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.     

Two different CC‐NBS‐LRR genes are required for Lr10‐mediated leaf rust resistance in tetraploid and hexaploid wheat

Comparative study of disease resistance genes in crop plants and their relatives provides insight on resistance gene function, evolution and diversity. Here, we studied the allelic diversity of the Lr10 leaf rust resistance gene, a CC‐NBS‐LRR coding gene originally isolated from hexaploid wheat, in 20 diploid and tetraploid wheat lines. Besides a gene in the tetraploid wheat variety ‘Altar’ that is identical to the hexaploid wheat Lr10, two additional, functional resistance alleles showing sequence diversity were identified by virus‐induced gene silencing in tetraploid wheat lines. In contrast to most described NBS‐LRR proteins, the N‐terminal CC domain of LR10 was found to be under strong diversifying selection. A second NBS‐LRR gene at the Lr10 locus, RGA2, was shown through silencing to be essential for Lr10 function. Interestingly, RGA2 showed much less sequence diversity than Lr10. These data demonstrate allelic diversity of functional genes at the Lr10 locus in tetraploid wheat, and these new genes can now be analyzed for agronomic relevance. Lr10‐based resistance is highly unusual both in its dependence on two, only distantly, related CC‐NBS‐LRR proteins, as well as in the pattern of diversifying selection in the N‐terminal domain. This indicates a new and complex molecular mechanism of pathogen detection and signal transduction.     

小麦NBS类抗病基因同源cDNA序列的克隆与特征分析

根据已克隆植物抗病(R)基因NBS保守结构域设计简并引物,采用RT-PCR和cDNA末端快速扩增技术(RACE),在小麦抗叶锈病近等基因系材料TcLr19中进行抗病同源基因cDNA全长的扩增。获得了1个通读的NBS类抗病同源基因S11A11cDNA序列,该序列全长2923bp,编码878个氨基酸序列。生物信息学分析结果表明,该片段含有NB-ARC保守结构域和多个LRR结构域。聚类分析表明,S11A11编码的蛋白与小麦抗叶锈病基因Lr1编码的蛋白亲缘关系较近,而与Lr10亲缘关系较远。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr19小麦中成功获得了抗病基因同源序列,为最终克隆小麦抗叶锈病目的基因奠定了基础。     

Divergent Evolution of Plant NBS-LRR Resistance Gene Homologues in Dicot and Cereal Genomes

The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well. Received: 17 May 1999 / Accepted: 25 September 1999     

Isolation, genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine ( Pinus monticola Dougl. ex. D. Don.)

Western white pine ( Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust ( Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.     

The genomic dynamics and evolutionary mechanism of the Pi2/9 locus in rice

The Pi2/9 locus contains at least four resistance specificities to Magnaporthe grisea and belongs to a gene complex comprised of multiple genes that encode highly homologous nucleotide binding site (NBS) and leucine rich repeat (LRR) proteins. To investigate the genetic events involved in the evolution of the Pi2/9 locus, we analyzed the Pi2/9 locus at the inter- and intralocus levels in five rice cultivars. The NBS-LRR genes in the five cultivars belong to the same phylogenetic clade among rice NBS-LRR genes, and all have a phase-2 intron at the N-terminus. However, the paralogs within each haplotype show a significant sequence divergence and their N-terminal intron and 5' regulatory regions are very different. On the contrary, the orthologs from different haplotypes are highly similar, indicating an obvious orthologous relationship has been maintained during the evolution of the Pi2/9 locus. These results suggest that sequence diversification in the 5' regulatory regions and N-terminal introns of the paralogs may have led to suppression of meiotic recombination between the paralogs within each haplotype, facilitating the maintenance of the orthologous relationship among rice cultivars. Our observations provide valuable insight into the genomic dynamics and evolutionary mechanism of an NBS-LRR resistance-gene complex in rice.     

Intrahaplotype polymorphism at the Brassica S locus

The S locus receptor kinase and the S locus glycoproteins are encoded by genes located at the S locus, which controls the self-incompatibility response in Brassica. In class II self-incompatibility haplotypes, S locus glycoproteins can be encoded by two different genes, SLGA and SLGB. In this study, we analyzed the sequences of these genes in several independently isolated plants, all of which carry the same S haplotype (S(2)). Two groups of S(2) haplotypes could be distinguished depending on whether SRK was associated with SLGA or SLGB. Surprisingly, SRK alleles from the two groups could be distinguished at the sequence level, suggesting that recombination rarely occurs between haplotypes of the two groups. An analysis of the distribution of polymorphisms along the S domain of SRK showed that hypervariable domains I and II tend to be conserved within haplotypes but to be highly variable between haplotypes. This is consistent with these domains playing a role in the determination of haplotype specificity.     

Three subfamilies of pheromone and receptor genes generate multiple B mating specificities in the mushroom Coprinus cinereus

The B mating type locus of the basidiomycete Coprinus cinereus encodes a large family of lipopeptide pheromones and their seven transmembrane domain receptors. Here we show that the B42 locus, like the previously described B6 locus, derives its unique specificity from nine multiallelic genes that are organized into three subgroups each comprising a receptor and two pheromone genes. We show that the three genes within each group are kept together as a functional unit by being embedded in an allele-specific DNA sequence. Using a combination of sequence analysis, Southern blotting, and DNA-mediated transformation with cloned genes, we demonstrate that different B loci may share alleles of one or two groups of genes. This is consistent with the prediction that the three subgroups of genes are functionally redundant and that it is the different combinations of their alleles that generate the multiple B mating specificities found in nature. The B42 locus was found to contain an additional gene, mfs1, that encodes a putative multidrug transporter belonging to the major facilitator family. In strains with other B mating specificities, this gene, whose functional significance was not established, lies in a region of shared homology flanking the B locus.     

Allelic series of four powdery mildew resistance genes at the Pm3 locus in hexaploid bread wheat

At the Pm3 locus in hexaploid wheat (Triticum aestivum), 10 alleles conferring race-specific resistance to powdery mildew (Blumeria graminis f. sp. tritici) are known. A cluster of genes encoding coiled-coil-nucleotide-binding site-leucine-rich repeat proteins spans the Pm3 locus on wheat chromosome 1A, and one member of this gene family has recently been identified as the Pm3b resistance gene. Using molecular markers closely linked to Pm3b, we performed haplotype analysis of 10 lines carrying different Pm3 alleles. All these lines have a conserved genomic region delimited by markers cosegregating with Pm3b and including a structurally conserved Pm3b-like gene. A polymerase chain reaction-based strategy allowed the amplification of one Pm3b-like sequence from lines carrying Pm3a, Pm3d, and Pm3f alleles. These candidate genes for Pm3a, Pm3d, and Pm3f conferred AvrPm3a-, AvrPm3d-, and AvrPm3f-dependent resistance, respectively, to wheat powdery mildew in a single cell transient transformation assay. A high level of amino acid similarity (97.8%) was found between the PM3A, PM3B, PM3D, and PM3F proteins. The coiled-coil domain was 100% conserved, whereas, in the nucleotide binding site region, sequence exchange was detected, indicating intragenic recombination or gene conversion between alleles. All these results indicate that Pm3a, Pm3b, Pm3d, and Pm3f form a true allelic series. The low level of sequence divergence between the four characterized alleles as well as the finding of a conserved Pm3 haplotype are in agreement with the hypothesis of a recent evolution of Pm3-based resistance, suggesting that some or most of the diversity found at the Pm3 locus in modern wheat has evolved after wheat domestication.     

Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination

Multiple alleles controlling different gene-for-gene flax rust resistance specificities occur at the L locus of flax. At least three distinct regions can be recognized in the predicted protein products: the Toll/interleukin-1 receptor homology (TIR) region, a nucleotide binding site (NBS) region, and a leucine-rich repeat (LRR) region. Replacement of the TIR-encoding region of the L6 allele with the corresponding regions of L2 or LH by recombination changed the specificity of the allele from L6 to L7. Replacement of the TIR and most of the NBS-encoding region of L10 with the equivalent region of L2 or L9 generated recombinant alleles having a novel specificity. However, replacement of the L10 TIR-encoding region with the TIR-encoding region of L2 gave rise to an allele with no detectable specificity. These data indicate that non-LRR regions can determine specificity differences between allelic gene products and that functional specificity involves interactions between coadapted polymorphic regions in the protein products of the alleles. Evidence for the action of diversifying selection on the TIR region is observed.     

The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells

The Linum usitatissimum (flax) L gene alleles, which encode nucleotide binding site-Leu rich repeat class intracellular receptor proteins, confer resistance against the Melampsora lini (flax rust) fungus. At least 11 different L resistance specificities are known, and the corresponding avirulence genes in M. lini map to eight independent loci, some of which are complex and encode multiple specificities. We identified an M. lini cDNA marker that cosegregates in an F2 rust family with a complex locus determining avirulence on the L5, L6, and L7 resistance genes. Two related avirulence gene candidates, designated AvrL567-A and AvrL567-B, were identified in a genomic DNA contig from the avirulence allele, whereas the corresponding virulence allele contained a single copy of a related gene, AvrL567-C. Agrobacterium tumefaciens-mediated transient expression of the mature AvrL567-A or AvrL567-B (but not AvrL567-C) proteins as intracellular products in L. usitatissimum and Nicotiana tabacum (tobacco) induced a hypersensitive response-like necrosis that was dependent on coexpression of the L5, L6, or L7 resistance gene. An F1 seedling lethal or stunted growth phenotype also was observed when transgenic L. usitatissimum plants expressing AvrL567-A or AvrL567-B (but not AvrL567-C) were crossed to resistant lines containing L5, L6, or L7. The AvrL567 genes are expressed in rust haustoria and encode 127 amino acid secreted proteins. Intracellular recognition of these rust avirulence proteins implies that they are delivered into host cells across the plant membrane. Differences in the three AvrL567 protein sequences result from diversifying selection, which is consistent with a coevolutionary arms race.     

Isolation,genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine (<Emphasis Type="Italic">Pinus monticola </Emphasis>Dougl. ex. D. Don.)

Western white pine (Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust (Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.Communicated by R. Hagemann     

小麦Mlo及NBS-LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因Mlo cDNA序列及一个来源于栽培一粒小麦(Triticum monococcum L.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选.结果获得两个表达基因的cDNA克隆.其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%.另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类.白粉菌诱导前后,该片段RT-PCR扩增产物存在差异,表明该片段可能与小麦抗病性相关.利用"中国春"缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上.     

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species.

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.     

Resistance gene analogues of chickpea ( Cicer arietinum L.): isolation,genetic mapping and association with a Fusarium resistance gene cluster

Resistance gene analogues (RGAs) of Cicer were isolated by different PCR approaches and mapped in an inter-specific cross segregating for fusarium wilt by RFLP and CAPS analysis. Initially, two pairs of degenerate primers targeting sequences encoded at nucleotide-binding sites (NBS), which are conserved in plant disease resistance genes such as RPS2, L6 and N, were selected for amplification. Cloning and sequence analysis of amplified products from C. arietinum DNA revealed eight different RGAs. Additionally, five RGAs were identified after characterisation of the presumptive RGA alleles from C. reticulatum. Therefore, a total of 13 different RGAs were isolated from Cicer and classified through pair-wise comparison into nine distinct classes with sequence similarities below a 68% amino acid identity threshold. Sequence comparison of seven RGA alleles of C. arietinum and C. reticulatum revealed polymorphisms in four RGAs with identical numbers of synonymous and non-synonymous substitutions. An NlaIII site, unique in the RGA-A allele of C. arietinum, was exploited for CAPS analysis. Genomic organisation and map position of the NBS-LRR candidate resistance genes was probed by RFLP analysis. Both single-copy as well as multi-copy sequence families were present for the selected RGAs, which represented eight different classes. Five RGAs were mapped in an inter-specific population segregating for three race-specific Fusarium resistances. All RGAs mapped to four of the previously established eight linkage groups for chickpea. Two NBS-LRR clusters were identified that could not be resolved in our mapping population. One of these clusters, which is characterised by RFLP probe CaRGA-D, mapped to the linkage group harbouring two of three Fusarium resistance genes characterised in the inter-specific population. Our study provides a starting point for the characterisation and genetic mapping of candidate resistance genes in Cicer that is useful for marker-assisted selection and as a pool for resistance genes of Cicer.     

Characterization of the rice blast resistance gene Pik cloned from Kanto51

To study similar, but distinct, plant disease resistance (R) specificities exhibited by allelic genes at the rice blast resistance locus Pik/Pikm, we cloned the Pik gene from rice cultivar Kanto51 and compared its molecular features with those of Pikm and of another Pik gene cloned from cv. Kusabue. Like Pikm, Pik is composed of two adjacent NBS-LRR (nucleotide-binding site, leucine-rich repeat) genes: the first gene, Pik1-KA, and the second gene, Pik2-KA. Pik from Kanto51 and Pik from Kusabue were not identical; although the predicted protein sequences of the second genes were identical, the sequences differed by three amino acids within the NBS domain of the first genes. The Pik proteins from Kanto51 and Kusabue differed from Pikm in eight and seven amino acids, respectively. Most of these substituted amino acids were within the coiled-coil (CC) and NBS domains encoded by the first gene. Of these substitutions, all within the CC domain were conserved between the two Pik proteins, whereas all within the NBS domain differed between them. Comparison of the two Pik proteins and Pikm suggests the importance of the CC domain in determining the resistance specificities of Pik and Pikm. This feature contrasts with that of most allelic or homologous NBS-LRR genes characterized to date, in which the major specificity determinant is believed to lie in the highly diverged LRR domain. In addition, our study revealed high evolutionary flexibility in the genome at the Pik locus, which may be relevant to the generation of new R specificities at this locus.     

Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping

The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals.     

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.     

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).