Pronounced intraspecific haplotype divergence at the RPP5 complex disease resistance locus of Arabidopsis

In Arabidopsis ecotype Landsberg erecta (Ler), RPP5 confers resistance to the pathogen Peronospora parasitica. RPP5 is part of a clustered multigene family encoding nucleotide binding-leucine-rich repeat (LRR) proteins. We compared 95 kb of DNA sequence carrying the Ler RPP5 haplotype with the corresponding 90 kb of Arabidopsis ecotype Columbia (Col-0). Relative to the remainder of the genome, the Ler and Col-0 RPP5 haplotypes exhibit remarkable intraspecific polymorphism. The RPP5 gene family probably evolved by extensive recombination between LRRs from an RPP5-like progenitor that carried only eight LRRs. Most members have variable LRR configurations and encode different numbers of LRRs. Although many members carry retroelement insertions or frameshift mutations, codon usage analysis suggests that regions of the genes have been subject to purifying or diversifying selection, indicating that these genes were, or are, functional. The RPP5 haplotypes thus carry dynamic gene clusters with the potential to adapt rapidly to novel pathogen variants by gene duplication and modification of recognition capacity. We propose that the extremely high level of polymorphism at this complex resistance locus is maintained by frequency-dependent selection.     

The tomato Cf-5 disease resistance gene and six homologs show pronounced allelic variation in leucine-rich repeat copy number.

The tomato Cf-2 and Cf-5 genes confer resistance to Cladosporium fulvum and map to a complex locus on chromosome 6. The Cf-5 gene has been isolated and is predicted to encode a largely extracytoplasmic protein containing 32 leucine-rich repeats (LRRs), resembling the previously isolated Cf-2 gene, which has 38 LRRs. Three haplotypes of this locus from Lycopersicon esculentum, L. pimpinellifolium, and L. esculentum var cerasiforme were compared, and five additional homologs of Cf-5 were sequenced. All share extensive sequence identity, particularly within the C-terminal portions of the predicted proteins. In striking contrast to the Cf-9 gene family, six of seven homologs in the Cf-2/Cf-5 gene family vary in LRR copy number, ranging from 25 to 38 LRRs. Cf-5 and one adjacent homolog differ by only two LRRs. Recombination events that vary the LRR copy number in this region could provide a mechanism for the generation of new specificities for recognition of different ligands. A recombination breakpoint between the Cf-2 and Cf-5 loci was fully characterized and shown to be intragenic.     

Plant disease resistance protein signaling: NBS-LRR proteins and their partners

Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide-binding site (NBS), and a putative amino-terminal signaling domain. They are termed NBS-LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the familiar hypersensitive response (HR). A total of 149 R genes are potentially expressed in the Arabidopsis genome, and plant cells must deal with the difficult task of assembling many of the proteins encoded by these genes into functional signaling complexes. Eukaryotic cells utilize several strategies to deal with this problem. First, proteins are spatially restricted to their sub-cellular site of function, thus improving the probability that they will interact with their proper partners. Second, these interactions are architecturally organized to avoid inappropriate signaling events and to maintain the fidelity and efficiency of the response when it is initiated. Recent results provide new insights into how the signaling potential of R proteins might be created, managed and held in check until specific stimulation following infection. Nevertheless, the roles of the R protein partners in these regulatory events that have been defined to date are unclear.     

Cloning of disease-resistance homologues in end sequences of BAC clones linked to Fom-2, a gene conferring resistance to Fusarium wilt in melon (Cucumis melo L.).

Disease resistance has not yet been characterized at the molecular level in cucurbits, a group of high-value, nutritious, horticultural plants. Previously, we genetically mapped the Fom-2 gene that confers resistance to Fusarium wilt races 0 and I of melon. In this paper, two cosegregating codominant markers (AM, AFLP marker; FM, Fusarium marker) were used to screen a melon bacterial artificial chromosome (BAC) library. Identified clones were fingerprinted and end sequenced. Fingerprinting analysis showed that clones identified by each marker assembled into two separate contigs at high stringency. GenBank searches produced matches to leucine-rich repeats (LRRs) of resistance genes (R genes); to retroelements and to cellulose synthase in clones identified by FM; and to nucleotide-binding sites (NBSs) of R genes, retroelements, and cytochrome P-450 in clones identified by AM. A 6.5-kb fragment containing both NBS and LRR sequences was found to share high homology to TIR (Toll-interleukin-1 receptor)-NBS-LRR R genes, such as N, with 42% identity and 58% similarity in the TIR-NBS and LRR regions. The sequence information may be useful for identifying NBS-LRR class of R genes in other cucurbits.     

Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein Avr4

The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9, Cf-4 lacks two LRRs and differs in 78 amino acid residues. To investigate the relevance of these differences for specificity, we exchanged domains between Cf-4 and Cf-9, and mutant constructs were tested for mediating the hypersensitive response by transient coexpression with either Avr4 or Avr9. We show that the number of LRRs is essential for both Cf-4 and Cf-9 function. In addition, Cf-9 specificity resides entirely in the LRR domain and appears to be distributed over several distant LRRs. In contrast, Cf-4 specificity determinants reside in the N-terminal LRR-flanking domain and three amino acid residues in LRRs 13, 14, and 16. These residues are present at putative solvent-exposed positions, and all are required for full Cf-4 function. Finally, we show that Cf-9 carrying the specificity determinants of Cf-4 has recognitional specificity for AVR4. The data indicate that diversifying selection of solvent-exposed residues has been a more important factor in the generation of Cf-4 specificity than has sequence exchange between Cf-4 progenitor genes. The fact that most variant residues in Cf-4 are not essential for Cf-4 specificity indicates that the diverse decoration of R proteins is not fully adapted to confer recognition of a certain avirulence determinant but likely provides a basis for a versatile, adaptive recognition system.     

Genetic and molecular analysis of tomato Cf genes for resistance to Cladosporium fulvum.

In many plant-pathogen interactions resistance to disease is controlled by the interaction of plant-encoded resistance (R) genes and pathogen-encoded avirulence (Avr) genes. The interaction between tomato and the leaf mould pathogen Cladosporium fulvum is an ideal system to study the molecular basis of pathogen perception by plants. A total of four tomato genes for resistance to C. fulvum (Cf-2, Cf-4, Cf-5 and Cf-9) have been isolated from two genetically complex chromosomal loci. Their gene products recognize specific C. fulvum-encoded avirulence gene products (Avr2, Avr4, Avr5 and Avr9) by an unknown molecular mechanism. Cf genes encode extracellular membrane-anchored glycoproteins comprised predominantly of 24 amino acid leucine-rich repeats (LRRs). Cf genes from the same locus encode proteins which are more than 90% identical. Most of the amino-acid sequence differences correspond to the solvent-exposed residues within a beta-strand/beta-turn structural motif which is highly conserved in LRR proteins. Sequence variability within this motif is predicted to affect the specificity of ligand binding. Our analysis of Cf gene loci at the molecular level has shown they comprise tandemly duplicated homologous genes, and suggests a molecular mechanism for the generation of sequence diversity at these loci. Our analysis provides further insight into the molecular basis of pathogen perception by plants and the organization and evolution of R gene loci.     

Role of extracytoplasmic leucine rich repeat proteins in plant defence mechanisms

Plant-pathogen interactions involve highly complex series of reactions in disease development. Plants are endowed with both, resistance and defence genes. The activation of defence genes after contact with avirulence gene products of pathogens depends on signals transduced by leucine-rich repeats (LRRs) contained in resistance genes. Additionally, LRRs play roles for various actions following ligand recognition. Polygalacturonase inhibiting proteins (PGIPs), the only plant LRR protein with known ligands, are pectinase inhibitors, bound by ionic interactions to the extracellular matrix (ECM) of plant cells. They have a high affinity for fungal endopolygalacturonases (endoPGs). PGIP genes are organised in families encoding proteins with similar physical characteristics but different specificities. They are induced by infection and stress related signals. The molecular basis of PG-PGIP interaction serves as a model to understand the evolution of plant LRR proteins in recognising non-self-molecules. Extensins form a different class of structural proteins with repetitive sequences. They are also regulated by wounding and pathogen infection. Linkage of extensins with LRR motifs is highly significant in defending host tissues against pathogen invasion. Overexpression of PGIPs or expression of several PGIPs in a plant tissue, and perhaps manipulation of extensin expression could be possible strategies for disease management.     

Expression of two soybean resistance gene candidates shows divergence of paralogous single-copy genes

The cloning of several plant genes directly involved in triggering a disease resistance response has shown that numerous resistance genes in the nucleotide binding site (NBS)/leucine-rich repeat (LRR) class have similar conserved amino acid sequences. In this study, we used a short soybean DNA sequence, previously cloned based on its conserved NBS, as a probe to identify full-length resistance gene candidates. Two homologous, but genetically independent genes were identified. One gene maps to the soybean molecular linkage group (MLG) F and a second is coded on MLG E. The first gene contains a 3,279 nucleotide open reading frame (ORF) sequence and possesses all the functional motifs characteristic of previously cloned NBS/LRR resistance genes. The N-terminal sequence of the deduced gene product is highly characteristic of other resistance genes in the subgroup of NBS/LRR genes which show homology to the Toll/Interleukin-1 receptor genes. The C-terminal region is somewhat more divergent as seen in other cloned disease resistance genes. This region of the F-linked gene contains an LRR region that is characterized by two alternatively spliced products which produce gene products with either a four-repeat or a ten-repeat LRR. The second cloned gene that maps to soybean MLG E contains 1,565 nucleotides of ORF in the N-terminal domain. Despite strong homology, however, the 3′ region of this gene contains several in-frame stop codons and apparent frame shifts compared to the F-linked gene, suggesting that its functionality as a disease resistance gene is questionable. These two disease resistance gene candidates are shown to be closely related to one another and to the members of the NBS/LRR class of disease resistance genes. Received: 29 November 1999 / Accepted: 22 December 1999     

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.     

Evolution of the number of LRRs in plant disease resistance genes

The largest group of plant resistance (R) genes contain the regions that encode the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains (NBS-LRR genes). To gain new resistance, amino acid substitutions and changes in number of the LRRs that recognize the presence of pathogens are considered important. In this study, we focus on the evolution of the number of LRRs and analyze the genome data of five plant species, Arabidopsis thaliana, Oryza sativa, Medicago truncatula, Lotus japonicus and Populus trichocarpa. We first categorized the NBS-LRR genes in each species into groups and subgroups based on the phylogenetic relationships of their NBS domain sequences. Then we estimated the evolutionary rate of the number of LRRs relative to the synonymous divergence in the NBS domain sequences by a maximum likelihood method assuming the single stepwise mutation model. The estimates ranged from 4.5 to 600 and differed between groups in the same species or between species. This indicated different roles played by different groups of the NBS-LRR genes within a species or the effects of various life history characteristics, such as generation time, of the species. We also tested the fit of the model to the data using the variance of number of LRRs in each subgroup. In some subgroups in some plants (16 out of 174 subgroups), the results of simulation using the estimated rates significantly deviated from the observed data. Those subgroups may have undergone different modes of selection from the other subgroups.     

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. I. Analysis of Leu-rich repeats of human luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

The luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) have an approximately 350-amino acid-long, N-terminal extracellular exodomain. This exodomain binds hormone with high affinity and specificity and contains eight to nine putative Leu-rich repeat (LRR) sequences. LRRs are known to assume the horseshoe structure in ribonuclease inhibitors, and the inner lining of the horseshoe consists of the beta-stranded Leu/Ile-X-Leu/Ile motif. In the case of ribonuclease inhibitors, these beta strands interact with ribonuclease. However, it is unclear whether the putative LRRs of LHR and FSHR play any role in the structure and function. In this work, the beta-stranded Leu/Ile residues in all LRRs of the human LHR and FSHR were Ala-scanned and characterized. In addition, the 23 residues around LRR2 of LHR were Ala-scanned. The results show that beta-stranded Leu and Ile residues in all LRRs are important but not equally. These Leu/Ile-X-Leu/Ile motifs appear to form the hydrophobic core of the LRR loop, crucial for the LRR structure. Interestingly, the hot spots are primarily in the upstream and downstream LRRs of the LHR exodomain, whereas important LRRs spread throughout the FSHR exodomain. This may explain the distinct hormone specificity despite the structural similarity of the two receptors.     

Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.     

Origin and Evolution of GALA-LRR,a New Member of the CC-LRR Subfamily: From Plants to Bacteria?

The phytopathogenic bacterium Ralstonia solanacearum encodes type III effectors, called GALA proteins, which contain F-box and LRR domains. The GALA LRRs do not perfectly fit any of the previously described LRR subfamilies. By applying protein sequence analysis and structural prediction, we clarify this ambiguous case of LRR classification and assign GALA-LRRs to CC-LRR subfamily. We demonstrate that side-by-side packing of LRRs in the 3D structures may control the limits of repeat variability within the LRR subfamilies during evolution. The LRR packing can be used as a criterion, complementing the repeat sequences, to classify newly identified LRR domains. Our phylogenetic analysis of F-box domains proposes the lateral gene transfer of bacterial GALA proteins from host plants. We also present an evolutionary scenario which can explain the transformation of the original plant LRRs into slightly different bacterial LRRs. The examination of the selective evolutionary pressure acting on GALA proteins suggests that the convex side of their horse-shoe shaped LRR domains is more prone to positive selection than the concave side, and we therefore hypothesize that the convex surface might be the site of protein binding relevant to the adaptor function of the F-box GALA proteins. This conclusion provides a strong background for further functional studies aimed at determining the role of these type III effectors in the virulence of R. solanacearum.     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

The broad-spectrum tospovirus resistance gene Sw-5 of tomato is a homolog of the root-knot nematode resistance gene Mi

We used a positional cloning approach to isolate the Sw-5 disease resistance locus of tomato. Complementation experiments with overlapping cosmid clones enabled us to demonstrate that Sw-5 is a single gene locus capable of recognizing several tospovirus isolates and species. Analysis of the predicted Sw-5 protein suggests that it is a cytoplasmic protein, with a potential nucleotide binding site (NBS) domain and a C-terminal end consisting of leucine-rich repeats (LRRs). Based on its structural features, Sw-5 belongs to the class of NBS-LRR resistance genes that includes the tomato Mi, 12, and Prf genes; the Arabidopsis RPM1 gene; and the plant potato virus X resistance gene Rx. The overall similarity between the Sw-5 and Mi proteins of tomato suggests that a shared or comparable signal transduction pathway leads to both virus and nematode resistance in tomato. The similarity also supports the hypothesis that Sw-5 provides resistance via a hypersensitive response. Sw-5 is a member of a loosely clustered gene family in the telomeric region of chromosome 9. Members of this family map to other regions of chromosome 9 and also to chromosome 12, where several fungal, virus, and nematode genes have been mapped, suggesting that paralogs of Sw-5 may have evolved to provide different resistance specificities.     

Heat shock protein 90 and its co-chaperone protein phosphatase 5 interact with distinct regions of the tomato I-2 disease resistance protein

Recent data suggest that plant disease resistance (R) proteins are present in multi-protein complexes. Tomato R protein I-2 confers resistance against the fungal pathogen Fusarium oxysporum. To identify components of the I-2 complex, we performed yeast two-hybrid screens using the I-2 leucine-rich repeat (LRR) domain as bait, and identified protein phosphatase 5 (PP5) as an I-2 interactor. Subsequent screens revealed two members of the cytosolic heat shock protein 90 (HSP90) family as interactors of PP5. By performing in vitro protein-protein interaction analysis using recombinant proteins, we were able to show a direct interaction between I-2 and PP5, and between I-2 and HSP90. The N-terminal part of the LRR domain was found to interact with HSP90, whereas the C-terminal part bound to PP5. The specific binding of HSP90 to the N-terminal region of the I-2 LRR domain was confirmed by co-purifying HSP90 from tomato lysate using recombinant proteins. Similarly, the interaction between PP5 and HSP90 was established. To investigate the role of PP5 and HSP90 for I-2 function, virus-induced gene silencing was performed in Nicotiana benthamiana. Silencing of HSP90 but not of PP5 completely blocked cell death triggered by I-2, showing that HSP90 is required for I-2 function. Together these data suggest that R proteins require, like steroid hormone receptors in animal systems, an HSP90/PP5 complex for their folding and functioning.     

The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map-based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine-rich repeats (LRRs); thus, Pib is a member of the NBS-LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N-terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness.     

An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease for potato cultivation. Here, we describe the positional cloning of the Rpi-blb1 gene from the wild potato species Solanum bulbocastanum known for its high levels of resistance to late blight. The Rpi-blb1 locus, which confers full resistance to complex isolates of P. infestans and for which race specificity has not yet been demonstrated, was mapped in an intraspecific S. bulbocastanum population on chromosome 8, 0.3 cM from marker CT88. Molecular analysis of a bacterial artificial chromosome (BAC) clone spanning the Rpi-blb1 locus identified a cluster of four candidate resistance gene analogues of the coiled coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) class of plant resistance (R) genes. One of these candidate genes, designated the Rpi-blb1 gene, was able to complement the susceptible phenotype in a S. tuberosum and tomato background, demonstrating the potential of interspecific transfer of broad-spectrum late blight resistance to cultivated Solanaceae from sexually incompatible host species. Paired comparisons of synonymous and non-synonymous nucleotide substitutions between different regions of Rpi-blb1 paralogues revealed high levels of synonymous divergence, also in the LRR region. Although amino acid diversity between Rpi-blb1 homologues is centred on the putative solvent exposed residues of the LRRs, the majority of nucleotide differences in this region have not resulted in an amino acid change, suggesting conservation of function. These data suggest that Rpi-blb1 is relatively old and may be subject to balancing selection.     

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.     

The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea

The rice blast resistance (R) genes Pi2 and Piz-t confer broad-spectrum resistance against different sets of Magnaporthe grisea isolates. We first identified the Pi2 gene using a map-based cloning strategy. The Pi2 gene is a member of a gene cluster comprising nine gene members (named Nbs1-Pi2 to Nbs9-Pi2) and encodes a protein with a nucleotide-binding site and leucine-rich repeat (LRR) domain. Fine genetic mapping, molecular characterization of the Pi2 susceptible mutants, and complementation tests indicated that Nbs4-Pi2 is the Pi2 gene. The Piz-t gene, a Pi2 allele in the rice cultivar Toride 1, was isolated based on the Pi2 sequence information. Complementation tests confirmed that the family member Nbs4-Piz-t is Piz-t. Sequence comparison revealed that only eight amino-acid changes, which are confined within three consecutive LRR, differentiate Piz-t from Pi2. Of the eight variants, only one locates within the xxLxLxx motif. A reciprocal exchange of the single amino acid between Pi2 and Piz-t did not convert the resistance specificity to each other but, rather, abolished the function of both resistance proteins. These results indicate that the single amino acid in the xxLxLxx motif may be critical for maintaining the recognition surface of Pi2 and Piz-t to their respective avirulence proteins.