The major resistance gene cluster in lettuce is highly duplicated and spans several megabases.

At least 10 Dm genes conferring resistance to the oomycete downy mildew fungus Bremia lactucae map to the major resistance cluster in lettuce. We investigated the structure of this cluster in the lettuce cultivar Diana, which contains Dm3. A deletion breakpoint map of the chromosomal region flanking Dm3 was saturated with a variety of molecular markers. Several of these markers are components of a family of resistance gene candidates (RGC2) that encode a nucleotide binding site and a leucine-rich repeat region. These motifs are characteristic of plant disease resistance genes. Bacterial artificial chromosome clones were identified by using duplicated restriction fragment length polymorphism markers from the region, including the nucleotide binding site-encoding region of RGC2. Twenty-two distinct members of the RGC2 family were characterized from the bacterial artificial chromosomes; at least two additional family members exist. The RGC2 family is highly divergent; the nucleotide identity was as low as 53% between the most distantly related copies. These RGC2 genes span at least 3.5 Mb. Eighteen members were mapped on the deletion breakpoint map. A comparison between the phylogenetic and physical relationships of these sequences demonstrated that closely related copies are physically separated from one another and indicated that complex rearrangements have shaped this region. Analysis of low-copy genomic sequences detected no genes, including RGC2, in the Dm3 region, other than sequences related to retrotransposons and transposable elements. The related but divergent family of RGC2 genes may act as a resource for the generation of new resistance phenotypes through infrequent recombination or unequal crossing over.     

Nucleotide polymorphism and molecular evolution of the LRR region in potato late blight resistance gene Rpi-blb2

Rpi-blb2, which is originally derived from Solanum bulbocastanum, is a broad-spectrum potato late blight resistance gene and belongs to the NBS-LRR family. Here, the LRR homologues of Rpi-blb2 were cloned with PCR method from 40 potato cultivars (including 20 resistant potato cultivars and 20 susceptible ones) and 7 wild potato populations. Then, the similarities of the sequences, polymorphic (segregating) sites, and nucleotide diversities were estimated by bioinformatic methods. The results showed that high nucleotide polymorphism and some hot-spot mutations existed in the LRR region of Rpi-blb2. The test of Ka/Ks ratio showed that the function of LRR was conserved because of the purifying selection, although different positions of the Rpi-blb2 LRR region were under different selection pressures. Moreover, the LRR region of Rpi-blb2 had no clear differentiation between the cultivated and wild potatoes.     

马铃薯抗晚疫病基因Rpi-blb2的LRR区域多态性及分子进化分析

马铃薯抗晚疫病基因Rpi-blb2是来源于马铃薯野生种Solanum bulbocastanum中的一个具有广谱抗性的NBS-LRR类抗病基因。文章用PCR的方法从20个高抗晚疫病的马铃薯栽培种和20个高感晚疫病的马铃薯栽培种以及7个马铃薯野生种中克隆了Rpi-blb2基因的LRR区段。采用生物信息学方法对这些序列的相似性、多态性位点、核酸多样性指数等参数进行了分析,发现Rpi-blb2的LRR区域在核酸水平上变异程度很高,而且存在多处热点突变位点;通过对该区域的Ka/Ks值进行估算,发现Rpi-blb2的LRR区域总体上受到纯化选择,功能保守,但是LRR区域的不同部位所受到的选择压力却不尽相同。同时,从核酸水平来看,Rpi-blb2基因的LRR区域在马铃薯栽培种和马铃薯野生种之间没有发现明显的分化。     

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.     

Dm3 is one member of a large constitutively expressed family of nucleotide binding site-leucine-rich repeat encoding genes

The major cluster of resistance genes in lettuce cv. Diana contains approximately 32 nucleotide binding site-leucine-rich repeat encoding genes. Previous molecular dissection of this complex region had identified a large gene, RGC2B, as a candidate for encoding the downy mildew resistance gene, Dm3. This article describes genetic and transgenic complementation data that demonstrated RGC2B is necessary and sufficient to confer resistance with Dm3 specificity. Ethylmethanesulphonate was used to induce mutations to downy mildew susceptibility in cv. Diana (Dm1, Dm3, Dm7, and Dm8). Nineteen families were identified with a complete loss of resistance in one of the four resistance specificities. Sequencing revealed a variety of point mutations in RGC2B in the six dm3 mutants. Losses of resistance were due to single changes in amino acid sequence or a change in an intron splice site. These mutations did not cluster in any particular region of RGC2B. A full-length genomic copy of RGC2B was isolated from a lambdaphage library and introduced into two genotypes of lettuce. Transgenics expressing RGC2B exhibited resistance to all isolates expressing Avr3 from a wide range of geographical origins. In a wildtype Dm3-expressing genotype, many of the RGC2 family members are expressed at low levels throughout the plant.     

Somatic variation plays a key role in the evolution of the Vf gene family residing in the Vf locus that confers resistance to apple scab disease

A cluster of four receptor-like genes has been previously identified in the Vf locus of the crabapple Malus floribunda clone 821 that confers resistance to five races of the fungal pathogen Venturia inaequalis, the casual agent of apple scab disease. Pairwise comparisons of the four Vf paralogs in both promoter and coding regions reveal their timeline evolutionary history. The four Vf paralogs have evolved from four ancient Vf members resulting from two sequential duplication events of a single Vf progenitor initially present in the Malus genome. The coding sequences of the four Vf paralogs are characterized with high numbers of unique polymorphic nucleotides, a number of short duplications/deletions, various deletions of complete LRR copy units, and a casual insert of a transposon-like element. Significant high ratios of nonsynonymous to synonymous substitutions, Ka/Ks, are observed in the putative ligand binding residues in the LRR domains. No sequence exchange between the four Vf paralogs is observed. Compared with promoter regions, only nucleotide substitutions are dramatically elevated in the coding regions. The results presented in this study strongly indicate that the Vf locus is under strong and steady horizontal selective pressures imposed by the fungal pathogen V. inaequalis, and divergent selection on somatic variations plays a key role in shaping the resistance specificity.     

A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes.

Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance (R) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways.     

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 chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

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     

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.     

Multiple genetic processes result in heterogeneous rates of evolution within the major cluster disease resistance genes in lettuce

Resistance Gene Candidate2 (RGC2) genes belong to a large, highly duplicated family of nucleotide binding site-leucine rich repeat (NBS-LRR) encoding disease resistance genes located at a single locus in lettuce (Lactuca sativa). To investigate the genetic events occurring during the evolution of this locus, approximately 1.5- to 2-kb 3' fragments of 126 RGC2 genes from seven genotypes were sequenced from three species of Lactuca, and 107 additional RGC2 sequences were obtained from 40 wild accessions of Lactuca spp. The copy number of RGC2 genes varied from 12 to 32 per genome in the seven genotypes studied extensively. LRR number varied from 40 to 47; most of this variation had resulted from 13 events duplicating two to five LRRs because of unequal crossing-over within or between RGC2 genes at one of two recombination hot spots. Two types of RGC2 genes (Type I and Type II) were initially distinguished based on the pattern of sequence identities between their 3' regions. The existence of two types of RGC2 genes was further supported by intron similarities, the frequency of sequence exchange, and their prevalence in natural populations. Type I genes are extensive chimeras caused by frequent sequence exchanges. Frequent sequence exchanges between Type I genes homogenized intron sequences, but not coding sequences, and obscured allelic/orthologous relationships. Sequencing of Type I genes from additional wild accessions confirmed the high frequency of sequence exchange and the presence of numerous chimeric RGC2 genes in nature. Unlike Type I genes, Type II genes exhibited infrequent sequence exchange between paralogous sequences. Type II genes from different genotype/species within the genus Lactuca showed obvious allelic/orthologous relationships. Trans-specific polymorphism was observed for different groups of orthologs, suggesting balancing selection. Unequal crossover, insertion/deletion, and point mutation events were distributed unequally through the gene. Different evolutionary forces have impacted different parts of the LRR.     

Unique evolutionary mechanism in R-genes under the presence/absence polymorphism in Arabidopsis thaliana

While the presence/absence polymorphism is commonly observed in disease resistance (R-) genes in Arabidopsis, only a few R-genes under the presence/absence polymorphism (R-P/A) have been investigated. To understand the mechanism of the molecular evolution of R-P/A, we investigated genetic variation of nine R-P/A in A. thaliana from worldwide populations. The number of possessed R-genes varied widely among accessions (two to nine, on average 4.3 +/- 1.6/accession). No pair of accessions shared the same haplotype, and no clear geographic differentiation was observed with respect to the pattern of presence/absence of the R-genes investigated. Presence allele frequencies also varied among loci (25-70%), and no linkage disequilibrium was detected among them. Although the LRR region in regular R-genes is known to be highly polymorphic and has a high Ka/Ks ratio in A. thaliana, nucleotide sequences of this region in the R-P/A showed a relatively low level of genetic variation (pi = 0.0002-0.016) and low Ka/Ks (0.03-0.70, <1). In contrast, the nucleotide diversities around the deletion junction of R-P/A were constantly high between presence and absence accessions for the R-genes (D(xy) = 0.031-0.103). Our results suggest that R-P/A loci evolved differently from other R-gene loci and that balancing selection plays an important role in molecular evolution of R-P/A.     

植物抗病基因同源序列及其在抗病基因克隆与定位中的应用

近10年来已有20多个植物抗病基因被克隆,测序,这些抗病基因所编码的蛋白中大多含有核苷酸结合位点,富含亮氨酸重复序列,蛋白激酶,亮氨酸拉链结构,跨膜结构域,Toll白介素－1区域等保守结构域。利用这些保守结构域合成PCR引物,已扩增出大量的植物抗病基因同源序列（RGA）。对RGA与抗病基因的关系进行了分析,讨论了RGA在研究抗病基因进化中的作用,指出RGA在抗病基因定位和转基因中具有重要意义。     

植物抗病基因的研究进展

近年来,已有１０多个植物抗病基因被克隆并定序。植物抗病基因编码的蛋白,大多含有富氨酸重复单位（ＬＲＲ）和核苷酸结合位点（ＮＢＳ）等结构。在植物与病原物的互作中,这些蛋白可作为受体识别由病原物无毒基因编码的激发子,从而激发一系列防卫反应,使植物表现出抗病性。克隆的植物抗病基因可用于培育基因工程植株而大大加快育种速度。本文对目前植物抗病基因研究中存在的问题及发展前景也进行了探讨。     

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.     

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.