The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana

We have used the naturally occurring plant-parasite system of Arabidopsis thaliana and its common parasite Peronospora parasitica (downy mildew) to study the evolution of resistance specificity in the host population. DNA sequence of the resistance gene, RPP13, from 24 accessions, including 20 from the United Kingdom, revealed amino acid sequence diversity higher than that of any protein coding gene reported so far in A. thaliana. A significant excess of amino acid polymorphism segregating within this species is localized within the leucine-rich repeat (LRR) domain of RPP13. These results indicate that single alleles of the gene have not swept through the population, but instead, a diverse collection of alleles have been maintained. Transgenic complementation experiments demonstrate functional differences among alleles in their resistance to various pathogen isolates, suggesting that the extreme amino acid polymorphism in RPP13 is maintained through continual reciprocal selection between host and pathogen.     

A genome-wide survey of R gene polymorphisms in Arabidopsis

We used polymorphism analysis to study the evolutionary dynamics of 27 disease resistance (R) genes by resequencing the leucine-rich repeat (LRR) region in 96 Arabidopsis thaliana accessions. We compared single nucleotide polymorphisms (SNPs) in these R genes to an empirical distribution of SNP in the same sample based on 876 fragments selected to sample the entire genome. LRR regions are highly polymorphic for protein variants but not for synonymous changes, suggesting that they generate many alleles maintained for short time periods. Recombination is also relatively common and important for generating protein variants. Although none of the genes is nearly as polymorphic as RPP13, a locus previously shown to have strong signatures of balancing selection, seven genes show weaker indications of balancing selection. Five R genes are relatively invariant, indicating young alleles, but all contain segregating protein variants. Polymorphism analysis in neighboring fragments yielded inconclusive evidence for recent selective sweeps at these loci. In addition, few alleles are candidates for rapid increases in frequency expected under directional selection. Haplotype sharing analysis revealed significant underrepresentation of R gene alleles with extended haplotypes compared with 1102 random genomic fragments. Lack of convincing evidence for directional selection or selective sweeps argues against an arms race driving R gene evolution. Instead, the data support transient or frequency-dependent selection maintaining protein variants at a locus for variable time periods.     

Two TIR:NB:LRR genes are required to specify resistance to Peronospora parasitica isolate Cala2 in Arabidopsis

Resistance responses that plants deploy in defence against pathogens are often triggered following a recognition event mediated by resistance (R) genes. The encoded R proteins usually contain a nucleotide-binding site (NB) and a leucine-rich repeat (LRR) domain. They are further classified into those that contain an N-terminal coiled coil (CC) motif or a Toll interleukin receptor (TIR) domain. Such R genes, when transferred into a susceptible plant of the same or closely related species, usually impart full resistance capability. We have used map-based cloning and mutation analysis to study the recognition of Peronospora parasitica (RPP)2 (At) locus in Arabidopsis accession Columbia (Col-0), which is a determinant of specific recognition of P. parasitica (At) isolate Cala2. Genetic mapping located RPP2 to a 200-kb interval on chromosome 4, which contained four adjacent TIR:NB:LRR genes. Mutational analysis revealed three classes of genes involved in specifying resistance to Cala2. One class, which resulted in pleiotropic effects on resistance to other P. parasitica (At) isolates, was unlinked to the RPP2 locus; this class included AtSGT1b. The other two classes were mapped within the interval and were specific to Cala2 resistance. Representatives of each of these classes were sequenced, and mutations were found in one or the other of two (RPP2A and RPP2B) of the four TIR:NB:LRR genes. RPP2A and RPP2B complemented their specific mutations, but failed to impart resistance when present alone, and it is concluded that both genes are essential determinants for isolate-specific recognition of Cala2. RPP2A has an unusual structure with a short LRR domain at the C-terminus, preceded by two potential but incomplete TIR:NB domains. In addition, the RPP2A LRR domain lacks conserved motifs found in all but three other TIR:NB:LRR class proteins. In contrast, RPP2B has a complete TIR:NB:LRR structure. It is concluded that RPP2A and RPP2B cooperate to specify Cala2 resistance by providing recognition or signalling functions lacked by either partner protein.     

Six amino acid changes confined to the leucine-rich repeat beta-strand/beta-turn motif determine the difference between the P and P2 rust resistance specificities in flax

At least six rust resistance specificities (P and P1 to P5) map to the complex P locus in flax. The P2 resistance gene was identified by transposon tagging and transgenic expression. P2 is a member of a small multigene family and encodes a protein with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains and an N-terminal Toll/interleukin-1 receptor (TIR) homology domain, as well as a C-terminal non-LRR (CNL) domain of approximately 150 amino acids. A related CNL domain was detected in almost half of the predicted Arabidopsis TIR-NBS-LRR sequences, including the RPS4 and RPP1 resistance proteins, and in the tobacco N protein, but not in the flax L and M proteins. Presence or absence of this domain defines two subclasses of TIR-NBS-LRR resistance genes. Truncations of the P2 CNL domain cause loss of function, and evidence for diversifying selection was detected in this domain, suggesting a possible role in specificity determination. A spontaneous rust-susceptible mutant of P2 contained a G-->E amino acid substitution in the GLPL motif, which is conserved in the NBS domains of plant resistance proteins and the animal cell death control proteins APAF-1 and CED4, providing direct evidence for the importance of this motif in resistance gene function. A P2 homologous gene isolated from a flax line expressing the P resistance specificity encodes a protein with only 10 amino acid differences from the P2 protein. Chimeric gene constructs indicate that just six of these amino acid changes, all located within the predicted beta-strand/beta-turn motif of four LRR units, are sufficient to alter P2 to the P specificity.     

Natural variation reveals key amino acids in a downy mildew effector that alters recognition specificity by an Arabidopsis resistance gene

RPP13, a member of the cytoplasmic class of disease resistance genes, encodes one of the most variable Arabidopsis proteins so far identified. This variability is matched in ATR13, the protein from the oomycete downy mildew pathogen Hyaloperonospora parasitica recognized by RPP13, suggesting that these proteins are involved in tight reciprocal coevolution. ATR13 exhibits five domains: an N-terminal signal peptide, an RXLR motif, a heptad leucine/isoleucine repeat, an 11-amino-acid repeated sequence and a C-terminal domain. We show that the conserved RXLR-containing domain is dispensable for ATR13-mediated recognition, consistent with its role in transport into the plant cytoplasm. Sequencing ATR13 from 16 isolates of H. parasitica revealed high levels of amino acid diversity across the entire protein. The leucines/isoleucines of the heptad leucine repeat were conserved, and mutation of particular leucine or isoleucine residues altered recognition by RPP13. Natural variation has not exploited this route to detection avoidance, suggesting a key role of this domain in pathogenicity. The extensive variation in the 11-amino-acid repeat units did not affect RPP13 recognition. Domain swap analysis showed that recognition specificity lay in the C-terminal domain of ATR13. Variation analyses combined with functional assays allowed the identification of four amino acid positions that may play a role in recognition specificity. Site-directed mutagenesis confirmed that a threonine residue is absolutely required for RPP13 recognition and that recognition can be modulated by the presence of either an arginine or glutamic acid at other sites. Mutations in these three amino acids had no effect on the interaction of ATR13 with a resistance gene unlinked to RPP13, consistent with their critical role in determining RPP13-Nd recognition specificity.     

Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines

The perception of downy mildew avirulence (Arabidopsis thaliana Recognized [ATR]) gene products by matching Arabidopsis thaliana resistance (Recognition of Peronospora parasitica [RPP]) gene products triggers localized cell death (a hypersensitive response) in the host plant, and this inhibits pathogen development. The oomycete pathogen, therefore, is under selection pressure to alter the form of these gene products to prevent detection. That the pathogen maintains these genes indicates that they play a positive role in pathogen survival. Despite significant progress in cloning plant RPP genes and characterizing essential plant components of resistance signaling pathways, little progress has been made in identifying the oomycete molecules that trigger them. Concluding a map-based cloning effort, we have identified an avirulence gene, ATR1NdWsB, that is detected by RPP1 from the Arabidopsis accession Niederzenz in the cytoplasm of host plant cells. We report the cloning of six highly divergent alleles of ATR1NdWsB from eight downy mildew isolates and demonstrate that the ATR1NdWsB alleles are differentially recognized by RPP1 genes from two Arabidopsis accessions (Niederzenz and Wassilewskija). RPP1-Nd recognizes a single allele of ATR1NdWsB; RPP1-WsB also detects this allele plus three additional alleles with divergent sequences. The Emco5 isolate expresses an allele of ATR1NdWsB that is recognized by RPP1-WsB, but the isolate evades detection in planta. Although the Cala2 isolate is recognized by RPP1-WsA, the ATR1NdWsB allele from Cala2 is not, demonstrating that RPP1-WsA detects a novel ATR gene product. Cloning of ATR1NdWsB has highlighted the presence of a highly conserved novel amino acid motif in avirulence proteins from three different oomycetes. The presence of the motif in additional secreted proteins from plant pathogenic oomycetes and its similarity to a host-targeting signal from malaria parasites suggest a conserved role in pathogenicity.     

Mutational analysis of the Arabidopsis RPS2 disease resistance gene and the corresponding pseudomonas syringae avrRpt2 avirulence gene

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.     

NBS-LRR resistance gene homologues in rice

Twenty three DNA fragments with a size of about 520 bp have been cloned from rice genome by PCR amplification using primers designed according to the conserved region of most plant resistance (R) genes which have Nucleotide Binding Site (NBS) and Leucine-Rich Repeat (LRR) domains. Homologous comparison showed that these fragments contained typical motifs of the NBS-LRR resistance gene class, kinase 1a, kinase 2a, kinase 3a and domain 2. Thus they were named R gene homologous sequences (RS). These RS were divided into 4 groups by clustering analysis and mapped onto chromosomes 1, 3, 4, 7, 8, 9, 10 and 11, respectively, by genetic mapping. Ten RS were located in the chromosomal intervals where known R genes had been mapped. Further RFLP analysis of an RS, RS13, near the bacterial blight resistance gene Xa4 locus on chromosome 11 among near isogenic lines and pyramiding lines of Xa4 showed that RS13 was possibly amplified from the gene family of Xa4.     

Genetic Variation and Evolution of the <Emphasis Type="Italic">Pi9</Emphasis> Blast Resistance Locus in the AA Genome <Emphasis Type="Italic">Oryza</Emphasis> Species

The rice nucleotide-binding site–leucine-rich repeat (NBS-LRR)-encoding resistance (R) gene Pi9 confers broad-spectrum resistance to the fungal pathogen Magnaporthe oryzae. The Pi9 locus comprises many NBS-LRR-like genes and is an ancient locus that is highly conserved in cultivated and wild rice species. To understand the genetic variation and molecular evolutionary mechanism of the Pi9 alleles in different rice species, we studied five AA genome Oryza species including two cultivated rice species (Oryza sativa and Oryza glaberrima) and three wild rice species (Oryza nivara, Oryza rufipogon, and Oryza barthii). A 2.9-kb fragment spanning the NBS-LRR core region of the Pi9 gene was amplified and sequenced from 40 accessions. Sequence comparison revealed that the Pi9 alleles had an intermediate-diversified nucleotide polymorphism among the AA genome Oryza species. Sequence variations were more abundant in the LRR region than in the NBS region, indicating that the LRR region has played a more important role for the evolution of the Pi9 alleles. Furthermore, positive selection was found to be the main force promoting the divergence of the Pi9 alleles, especially in the LRR region. Our results reveal the characteristics and evolutionary dynamics of the Pi9 alleles among the two cultivated and three wild rice species.     

Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley

 The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach. The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2), 5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with regions carrying resistance to CCN and corn leaf aphid. Received: 6 January 1998 / Accepted: 1 April 1998     

NBS-LRR resistance gene homologues in rice

Twenty three DNA fragments with a size of about 520 bp have been cloned from rice genome by PCR amplification using primers designed according to the conserved region of most plant resistance (R) genes which have Nucleotide Binding Site (NBS) and Leucine-Rich Repeat (LRR) domains. Homologous comparison showed that these fragments contained typical motifs of the NBS-LRR resistance gene class, kinase 1a, kinase 2a, kinase 3a and domain 2. Thus they were named R gene homologous sequences (RS). These RS were divided into 4 groups by clustering analysis and mapped onto chromosomes 1, 3, 4, 7, 8, 9, 10 and 11, respectively, by genetic mapping. Ten RS were located in the chromosomal intervals where known R genes had been mapped. Further RFLP analysis of an RS, RS13, near the bacterial blight resistance geneXa4 locus on chromosome 11 among near isogenic lines and pyramiding lines ofXa4 showed that RS13 was possibly amplified from the gene family ofXa4.     

Allelic Specificity at the Het-C Heterokaryon Incompatibility Locus of Neurospora Crassa Is Determined by a Highly Variable Domain

In filamentous fungi, the ability to form a productive heterokaryon with a genetically dissimilar individual is controlled by specific loci termed het loci. Only strains homozygous for all het loci can establish a heterokaryon. In Neurospora crassa, 11 loci, including the mating-type locus, regulate the capacity to form heterokaryons. An allele of the het-c locus (het-c(OR)) of N. crassa has been previously characterized and encodes a nonessential 966 amino acid glycine-rich protein. Herein, we describe the genetic and molecular characterization of two het-c alleles, het-c(PA) and het-c(GR), that have a different specificity from that of het-c(OR), showing that vegetative incompatibility is mediated by multiple alleles at het-c. By constructing chimeric alleles, we show that het-c specificity is determined by a highly variable domain of 34-48 amino acids in length. In this regard, het-c is similar to loci that regulate recognition in other species, such as the (S) self-incompatibility locus in plants, the sexual compatibility locus in basidiomycetes and the major histocompatibility complex (MHC) genes in vertebrates.     

Autoactive alleles of the flax L6 rust resistance gene induce non-race-specific rust resistance associated with the hypersensitive response

L6 is a nucleotide binding site-leucine rich repeat (NBS-LRR) gene that confers race-specific resistance in flax (Linum usitatissimum) to strains of flax rust (Melampsora lini) that carry avirulence alleles of the AvrL567 gene but not to rust strains that carry only the virulence allele. Several mutant and recombinant forms of L6 were made that altered either the methionine-histidine-aspartate (MHD) motif conserved in the NBS domain of resistance proteins or exchanged the short domain C-terminal to the LRR region that is highly variable among L allele products. In transgenic flax some of these alleles are autoactive; they cause a gene dosage-dependent dwarf phenotype and constitutive expression of genes that are markers for the plant defense response. Their effects and penetrance ranged from extreme to mild in their degree of plant stunting, survival, and reproduction. Dwarf plants were also resistant to flax rust strains virulent to wild-type L6 plants, and this nonspecific resistance was associated with a hypersensitive response (HR) at the site of rust infection. The strongest autoactive allele, expressed in Arabidopsis from an ethanol-inducible promoter, gave rise to plant death dependent on the enhanced disease susceptibility 1 (EDS1) gene, which indicates that the mutant flax (Linaceae) L6 gene can signal cell death through a defined disease-resistance pathway in a different plant family (Brassicaceae).     

A gain-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1

Plants have evolved sophisticated defense mechanisms against pathogen infections, during which resistance (R) genes play central roles in recognizing pathogens and initiating defense cascades. Most of the cloned R genes share two common domains: the central domain, which encodes a nucleotide binding adaptor shared by APAF-1, certain R proteins, and CED-4 (NB-ARC), plus a C-terminal region that encodes Leu-rich repeats (LRR). In Arabidopsis, a dominant mutant, suppressor of npr1-1, constitutive 1 (snc1), was identified previously that constitutively expresses pathogenesis-related (PR) genes and resistance against both Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2. The snc1 mutation was mapped to the RPP4 cluster. In snc1, one of the TIR-NB-LRR-type R genes contains a point mutation that results in a single amino acid change from Glu to Lys in the region between NB-ARC and LRR. Deletions of this R gene in snc1 reverted the plants to wild-type morphology and completely abolished constitutive PR gene expression and disease resistance. The constitutive activation of the defense responses was not the result of the overexpression of the R gene, because its expression level was not altered in snc1. Our data suggest that the point mutation in snc1 renders the R gene constitutively active without interaction with pathogens. To analyze signal transduction pathways downstream of snc1, epistasis analyses between snc1 and pad4-1 or eds5-3 were performed. Although the resistance signaling in snc1 was fully dependent on PAD4, it was only partially affected by blocking salicylic acid (SA) synthesis, suggesting that snc1 activates both SA-dependent and SA-independent resistance pathways.     

The Arabidopsis RPS4 bacterial-resistance gene is a member of the TIR-NBS-LRR family of disease-resistance genes

Plant-disease resistance (R) genes mediate the specific recognition of invading pathogens carrying cognate avirulence (avr) determinants. RPS4 is a disease-resistance locus on chromosome 5 of Arabidopsis thaliana specifying resistance to strains of Pseudomonas syringae pv. tomato expressing avrRps4. We have isolated the RPS4 gene using a map-based cloning approach. RPS4 encodes a predicted protein of 1217 amino acids that contains an N-terminus with homology to the intracellular domains of the Drosophila Toll protein and the mammalian interleukin-1 receptor (TIR domain), a tripartite nucleotide-binding site (NBS), and leucine-rich repeats (LRR). Incomplete splicing of the RPS4 mRNA was observed, which may give rise to truncated protein products consisting mainly of the TIR and NBS domains. These features classify RPS4 as a member of the TIR-NBS-LRR R gene family founded by N, L6 and RPP5, which determine resistance to viral, fungal and oomycete pathogens, respectively. Previous work has shown that RPS4, like other Arabidopsis TIR-NBS-LRR R genes specifying resistance to oomycetes, is dependent on a functional EDS1 allele for disease-resistance signaling. The characterization of RPS4 presented here thus establishes a role for TIR-NBS-LRR R genes in resistance to bacterial pathogens, and provides evidence for the model that dependence of R genes on EDS1 is determined by R protein structure, and not by pathogen type. The cloning of RPS4 and the previous isolation of avrRps4 provide the molecular tools for a genetic and molecular dissection of the TIR-NBS-LRR R gene signaling pathway in Arabidopsis.     

Cloning and characterization of a non-TIR-NBS-LRR type disease resistance gene analogue from peach.

Cloning of plant disease resistant genes is greatly helpful for disease resistant breeding in plants and the insight of resistance mechanism. However, there are less relevant researches in peach [prunus persica (L.) Batch]. In this study, four NBS-LRR type resistance gene analogs (RGAs) were cloned from genomic DNA of peach. The PNBS2 fragment was also amplified from peach cDNA and the full-length cDNA of PNBS2 (PRPM1, GenBank accession no. AY599223) has been cloned. Sequence analysis indicated that the cDNA of PRPM1 is 3007 bp in length and that the contained ORF encodes for a polypeptide of 917 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has typical structure of non-TIR-NBS-LRR genes, with NB-ARC, LZ, LRR and transmembrane domains. Southern analysis indicated that the PRPM1 gene might be a single copy in peach genome. Northern blot and RT-PCR analysis showed that the expression of PRPM1 was not induced by salicylic acid (SA) in peach young leaves. The isolation of putative resistance genes from peach provided useful bases for studying the structure and function of peach disease-resistance relating genes and disease resistant genetic breeding in peach.     

Fine-mapping and molecular marker development for <Emphasis Type="Italic">Pi56(t),</Emphasis> a NBS-LRR gene conferring broad-spectrum resistance to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in rice

The major quantitative trait locus qBR9.1 confers broad-spectrum resistance to rice blast, and was mapped to a ~69.1 kb region on chromosome 9 that was inherited from resistant variety Sanhuangzhan No 2 (SHZ-2). Within this region, only one predicted disease resistance gene with nucleotide binding site and leucine-rich repeat (NBS-LRR) domains was found. Specific markers corresponding to this gene cosegregated with blast resistance in F₂ and F₃ populations derived from crosses of susceptible variety Texianzhan 13 (TXZ-13) to SHZ-2 and the resistant backcross line BC-10. We tentatively designate the gene as Pi56(t). Sequence analysis revealed that Pi56(t) encodes an NBS-LRR protein composed of 743 amino acids. Pi56(t) was highly induced by blast infection in resistant lines SHZ-2 and BC-10. The corresponding allele of Pi56(t) in the susceptible line TXZ-13 encodes a protein with an NBS domain but without LRR domain, and it was not induced by Magnaporthe oryzae infection. Three new cosegregating gene-specific markers, CRG4-1, CRG4-2 and CRG4-3, were developed. In addition, we evaluated polymorphism of the gene-based markers among popular varieties from national breeding programs in Asia and Africa. The presence of the CRG4-2 SHZ-2 allele cosegregated with a blast-resistant phenotype in two BC₂F₁ families of SHZ-2 crossed to recurrent parents IR64-Sub1 and Swarna-Sub1. CRG4-1 and CRG4-3 showed clear polymorphism among 19 varieties, suggesting that they can be used in marker-assisted breeding to combine Pi56(t) with other target genes in breeding lines.     

Recombination between paralogues at the Rp1 rust resistance locus in maize

Rp1 is a complex rust resistance locus of maize. The HRp1-D haplotype is composed of Rp1-D and eight paralogues, seven of which also code for predicted nucleotide binding site-leucine rich repeat (NBS-LRR) proteins similar to the Rp1-D gene. The paralogues are polymorphic (DNA identities 91-97%), especially in the C-terminal LRR domain. The remaining family member encodes a truncated protein that has no LRR domain. Seven of the nine family members, including the truncated gene, are transcribed. Sequence comparisons between paralogues provide evidence for past recombination events between paralogues and diversifying selection, particularly in the C-terminal half of the LRR domain. Variants selected for complete or partial loss of Rp1-D resistance can be explained by unequal crossing over that occurred mostly within coding regions. The Rp1-D gene is altered or lost in all variants, the recombination breakpoints occur throughout the genes, and most recombinant events (9/14 examined) involved the same untranscribed paralogue with the Rp1-D gene. One recombinant with a complete LRR from Rp1-D, but the amino-terminal portion from another homologue, conferred the Rp1-D specificity but with a reduced level of resistance.