e., a worse fit to the data) on the basis of theKishino-Hasegawa test, rejecting the hypothesis thatA

alleles have a single origin (P, 0.0001; Kishino andHasegawa 1989). Unsurprisingly, no single amino acidchange or group of changes united the A alleles anddistinguished them from the A1 alleles (Figure 2).

Functional effects of amino acid substitutions in Pto:Since each A allele was unique, the sequence of eachallele was compared in turn to the 11 amino acid se-quences of the A1 genes. Substitutions unique to the

A

alleles are candidates for mutations that directlyaffect pathogen recognition and resistance in theseindividuals or indirectly affect the phenotype by alteringprotein stability. These substitutions and their positionsare summarized in Table 2. The positions of thesesubstitutions on the predicted three-dimensional struc-ture of the Pto protein are illustrated in supplementaryFigure S1 (http://www.genetics.org/supplemental/).The chi558 allele has three unique substitutions: twoconservative and one nonconservative. The nonconser-

vative substitution at site 135 from a serine (polar) toa phenylalanine (nonpolar) occurs at the junctionbetween domains V and VIa. This region forms an ex-

tended chain that connects two a-helices and is impor-tant for anchoring the ATP molecule (Hanks andHunter1995). A second A allele, peru592, has nearlythe same amino acid sequence as chi558. In addition tothe nonconservative change at site 135, this protein hasanonconservative change at site 217 [arginine (1charge)to a glycine (polar)]. This substitution occurs in domainIX along an exposed part of the activation domain.Peru570 has two nonconservative substitutions: site 168isoleucine (nonpolar) to threonine (polar) and 178 pro-line (nonpolar) to threonine (polar). The 168 substitu-tion is the first amino acid following the catalytic loopregion. This region is involved in phosphoryl-catalysisand transfer. The second substitution occurs in domain

VII, which is made up of two b-sheets and an interveningloop. This substitution is embedded in theb-sheet, priorto the activation domain.

The fourth A allele, chi487, has only one uniquesubstitution relative to the A1 sequences. Alanine 313(nonpolar) is replaced by a glutamic acid (negativecharge). This substitution lies outside the 11 conservedkinase domains close to the C terminus. The structureand function of this regionhave not been defined (Hanksand Hunter 1995). Site-directed mutagenesis of the

wild-type Pto replacing alanine 313 with an aspartic acid

(chemically similar to glutamic acid) results in the loss ofHR induction in transient assays in planta(Bernal et al.2005). Also, the gain-of-function phenotype is lost whenthe same change is made in the constitutively active PtoL205D allele, indicatingthat a negatively chargedaminoacid at position 313 either alters protein stability ordisrupts downstream signaling rather than recognitionof AvrPto per se(Bernal et al. 2005).

Hir46 had three unique amino acid substitutions ofwhich one change was nonconservative: at site 104, a cys-teine (polar) is changed to a phenylalanine (nonpolar).

Figure 3.Summary of results from plants tested for resis-tance to Pst expressing AvrPto and from which the allelestested in the transient assay were derived. The top numberis the number of observations per category and the bottomnumber is the percentage of observations per category. Openblocks show a 1:1 correspondence between the resistance as-say and transformation assay. Shaded blocks indicate those ob-servations that suggest that additional loci are involved indisease resistance.


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This substitution is in subdomain IV of the proteinkinase, which forms a large hydrophobic b-strand in thesmall lobe of the protein. The substitution of a bulkynonpolar amino acid at this site may distort the protein

structure sufficiently to eliminate function. This allelealso has an isoleucine-to-leucine substitution at posi-tion 155 and a leucine-to-isoleucine substitution at po-sition 185, but due to the conservative nature of thesesubstitutions, it is less likely that these changes wouldimpair function. Yeast two-hybrid analysis of the hir46allele demonstrated that this allele failed to bind the

AvrPto ligand in yeast. This allele is most similar tohir183, which elicited an AvrPto-specific HR in the tran-sient assay and interacted with AvrPto in the yeast two-hybrid assay. Three of the seven amino acid differences

between these two alleles were unique to the non-functional hir46 allele compared to the larger data setof Pto alleles. Assuming that recognition of AvrPto hasbeen conserved among alleles of Pto, then the loss of

function in hir46 may have been specifically causedby one or more of these substitutions affecting either

AvrPto recognition or protein stability rather than down-stream signaling.

The presence of polymorphic residues among the A1alleles indicates that at least some variation is toleratedat these positions. Thirty sites and one indel were poly-morphic within the A1 class, indicating that these sitesdo not affect AvrPto recognition and downstream sig-naling (Figure 2). Of the 30 polymorphic sites, twocodons hadthree amino acids segregating, while the rest

Figure 4.One of 100 equally mostparsimonious trees of the nucleotide se-quences of alleles of Pto. The tree wasrooted using the Pth2, Pth4, and Fengenes. Alleles are named by a short ab-breviation of the species name followedby an arbitrary number. The source ofthe allele according to population is in-dicated further to the right by the LA

and PI accession numbers. The re-sponses of alleles tested in the transientassay are indicated by HR (hypersensi-tive response) or no HR. The resistanceresponses to Pst expressing AvrPto ofthe plants from which each allele wasderived are indicated (R, resistant;S, susceptible).


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segregated only two amino acids. Eighteen were non-conservative substitutions located throughout the pro-tein (supplementary Figure S1 at http://www.genetics.org/supplemental/).

Pseudogenes of Pto: Individuals of two differentspecies of Lycopersicon had frameshift mutations intheir alleles of Pto (supplementary Table S2 at http://

www.genetics.org/supplemental/). The pim114c allelefrom L. pimpinellifolium had a 4-bp deletion at position168 of the nucleotide alignment, in addition to 14 otherunique mutations. Phenotypic assays confirmed thatthis plant was susceptible to Pst, consistent with thisplant lacking a functional allele of Pto. Phylogeneticanalysis showed that this allele shared its most recentcommon ancestor with alleles from L. parviflorumand L. chmielewskii rather than with alleles from otherL. pimpinellifoliumpopulations, a result that, taken withthe high number of observed unique variants, indicatesthat this is an ancient pseudogene (Figure 4). A singleindividual from L. peruvianumpopulation LA 2744 hadtwo distinct pseudogenes (peru602c and peru505c),

each containing a different frameshift mutation. Thiswas the only individual of seven studied from this pop-ulation from which putative pseudogenes were identi-fied. Ptoalleles derived from all other individuals in thispopulation do not contain frameshift or nonsense mu-tations and the two alleles tested from this populationelicited HR in the transient assay. The Pto allele fromSolanum ochranthum, a close relative of Lycopersicon,also has a frameshift mutation caused by a 10-bp de-letion with respect to the other Ptoalleles. This allele didnot elicit the HR when tested in transient assays of

N. benthamiana. In addition, two alternate approaches torestore the open reading frame using site-directed mu-tagenesis (one involving an insertion of 2 bp to restore asingle missing codon and the other involving the in-sertion of the 10 bp to restore the four missing codons

with respect to the other alleles) failed to restore this HR-inducing activity, indicating that at least one of the other16 unique amino acid substitutions impairs function.

Loss of the Pto gene: Attempts were made to amplifyPto alleles from all Lycopersicon species. However,multiple PCRs on some individuals of L. pennellii,L. parviflorum, and L. hirsutum never yielded alleles ofPto. Alleles of paralogs of Pto were amplified and se-quenced from the same individuals, indicating that

while other members of the gene family were present,the Pto gene may have been specifically absent. Thethree individuals of L. pennellii from different popula-tions and the L. parviflorum LA247 individual weresusceptible to Pst with and withoutavrPto, which is con-sistent with the absence of a functional Pto gene.Individuals from LA2133 (L. parviflorum), LA1775

(L. hirsutum), and LA1777 (L. hirsutum) also were miss-ing Pto but were resistant to Pst both with and withoutavrPto, suggesting that resistance to Pst was not due toPtoin these individuals.

To determine if the failure to amplifyPtofrom specificindividuals was correlated with the absence of specificfragments in genomic Southern hybridizations, band-ing patterns were compared between individuals withina given species that had Pto to those lacking Pto fromthe same species (Figure 5; supplementary Table S3 athttp://www.genetics.org/supplemental/). In L. parviflorum,

TABLE 2

Summary of unique substitutions in alleles that were A in the transient expression assay

Allele Individual

No. and type ofunique

substitutionsPosition of nonconservative

substitutions

Putative function of subdomainin which nonconservative

substitution was found

chi558 LA2750 (1) 2 C,a 1 Nb S135F: junction of domains V andVIa

Region forms an extended chainbetween two a-helices, important

for anchoring the ATP moleculechi487 LA2884 (7177) 1 N A313Ec: outside domain XI, close toC terminus

The structure and function of thisregion is undefinede

peru592 LA3636 (7252) 1 C, 2 N R217G: domain IXS135Fd

Exposed part of the activationdomain

peru570 LA3636 (7258) 2 N I168T: catalytic loop regionP178T: domain VII in the b-sheet,

upstream of activation domain

Phosphoryl-catalysis and transfer

hir46f LA1775 (7219) 2 C, 1 N C104F: domain IV Forms a large hydrophobic b-strandin the small lobe of the protein

a Conservative amino acid substitution.b Nonconservative amino acid substitution.c The introduction of aspartic acid at this position in the functional Ptoallele from L. pimpinellifoliumresults in the loss of HR in the

transient assay. Also the gain-of-function phenotype is lost when this substitution is made in the constitutively active form of Pto (L205D).d

See chi558 allele.e Hanks and Hunter (1995).fYeast-two-hybrid analysis demonstrated that this allele failed to bind the AvrPto ligand in yeast.


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there was a correlation between band number and thepresence or absence of Pto. The two individuals fromLA2200 and LA1322 both had alleles ofPtoand had sixstrongly hybridizing bands. The individual from LA2133that lacked Pto had five rather than six fragments. Theindividual from LA247 also lacked Pto and had only

four hybridizing bands. Although this species is self-compatible, three different banding patterns were iden-tified in the four individuals from different populations,reinforcing the observation that substantial structuralpolymorphism exists at this locus, even within the self-compatible species. Southern analysis ofL. parviflorumtherefore supported the hypothesis that Pto was absentfrom these genotypes.

A correlation between banding pattern and the pres-ence or absence ofPto was not detected in L. hirsutum.

All individuals had five bands even though two individ-

uals had Pto and the other two did not. The bandingpatterns of all three individuals ofL. pennellii were sur-

veyed because none of these individuals yielded a copyofPtoand all were susceptible to both strains ofPst. Eachbanding pattern was different and there were at least sixstrongly hybridizing bands per individual. Southernhybridizations therefore confirmed that the Pto familyis present in these two species. The lack of amplification

of a Pto allele could have been due to its absence orsequence divergence at a primer binding site.

DISCUSSION

Up until now, studies of the molecular evolution ofresistance genes in plants have been limited to a fewmodel plant species and their closest relatives (Caicedoet al. 1999; Stahl et al. 1999; van derHoorn et al. 2001;Tian etal. 2002; Mauricio et al. 2003). Here we describethe sequence variability at the Pto locus among sevenclose relatives of the model plant species L. esculentum.

We characterized the phenotypic variability in these

plant species and related the amino acid sequencevariation at the Pto locus to differences in recognitionof a pathogen molecule and activation of downstreamresistance response. This is currently one of the mostextensive studies of diversity in the function of alleles ata single R-gene across species.

Plants of different Pto genotypes show functionaldifferences in their resistance to Pstexpressing AvrPto.

We observed the cooccurrence ofPtoalleles capable ofAvrPto recognition, those incapable of AvrPto recogni-tion, and pseudogenes not only within species, but alsosegregating within single populations (e.g., L. peruvianumand L. chilense). The transient expression of 16 alleles insusceptible plants allowed us to characterize the func-tional effects of 41 substitutions (i.e., 12.7% of the totalprotein) that were distributed across the molecule(supplementary Figure S1 at http://www.genetics.org/supplemental/). Amino acid variation in the alleleslacking AvrPto recognition was not localized to regionsknown to be involved in ligand binding or downstreamsignaling (e.g., the activation domain) and there wereno obvious differences in the patterns of variation be-tween the alleles with and without AvrPto recognition.This lack of differentiation could be due in part to ourincomplete understanding of the functional regions of

the protein. Although the Pto gene is one of the best-characterized R-genes and previous domain swap, mu-tational analyses, and DNA shuffling experiments havecharacterized some of the functional domains of Pto,the precisecrystalstructure is not known anda thoroughmolecular dissection of Pto is still underway (Wu et al.2004, Bernal et al. 2005). However, the observed distri-bution of the naturally occurring substitutions that af-fect function suggests that there are several regionscritical to function in addition to those previouslyimplicated.

Figure 5.Southern blot hybridization of 26 accessions ofLycopersicon and Solanum species using the entire openreading frame ofPtoas a probe. See materials and methods

for details. Above each lane is the abbreviated name of thespecies and the population or individual number of eachplant sample. Pim (76R) is the L. esculentum cultivar contain-ing the Pto region introgressed from L. pimpinellifolium.F1 (LA400 3 LA114) is an F1 individual of a cross betweenthe LA400 and LA114 individuals.


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Amino acid substitutions that are restricted to allelesthat did not elicit an HR in response to AvrPto are can-didates for sites that may alter protein stability, disruptligand binding, or interfere with downstream signaling,

while polymorphisms among alleles that confer AvrPtorecognition identify substitutions that are nonessentialfor AvrPto-dependent resistance. There was as much

variation among Ptoalleles that elicit an AvrPto-specific

HR (A1

alleles) as there was between this group and thegroup of alleles that did not recognize AvrPto in planta(A1 vs. A alleles). The 5 sequences that failed to con-fer an AvrPto-dependent response appear to be inde-pendently derived, each differing by only a few aminoacids from one of the 11 different sequences that con-ferred an AvrPto-dependent HR. Ten substitutions werelimited to the class of alleles that lacked AvrPto rec-ognition and downstream signaling, none of which hadbeen identified in previous structure-function studies ofPto utilizing induced and site-directed mutation ana-lyses and domain swaps.

Previous work by Rathjen et al. (1999) demonstrated

that specific amino acid substitutions in the P 1 1 loopof the activation segment of Pto lead to a constitutivegain-of-function (CGF) phenotype (induction of HR) inthe absence of AvrPto. More recently, Wu et al. (2004)used structural modeling of Pto to predict which aminoacid residues of the Pto protein are in close proximity totheP1 1 loop and therefore might also play a role in theregulation and function of Pto. Residues in close prox-imity to the activation loop were systematically mutatedto investigate the extent of the negative regulatorypatch. In total, Wu et al. mutated 38 residues and testedthese molecules for the CGF phenotype and the abilityto bind AvrPto and AvrPtoB in yeast. The vast majority ofthe residues investigated by Wu et al. (33 of 38) wereinvariant among our 16 alleles derived from naturalpopulations. The high level of protein conservation atthese positions can be explained in part by the fact thatmutations in these residues are likely to have deleteriouseffects in nature. In particular, 12 of these mutationslead to the CGF phenotype and substitutions leading touncontrolled cell death of host tissue are unlikely to befound in natural plant populations. Therefore over one-third of the mutations tested by Wu et al. may be stronglydeleterious and would be removed by natural selection.

Among our 16 alleles, we detected natural variation at

five sites mutated by Wu et al. However, none of thesenatural variants showed the same amino acid residue astested by Wu et al. At three positions, the variationobserved was found only among A1 alleles in our study,making these unlikely candidates for affecting AvrPtorecognition and signaling of HR in natural populations.Two positions that were predicted to be exposed res-idues by Wu et al. (isoleucine at position 168 andisoleucine at position 185) showed polymorphismsexclusively in our A alleles, bolstering the hypothesisthat these are functionally important residues; however,

the specific mutations studied by Wu et al. did not matchthose amino acid substitutions that were segregating inour natural populations. The fact that the functionallyimportant residues defined in these two studies do notshow greater overlap is perhaps not surprising consid-ering that each work addressed different aspects of Ptofunction. In our study of natural variants, we anticipatedrecovering at least some variants that differed in rec-

ognition specificity, but we could not predict a prioriwhich types of variants we would recover. In contrast, theWu et al. study specifically targeted sites predicted to beinvolved in negative regulation of the protein. There-fore these studies provide complementary data sets forthe dissection of the structure and function of resistanceproteins.

In parallel to the current study, we used DNA shuf-fling, a PCR-based combinatorial method for generat-ing large numbers of variant progeny molecules from aset of related template molecules in vitro, to investigatefunctionally important regions of the Pto molecule(Bernal et al. 2005). Pto was shuffled with four of its

paralogs and the resulting recombinant molecules weretested for their interaction with AvrPto and AvrPtoB in

yeast two-hybrid assays and their activation of HR in anAvrPto- or AvrPtoB-dependent manner in planta. Ninecandidate regions important for binding to AvrPto orfor signaling downstream were identified by statisticalcorrelations between individual amino acid positionsand phenotype. Residues correlated with Pto function

were further investigated by site-directed mutationalanalyses. Since the source of variation among the recom-binant molecules was Pto and its paralogs, most of

which are expressed and encode potentially functionalprotein kinases (Chang et al. 2002), we might have ex-pected considerable overlap between the residues iden-tified as functionally important in the study of allelic

variation of Pto and the shuffling experiment, assumingthat variation in alleles/orthologs of Pto and paralogs ofPto are shaped by similar evolutionary forces. However,nearly all positions implicated and tested in the shufflingexperiment were invariant among the 16 alleles of Pto westudied. The one exception was residue 313. This wasselected for site-directed mutagenesis because (1) it fell

within a large region identified as functionally importantby the shuffling experiment, (2) it was the only residuethat was polymorphic among our 16 Pto alleles, and (3) it

was found exclusively in an A

allele. Therefore, theselection of this site for site-directed mutagenesis wasinformed by the population study. Due to the greaterdivergence between Pto and its paralogs as compared tothat between alleles of Pto, different subsets of function-ally important residues were identified using the shuf-fling experiment compared to our analysis of alleles andorthologs.

The lack of an AvrPto-dependent elicitation of HRin different accessions was due to a variety of indepen-dent genetic events, as indicated by the integration of


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inoculation, sequence, and transient assay data. Fouralleles were putative pseudogenes and no Pto genescould be isolated from seven individuals of differentLycopersicon species. The presence of pseudogenesand null alleles is not uncommon in multigene familiesincluding other R-gene loci. At the Rpp5 cluster inA. thaliana, out of a total of 18 genes observed in twoecotypes, only 3 genes encode intact, full-length open

reading frames, 1 of which confers resistance to thePeronospora parasitica pathogen (Noel et al. 1999).Several paralogs at the Dm3locus in lettuce have frame-shift mutations and/or are truncated (Meyers et al.1998). At the Xa21 locus in rice, of seven paralogs, onlythree are expressed (Wang et al. 1998). In Arabidopsisthaliana, some individuals have a functional RPM1gene encoding resistance to Ps. pv. maculicolaexpressingavrRpm1, while all susceptible individuals lacked thisgene entirely (Grant et al. 1998; Stahl et al. 1999).

The presence of null alleles at R-gene loci could bedue to a number of reasons. If the distribution of thepathogen is heterogeneous in space and time, as is

likely, the loss of a resistance gene in a host populationcould occur by random chance due to drift. In selfingspecies including several of the Lycopersicon species,stochastic forces such as genetic drift, founder effects,and/or hitchhiking may lead to the fixation of nullalleles or pseudogenes in the populations not exposedcontinuously to the pathogen. However, even within theself-incompatible Lycopersicon species we observedthat some individuals within a single population havea functional Pto gene while some lack the Pto geneentirely or have a pseudogene. An alternative explana-tion is that the loss of function is driven by selectionand maintained within populations through negativefrequency-dependent selection. This would occur ifthere were a cost of resistance conferring an advantageto the loss of function. R-genes, although generally ex-pressed at low levels, may be associated with fitness costs(Tian et al. 2003). Such costs need not be strictlymetabolic, but could occur because a resistance proteinsuch as Pto could be a target of virulence effectors forsome pathogens. These costs would be evident only inthe presence of a pathogen strain possessing the

virulence effector. However, experiments designed tostudy costs generally have excluded pathogens and assuch would have failed to detect this particular cost.

Not all resistance observed in this study was deter-mined by Pto. However, nearly all of the individuals thatare resistant to Pst expressing avrPto had alleles of Ptothat conferred an AvrPto-dependent HR. This is consis-tent with the conservation of AvrPto recognition con-ferred by the Ptogene in these species. However, .40%of the individuals tested from these seven species wereresistant to the Pst strain lacking avrPto. Presumablyother resistance genes in these accessions conferredrecognition of additional Psteffector proteins. Previousgenetic studies have indicated that resistance to Psthas

evolved at several other loci in different Lycopersiconspecies (Pilowsky and Zutra 1986; Stockinger and

Walling 1994). Pseudomonas spp. secrete many dif-ferent effector molecules into the plant cell; each ofthese is a potential avirulence protein subject to co-evolution with resistance genes (Collmer et al. 2002;Guttman et al. 2002; Deng et al. 2003). Evolution ofPstrecognition at independent loci may also make the

maintenance of a functional version of Pto redundant.Therefore mutations within this gene or deletion maynot be deleterious in all host backgrounds and thiscould account for the repeated loss-of-function muta-tions at the Ptolocus.

We are grateful to H. Akashi, J. Chang, D. Lavelle, T. Long, J. Parsch,

J. Rathjen, R. Ree, Y.-S. Tai, and A. Wu for many valuable discussions.We acknowledge C. Rick for help and inspiration during this project.The plant seeds were provided by the C. M. Rick Tomato Genetics

Resource Center and the U.S. Department of Agriculture Plant

Genetic Resources Unit. We also acknowledge expert technical assis-tance provided by the University of California (UC) Davis greenhouse

and controlled environment facility staff and the Center for Engi-neering Plants Resistant Against Pathogens (CEPRAP) and UC Davis

Division of BiologicalSciences DNA sequencingfacilitystaff. Thisworkwas supported by a National Science Foundation (NSF) dissertation

improvement grant (9902342) and a Jastro Shields graduate researchaward to L.R. and by NSF Cooperative Agreement (BIR-8920216) toCEPRAP.

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