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Abstract
Eleven distinct families of resistance gene analogs (RGAs) with the characteristic nucleotide-binding sequence (NBS) were identified in two wild apple species, Malus prunifolia and M. baccata, and two cultivated apple cultivars, M. domestica cv. Fuji and M. domestica cv. Hong-ok, using PCR approaches with degenerate primers based on two conserved motifs of known NBS-LRR resistance genes. These RGA families were found to be represented in all the apple species tested, including wild and cultivated species. However, their sequences are very divergent from each other. Furthermore, the low level of recombination detected within their RGA families supports the idea that the evolution of NBS-encoding sequences in apple species involves the gradual accumulation of mutations. Despite the high diversity of the RGA families found in all apple species, the apparent lack of differentiation between wild and cultivated forms suggests that other factors, such as the capacity to tolerate pathogens, might play an important role in the survival of wild-type species.
... About five classes of R genes have been identified in plants and among the core of their function is the ability to scan and recognise microbial type III effectors. These R gene classes include the NBS-LRR protein kinases, extracytoplasmic-LRR transmembrane proteins (eLRR-TM), LRR, and toxin reductase (Lee, Seo, Rodriguez-Lanetty and Lee, 2003). The first three classes in this list are discussed in sections 1.4.1 through 1.4.3 of this review. ...
... Some authors talk of a leucine zipper (LZ) molecule as the amino-terminal region found in the non-TIR subfamily (Dinesh- Kumar et al., 2000;Lee et al., 2003). These two Nterminal regions participate in pathogen recognition and also determine which signal transduction pathway is used in the activation of the defence arsenal. ...
... A number of studies have confirmed that the NBS-encoding region of RGAs contains valuable sequence data that allows construction of informative phylogenetic analyses (Calenge et al., 2005). Not much has been done on the comparative analysis of expression profiles for cloned RGAs although phylogenetic analyses to determine the clustering pattern have been done though with smaller datasets (Lee et al., 2003;Baldi et al., 2004). Research has confirmed that RGAs constitute the best markers for resistance genes. ...
PhD thesis submitted in 2008 and resulted in a doctorate graduation in 2009.
... Motifs in the NBS domains have been used to isolate RGAs from various families of plants, including legumes such as soybean (52) and common bean (38), as well as from maize (8) and other cereals, including rice, wheat, and barley (7,10). Other fruit and vegetable crops with characterized RGAs include potato, lettuce, apple (24)(25)(26)36), raspberry (41,51), strawberry (34), and melon (5). More specifically, in the Rosaceae family, RGA motifs have been used for phylogenetic and evolutionary analyses (51), marking resistance regions, and identifying parental materials in germplasm collections (3,6,13,33). ...
... Results thus far indicate that, in the Andean blackberry genome as in the common bean genome, sequences of non-TIR types may predominate over TIR types. This is the first report of RGAs for Andean blackberry in Colombia and it contributes significantly to the total of RGAs cloned and identified for different plant species, including several within the Rosaceae family (2,3,13,25,33,41,(49)(50)(51). Most of the RGAs detected in Andean blackberry are related to known sequences of R genes and RGAs in species such as Vitis vinifera (n = 2), Malus × domestica (n = 11), M. prunifolia (n = 3), M. floribunda (n = 2), Rosa hybrid cultivar (n = 5), R. roxburghii (n = 3), Populus trichocarpa (n = 4), P. tomentosa × P. bolleana, P. tomentosa var. ...
... Most of the RGA classes identified were distributed equally between all genotypes evaluated, except for the TIR types, which were present only in the wild and two susceptible genotypes. These results agree with those reported by Lee et al. (25). By comparing apple RGAs, they found that no differences appeared for RGA diversity between species and that the RGAs were consistently distributed among wild and cultivated species. ...
Five Andean blackberry Rubus genotypes, three resistant and two susceptible to anthracnose, were used to identify regions in the Rubus genome with homology to disease-resistance genes found in other plant species. Polymerase chain reaction amplification with 12 pairs of primers and fragment cloning yielded 520 clones, of which 151 showed inserts between 500 and 700 bp long. When sequenced, 47 clones showed homology with two types of resistance genes, non-Toll/interleukin-1 receptor (TIR) nucleotide binding site (NBS) leucine-rich repeat (LRR) and TIR-NBS-LRR, thereby confirming their designation as resistance gene analogs (RGAs). The number of RGAs detected per Rubus genotype ranged from 7 to 11, with the highest in a wild resistant and a cultivated susceptible genotype. Rubus RGAs were also homologous with several non-TIR- and TIR-type RGAs found in other members of the Rosaceae family (Rosa hybrid cultivar, Rosa roxburghii, Malus × domestica, M. prunifolia, M. baccata, M. floribunda, Pyrus communis, Prunus persica, P. kansuensis, P. avium, and Fragaria vesca). Three RGAs shared identity with two Rosaceae RGAs associated with the CRPM1 locus for powdery mildew resistance in R. roxburghii and the Rosa hybrid cultivar. This is the first report on RGAs present in the Andean blackberry in Colombia.
... The PCR approach with degenerate primers is based on conserved amino acid motifs of a known NBS-LRR gene. This method has been widely used to find NBS-encoding disease resistant gene analogs (RGAs) from a variety of species, including Arabidopsis thaliana (Aarts et al., 1998), tomatoes (Leister et al., 1996), soybeans (Yu et al., 1996), and lettuce (Shen et al., 1998), as well as plants such as Rosaceae fruit crops, Prunus (Lalli et al., 2005;Soriano et al., 2005), apples (Lee et al., 2003;Baldi et al., 2004), and strawberries (Martinez Zamora et al., 2004). ...
... Two R-gene-specific degenerate oligonucleotide primers were used. The first selected primers, OLE 1121 (5'-GGWATGGGWGGWRTHGGWAARACHAC-3') and OLE 1122 (5'-ARNWYYTTVARDGCVARWGGVARWCC-3'), were designed according to the conserved P-loop (GGVGKTT) and hydrophobic domain (GLPLAL) from the RPS2, RPS5, and L6 Xa1 genes of the NBS-LRR class, which is specific against pathogens (Lee et al., 2003). The second selected primers, BP2f (5'-GGNGGDGTDGGSAARAC-3') and BP2r (5'-GCTAGTGGCAMNCCWCC-3'), were designed according to Baldi et al. (2004). ...
... By using two pairs of degenerate primers matching conserved motifs from the NBS domains of NBS-LRR R-genes from various species, we successfully isolated and characterized 124 RGAs from Korean wild apple germplasm collections and M. baccata in this study. The 124 clones we found were truly RGA sequences due to these sequencing-containing uninterrupted ORFs and the conserved motif characteristics of NBS-LRR R-genes (Lee et al., 2003). Also, it was found that apple germplasm RGAs were closely related to the NBS sequences of A. thaliana (Aarts et al., 1998) and tomatoes (Leister et al., 1996). ...
Several plant disease resistance gene (R-gene) classes have been identified on the basis of specific conserved functional domains. Cloning of disease-resistance apple genes would be useful for breeding programs and for studying resistance mechanisms. We used a PCR approach with degenerate primers designed from conserved NBS-LRR (nucleotide binding site-leucine-rich repeat) regions of known R-genes to amplify and clone homologous sequences from six Korean wild apple germplasm collections and an individual plant of the Siberian wild apple, Malus baccata. One hundred and twenty-four sequenced clones showed high similarity at multiple NBS motifs with the R-genes of other plants. The clones OLE 2-9, BP 6-11, OLE 1-22, and OLE 5-13 shared 45% identity with the R-gene of other plants. The conserved sequence, which plays an important role in resistance, was found in our isolated resistance gene analogs (RGAs). The sequences of isolated apple RGAs showed more similarity to Toll/interleukin-1 receptor (TIR)-NBS-LRR than non-TIR-NBS-LRR. We suggest using a marker for this resistance gene region as well as for identifying potential material for disease-resistant breeding among Korea wild apple germplasms. This is the first step in preparing a comprehensive analysis of the RGAs in Korean wild apple germplasm.
... These well-conserved regions within the NBS domain offer a great opportunity to design degenerate primers to amplify the resistance gene analogs (RGA) from genomic DNA of a wide variety of plant species (Shen et al. 1998, Speulman et al. 1998, Timmerman-Vaughan et al. 2000. Numerous RGAs have been isolated from many plant species including soybean (Kanazin et al. 1996, Yu et al. 1996, Arabidopsis (Aarts et al. 1998), potato (Leister et al. 1996), lettuce (Shen et al. 1998), cereals (Leister et al. 1998), coffee trees (Noir et al. 2001), bean (Lopez et al. 2003), apple (Lee et al. 2003), and strawberry (Martinez Zamora et al. 2004). Some RGAs from Arabidopsis, potato, and maize have been shown to be mapped closely to known disease resistance gene loci (Leister et al. 1996, Aarts et al. 1998, Collins et al. 1998, therefore this PCR-based approach can be an effective alternative strategy to isolate candidate genes for disease resistance. ...
... Based on the analysis for the number of potential recombination events among ryegrass RGA sequences, we found only one pair of inner sequences which meant that there was evidence of a possible gene conversion event due to recombination, suggesting recombination might not be the primary source of variation. This result was the same as that from the studies in coffee tree, apple, and strawberry (Noir et al. 2001, Lee et al. 2003, Martinez Zamora et al. 2004) in which point mutation has been suggested as the main source of variation for RGAs isolated from these crops. So far, there was no report on the analysis of the source of variation for NBS-LRR genes and RGA in monocots. ...
Recently, a number of disease-resistance genes related to a diverse range of pathogens were isolated from a wide variety of plant species. The majority of plant disease-resistance genes encoded a nucleotide-binding site (NBS) domain. According to the comparisons of the NBS domain of cloned R-genes, it has shown highly conserved amino acid motifs in this structure, which made it possible to isolate resistance gene analogs (RGAs) by PCR using degenerate primers. We have designed three pairs of degenerate primers based on two conserved motifs in the NBS domain of resistance proteins encoded by R-genes to amplify genomic sequences from ryegrass (Lolium sp.). Sixteen NBS-like RGAs were isolated from turf and forage type grasses. The sequence analysis of these RGAs revealed that there existed a high similarity (up to 85%) between RGA sequences among ryegrass species and other plants. The alignment of the predicted amino acid sequences of RGAs showed that ryegrass RGAs contained four conserved motifs (P-Loop, kinase-2, kinase-3a, GLPL) present in other known plant NBS-leucine rich repeat resistance genes. These ryegrass RGAs all belonged to non-toll and interleukin-1 receptor subclass. Phylogenetic analysis of ryegrass RGAs and other cloned R-genes indicated that gene mutation was the predominant source of gene variations, and the sequence polymorphism was due to purifying selection rather than diversifying selection. We further analyzed the source of gene variation in other monocots, rice, barley, wheat, and maize based on the data published before. Our analysis indicated that the source of RGA diversity in these monocots was the same as in ryegrass. Thus, monocots were probably the same as dicots in the source of RGA diversity. Ryegrass RGAs in the present paper represented a large group of resistance gene homologs in monocots. We discussed the origin and the evolution of R-genes in grass species.
... restricted RGA fragments as a probe. The fragments were derived from the conserved NBS domain, as described by Lee et al. (2003). Positive clones were examined by dye-labeled primer and terminator sequencing, on an automatic DNA sequencer (Bionex Inc. and Core Bio System Inc., Korea). ...
... The probes comprised five RGA clones that contained an NBS domain in that species. The nucleotide sequences of the RGA clones were deposited in the GenBank database under accession numbers AF516645, AF516646, AF516647, AF516648, and AF516650 (Lee et al., 2003). Nucleotide sequences of the clones were determined and analyzed using BlastX algorithms in GenBank. ...
A single disease resistance gene candidate,MbR4, was isolated from the wild-type apple speciesMalus baccta. This gene was predicted to encode motifs characteristic of the Toll Interleukin 1 Receptor (TIR) — Nucleotide Binding Site
(NBS) of theR gene. Starting with an isolated cDNA clone, genomic clones were obtained via inverse polymerase chain reaction (IPCR). TheMbR4 gene has a single open reading frame (ORF) of 2178 nucleotides, a 41-b untranslated 5’ region, a 21-b untranslated 3’ region,
and a predicted protein of 726 amino acids (82 kDa). Its deduced amino acid sequence resembles the N protein of tobacco and
the NL25 protein of potato. Ectopic expression ofMbR4 induced enhanced resistance in transgenicArabidopsis plants against the virulent pathogen,Pseudomonas syringae pv.tomato DC3000. Microarray analysis confirmed the induction of defense-related gene expression in pathogen-free 35S::MbR4 heterologousArabidopsis plants, thereby indicating that theMbR4 gene likely activates a pathogen-independent resistance pathway, rather than a gene-for-gene pathway. Our results suggest
thatMbR4 plays a role in theR gene, and may be a source of resistance for cultivated apple species.
... This approach has been successfully used in potato (Leister et al. 1996), soybean (Yu et al. 1996, Kanazin et al. 1996, lettuce (Shen et al. 1998), Arabidopsis thaliana (Aarts et al. 1998;Speulman et al. 1998), Brassica spp. (Joyeux et al. 1999), apple (Lee et al. 2003) and in several other plant species. Genetic analysis showed that RGAs often map to known disease resistance loci where they tend to occur in clusters (Leister et al. 1996). ...
... The use of a pair of non-exhaustive degenerate primers matching the P-Loop and GLPL motifs from NBS domains similar to those used in strawberry has permitted the identification of TIR and non-TIR types of RGAs in apple (Lee et al. 2003), alfalfa (Cordero and Skinner 2002) and chickpea (Huettel et al. 2002), among others. But in soybean, Kanazin et al. (1996), using a similar pair of primers, detected only sequences that clearly belong to the TIR type of R genes, as we have detected in strawberry. ...
Degenerate oligonucleotide primers, designed based on conserved regions of Nucleotide Binding Site (NBS) domains from previously cloned plant resistance genes, were used to isolate Resistance Gene Analogues (RGAs) from wild and cultivated strawberries. Seven distinct families of RGAs of the NBS-LRR type were identified from two related wild species, Fragaria vesca and F. chiloensis, and six different Fragaria x ananassa cultivars. With one exception (GAV-3), the deduced amino acid sequences of strawberry RGAs showed strong similarity to TIR (Toll Interleukin I Receptor)-type R genes from Arabidopsis, tobacco and flax, suggesting the existence of common ancestors. GAV-3 seemed to be more closely related to the non-TIR type. Further studies showed that the recombination level and the ratio of non-synonymous to synonymous substitutions within families were low. These data suggest that NBS-encoding sequences of RGAs in strawberry are subject to a gradual accumulation of mutations leading to purifying selection, rather than to a diversifying process. The present paper is the first report on RGAs in strawberry.
... These domains are involved in pathogen recognition, signaling, and plant innate immunity responses [26,27,29,[31][32][33][34][35]. R-genes have been identified in the genomes of plant species including watermelon [36], cucumber [25], rice [37,38], Chinese cabbage [39], maize [40], wheat [41], Arabidopsis thaliana [42], and apple [43]. ...
... These domains are involved in pathogen recognition, signaling, and plant innate immunity responses [26,27,29,[31][32][33][34][35]. R-genes have been identified in the genomes of plant species including watermelon [36], cucumber [25], rice [37,38], Chinese cabbage [39], maize [40], wheat [41], Arabidopsis thaliana [42], and apple [43]. ...
Background:
Bacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli, results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance (R)-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R-genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB.
Results:
We identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R-genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli. Six R-genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession.
Conclusion:
We identified six putative candidate R-genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.
... A. citrulli: collection, culture, and inoculum preparation A. citrulli strain NIHHS15-280 was obtained from the National Institute of Horticultural and Herbal Science (NIHHS), South Korea. The bacterium was cultured on Petri plates containing 20 ml King's B (KB) medium supplemented with 100 μg ml −1 ampicillin for [36][37][38][39][40][41][42][43][44][45][46][47][48] h at 28°C [84] until bacterial colonies formed. For all inoculations, a bacterial suspension was prepared by covering the culture plates with 5 ml of sterile, double distilled (DD) water and gently scraping the surface of the KB medium using an L-shaped rubber spreader to an optical density (OD) of 1.0 at 600 nm, as measured using a NanoDrop ND-1000 Spectrophotometer. ...
Background: Bacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli, results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance (R)-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R-genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB.
Results: We identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R-genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli. Six R-genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession.
Conclusion: We identified six putative candidate R-genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.
... Watermelon contains 44 NBS-LRR genes, as revealed by genomic analysis [1]. R genes have been identified in a number of plant species, including Arabidopsis thaliana [16], rice [17,18], melon [19], cucumber [20] and apple [21]. Initially, we identified GSB-resistant watermelon lines through an extensive bioassay. ...
Watermelon (Citrullus lanatus) is a nutritionally rich and economically important horticultural crop of the Cucurbitaceae family. Gummy stem blight (GSB) is a major disease of watermelon, which is caused by the fungus Didymella bryoniae, and results in substantial economic losses in terms of yield and quality. However, only a few molecular studies have focused on GSB resistance in watermelon. Nucleotide binding site (NBS)-encoding resistance (R) genes play important roles in plant defense responses to several pathogens, but little is known about the role of NBS-encoding genes in disease resistance in watermelon. The analyzed NBS-encoding R genes comprises several domains, including Toll/interleukin-1 receptor(TIR), NBS, leucine-rich repeat (LRR), resistance to powdery mildew8(RPW8) and coiled coil (CC), which are known to be involved in disease resistance. We determined the expression patterns of these R genes in resistant and susceptible watermelon lines at different time points after D. bryoniae infection by quantitative RT-PCR. The R genes exhibited various expression patterns in the resistant watermelon compared to the susceptible watermelon. Only six R genes exhibited consistent expression patterns (Cla001821, Cla019863, Cla020705, Cla012430, Cla012433 and Cla012439), which were higher in the resistant line compared to the susceptible line. Our study provides fundamental insights into the NBS-LRR gene family in watermelon in response to D. bryoniae infection. Further functional studies of these six candidate resistance genes should help to advance breeding programs aimed at improving disease resistance in watermelons.
... The NBS-LRR genes are classified into two subfamilies: (i) TIR-NB-LRR proteins that contain Toll/interleukin-1 receptor (TIR) domains, and (ii) CC-NB-LRR proteins that contain coiled-coil (CC) domains [28,35,36]. Such R genes have been identified in a number of plant species, including Arabidopsis [35], rice [37,38], Chinese cabbage [36,39], cucumber [40], potato [41], papaya [42], grape [43], and apple [44]. ...
Heading cabbage is a nutritionally rich and economically important cruciferous vegetable. Black rot disease, caused by the bacterium Xanthomonas campestris pv. campestris, reduces both the yield and quality of the cabbage head. Nucleotide binding site (NBS)-encoding resistance (R) genes play a vital role in the plant immune response to various pathogens. In this study, we analyzed the expression and DNA sequence variation of 31 NBS-encoding genes in cabbage (Brassica oleracea var. capitata). These genes encoded TIR, NBS, LRR and RPW8 protein domains, all of which are known to be involved in disease resistance. RNA-seq revealed that these 31 genes were differentially expressed in leaf, root, silique, and stem tissues. Furthermore, qPCR analyses revealed that several of these genes were more highly expressed in resistant compared to susceptible cabbage lines, including Bol003711, Bol010135, Bol010559, Bol022784, Bol029866, Bol042121, Bol031422, Bol040045 and Bol042095. Further analysis of these genes promises to yield both practical benefits, such as molecular markers for marker-assisted breeding, and fundamental insights to the mechanisms of resistance to black rot in cabbage.
... It acts as a mixture of similar primer sequences with a number of possible bases in the respective coded nucleotides (Linhart and Shamir, 2005). These primers were derived from Bertioli et al. (2003), Hunger et al. (2003), Lee et al. (2003), Yaish et al. (2004) and Weng et al. (2009) The PCR products were resolved on Ethidium bromidestained 1.0 % agarose gel and electrophoresed in 1 x TBE buffer at 120 V until the bands were clearly separated. The clear and resolved bands (ranging from ~250-700 bp) were individually gel-purified using the MinElute gel extraction kit (Qiagen Inc.) and ligated into pGEM-T Easy vector (Promega) as per the manufacturers' instruction. ...
Nucleotide binding site leucine rich repeat protein (NBS-LRR) is a multi-member gene family in plants encoding important disease resistance proteins to fend against various pathogen infections. Recent advances in genetics and genomics facilitated discovery and functional analysis of NBS-LRR genes or resistance gene homologs (RGHs) in various plant species. In the present work, we have cloned 57 non-identical NBS-LRR sequences from a blast disease resistant finger millet genotype IE1012. We assembled the 57 NBS-LRR sequences to the existing finger millet NBS-LRRs from NCBI Genbank through CAP3 program to obtain a total of 28 sequences. The hidden Markov models (HMMs) analysis further identified 16 NBS-LRRs as EcRGHs with uninterrupted open reading frames (ORFs), of which nine EcRGHs was added from the present work. The secondary structure analysis of protein sequences revealed characteristic conserved motifs, P-loop, Kinase2 and GLPL and other motifs with significant variations. The phylogenetic classification clustered the 16 EcRGH proteins, mostly in the non-TIR group. BLAST-P analysis showed homology of the 16 EcRGHs to NBS-LRR proteins reported across other grass species. The EcRGHs analysed here providing a useful genomic resource for genetic studies of resistance genes in finger millet and exploit them in resistance breeding programs.
... The NBS domain is characterized by several distinct motifs: P-loop/kinase-1a, kinase-2, kinase-3a, and GLPL, and all of them are highly conserved among species (MEYERS et al., 1999;PAN et al., 2000). These well-conserved regions within the NBS domain have been used to isolate resistance gene analogs (RGAs) in a broad range of plant species, such as soybean (KANAZIN et al., 1996), potato (LEISTER et al., 1996, rice (BAI et al., 2002), Arabidopsis thaliana (MEYERS et al., 1999;MEY-ERS et al., 2003), grapevine (DONALD et al., 2002), apple (LEE et al., 2003), strawberry (MARTINEZ ZAMORA et al., 2004), oats (IRIGOYEN et al., 2006), apricot (SORIANO et al., 2005), rose (HATTENDORF and DEBENER, 2007), banana (MILLER et al., 2008) and sugarcane (GLYNN et al., 2008). These RGAs are useful in physical mapping and as gene candidates in positional cloning. ...
The majority of verified plant disease resistance genes (R genes) isolated to date was of the nucleotide binding site-leucine rich repeat (NBS-LRR) class. The conservation between different NBS-LRR R genes opens the avenue for the use of PCR based strategies in isolating and cloning other R gene family members or analogs (resistance gene analogue, RGA) using degenerate primers for these conserved regions. In this study, to better understand the R gene in European aspen (Populus tremula), a perennial tree, we used degenerate primers to amplify RGA sequences from European aspen. Cloning and sequence characterization identified 37 European aspen RGAs, which could be phylogenetically classified into seven subfamilies. Deduced amino acid sequences of European aspen RGAs showed strong identity, ranging from 30.41 to 46.63%, to toll interleukin receptor (TIR) R gene subfamily. BLAST searches with reference to the genomic sequence of P. trichocarpa found 209 highly homologous regions distributed in 28 genomic loci, suggesting the abundance and divergence of NBS-encoding R genes in European aspen genome. Although, numerous studies have reported that plant R genes are under diversifying selection for specificity to evolving pathogens, non-synonymous to synonymous nucleotide substitution (dN/d s) ratio were <1 for NBS domains of European aspen RGA, showing the evidence of purifying selection in this perennial tree. In further analysis, many intergenic exchanges were also detected among these RGAs, indicating a probable role in homogenising NBS domains. The present study permits insights into the origin, diversification, evolution and function of NBS-LRR R genes in perennial species like European aspen and will be useful for further R gene isolation and exploitation.
... A polymerase chain reaction (PCR)-based approach has been developed to clone NBSÀLRR-containing R-genes on the basis of degenerate primers designed from the conserved regions of the NBS domain. This method has been applied successfully in many species, such as potato, [10] soybean, [11] maize, [12] sunflower, [13] lettuce, [14] Brassica napus, [15] rice, [16] common bean, [17] citrus, [18] coffee, [19] chickpea, [20] grapevine, [21] apple, [22] wheat, [23] chicory [24] and sorghum. [25] NBSÀLRR resistance (R) genes have been also used as polymorphic markers to locate disease resistance genes in Arabidopsis thaliana, [26] wheat, [27] melon, [28] cowpea, [29] tomato plants (Lycopersicon esculentum Mill), [30] common bean, [31] cocoa [32] and cotton. ...
The nucleotide-binding site (NBS)–leucine-rich repeat (LRR) gene family is a class of R genes in plants. NBS genes play a very important role in disease defence. To further study the variation and homology of mango NBS–LRR genes, 16 resistance gene analogues (RGAs) (GenBank accession number HM446507-22) were isolated from the polymerase chain reaction fragments and sequenced by using two degenerate primer sets. The total nucleotide diversity index Pi was 0.362, and 236 variation sites were found among 16 RGAs. The degree of homology between the RGAs varied from 44.4% to 98.5%. Sixteen RGAs could be translated into amino sequences. The high level of this homology in the protein sequences of the P-loop and kinase-2 of the NBS domain between the RGAs isolated in this study and previously characterized R genes indicated that these cloned sequences belonged to the NBS–LRR gene family. Moreover, these 16 RGAs could be classified into the non-TIR–NBS–LRR gene family because only tryptophan (W) could be claimed as the final residual of the kinase-2 domain of all RGAs isolated here. From our results, we concluded that our mango NBS–LRR genes possessed a high level of variation from the mango genome, which may allow mango to recognize many different pathogenic virulence factors.
... The NBS domain is believed to participate in signal transduction, while the LRR domain is thought to be involved in ligand binding and pathogen recognition (Young, 2000). P-loop, RNBS-A, kinase 2, RNBS-B, RNBS-C, GLPL, and RNBS-D are also highly conserved motifs generally present in the NBS domain of the R genes (Lee et al., 2003). The use of resistance genes is an effective, economical and ecological method to control epidemics of leaf rust disease. ...
Leaf (brown) rust caused by Puccinia triticina is a fungal disease of wheat (Triticum aestivum L.) that causes significant yield losses annually in many wheat-growing regions of the world. Host plant resistance is the most economically viable and environmentally responsible method for controlling Puccinia triticina, the causal agent of leaf rust in wheat. The identification and utilization of new resistance sources is critical to continue the development of improved cultivars. The objective of this work was to identify defense-related genes against rust in the Egyptian rust resistant cultivar Giza168. Specific primers were designed on the basis of converse motifs of cloned resistance genes of the resistance gene analog (RGA) and leaf rust resistance gene (Lr21) in wheat (Triticum aestivum L.). The designed PCR primers were subsequently used for RT-PCR using RNA isolated from a resistant variety to amplify fragments of 445 bp and 235 bp for RGA and Lr21 genes, respectively. The amplified products were cloned, sequenced and submitted to the GenBank. The nucleotide sequences of the amplified fragments were aligned with their corresponding genes using the BLAST. The expressions of the two genes in the infected and healthy plants were studied using RT-PCR. The RGA expression was induced and detected by RT-PCR, which is up-regulated by fungal infection. The Lr21 expression was detected on both healthy and infected plants, although the expression was higher in infected plants.
... Additional motifs found in the NBS region of plant resistance genes are the kinase-3a and the GLPL (also called "hydrophobic domain"), a putative membrane spanning domain (Baldi et al. 2004). By making use of these conserved domains, NBS-sequences were obtained by PCR-mediated approach and provided candidate resistance genes (or Resistance Gene Analogs) in several crops such as potato (Leister et al. 1996), soybean (Kanazin et al. 1996;Yu et al. 1996;Peñuela et al. 2002;He et al. 2003), maize (Collins et al. 1998), sunflower (Gentzbittel et al. 1998, lettuce (Shen et al. 1998), Brassica (Joyeux et al. 1999), rice (Mago et al. 1999), common bean (Rivkin et al. 1999), citrus (Deng et al. 2000), coffee (Noir et al. 2001), chickpea (Huettel et al. 2002), grapewine (Donald et al. 2002), apple (Lee et al. 2003), wheat (Lacock et al. 2003), chicory (Plocik et al. 2004) and sorghum (Totad et al. 2005). The RGA fragments were also used as molecular markers for tagging the disease resistance loci in Arabidopsis thaliana (Aarts et al. 1998), wheat (Chen et al. 1998), rice (Ilag et al. 2000), melon (Mas et al. 2001), cowpea (Gowda et al. 2002), Lycopersicon (Zhang et al. 2002), common bean (Lopez et al. 2003), cocoa (Lanaud et al. 2004) and cotton (Hinchliffe et al. 2005). ...
Genomic DNA sequences sharing homology with NBS
region of resistance gene analogs were isolated and
characterized from Pongamia glabra, Adenanthera
pavonina, Clitoria ternatea and Solanum trilobatum
using PCR based approach with primers designed from
conserved regions of NBS domain. The presence of
consensus motifs viz., kinase 1a, kinase 2, kinase 3a and
hydrophobic domain provided evidence that the cloned
sequences may belong to the NBS-LRR gene family.
Conservation of tryptophan as the last residue of
kinase-2 motif further confirms their position in non-
TIR NBS-LRR family of resistance genes. The
Resistance Gene Analogs (RGAs) cloned from P. glabra,
A. pavonina, C. ternatea and S. trilobatum clustered
together with well- characterized non -TIR-NBS-LRR genes leaving the TIR-NBS-LRR genes as a separate
cluster in the average distance tree constructed based on
BLOSUM62. All the four RGAs had high level of
identity with NBS-LRR family of RGAs deposited in the
GenBank. The extent of identity between the sequences
at NBS region varied from 29% (P. glabra and S.
trilobatum) to 78% (A. pavonina and C. ternatea), which
indicates the diversity among the RGAs.
... This could also contribute to a possible deviation in the R gene diversity in perennials. Using degenerate primers large numbers of the NBS-LRR proteins have been cloned from several woody perennials including Rosaceae (Samuelian et al., 2008;Hattendorf and Debener, 2007;Martinez Zamora et al., 2004;Lee et al., 2003;Baldi et al., 2004;Lalli et al., 2005;Soriano et al., 2005;Xu et al., 2005). The Vf gene locus from apple was also sequenced and analyzed (Xu and Korban, 2002). ...
Sequence analysis of the locus carrying the Rdr1 gene conferring resistance to the black spot disease in roses was conducted. A 204-b and 249-b sequence from the resistant Rosa multiflora and susceptible Rosa rugosa loci, respectively, were compared to understand the mechanism involved in the diversification of the Rdr1 gene cluster. Sequence similarity between the nine Rdr1 paralogues of R. multiflora and 11 orthologues of R. rugosa up to 99% was observed. In some cases the similarity was higher among the Rdr1 orthologues than among the paralogues. Recombination, gene conversion and point mutation events were detected within the Rdr1 orthologues. The analysis showed that similar R gene diversifying mechanisms would take place in woody perennial rose plants as already indicated for herbaceous annuals. In relation to the R genes in annual plants, recent duplications might have generated the Rdr1 gene cluster in rose plants.
... Four pairs of degenerate primers targeting the NBS domain [62,63] were used to amplify RGA sequences from 26 different Malus accessions present in the USDA apple germplasm collection at Geneva (NY, USA) (www.ars-grin.gov/npgs/index.html; Table S2). ...
... Four pairs of degenerate primers targeting the NBS domain [62,63] were used to amplify RGA sequences from 26 different Malus accessions present in the USDA apple germplasm collection at Geneva (NY, USA) (www.ars-grin.gov/npgs/index.html; Table S2). ...
The family of resistance gene analogues (RGAs) with a nucleotide-binding site (NBS) domain accounts for the largest number of disease resistance genes and is one of the largest gene families in plants. We have identified 868 RGAs in the genome of the apple (Malus × domestica Borkh.) cultivar 'Golden Delicious'. This represents 1.51% of the total number of predicted genes for this cultivar. Several evolutionary features are pronounced in M. domestica, including a high fraction (80%) of RGAs occurring in clusters. This suggests frequent tandem duplication and ectopic translocation events. Of the identified RGAs, 56% are located preferentially on six chromosomes (Chr 2, 7, 8, 10, 11, and 15), and 25% are located on Chr 2. TIR-NBS and non-TIR-NBS classes of RGAs are primarily exclusive of different chromosomes, and 99% of non-TIR-NBS RGAs are located on Chr 11. A phylogenetic reconstruction was conducted to study the evolution of RGAs in the Rosaceae family. More than 1400 RGAs were identified in six species based on their NBS domain, and a neighbor-joining analysis was used to reconstruct the phylogenetic relationships among the protein sequences. Specific phylogenetic clades were found for RGAs of Malus, Fragaria, and Rosa, indicating genus-specific evolution of resistance genes. However, strikingly similar RGAs were shared in Malus, Pyrus, and Prunus, indicating high conservation of specific RGAs and suggesting a monophyletic origin of these three genera.
... This classification is supported by high bootstrap resembling (Fig. 2). The number of subfamilies is similar to that reported in other species such as soybean (Kanazin et al., 1996; Yu et al., 1996), apple (Lee et al., 2003), Medicago truncatula (Zhu et al., 2002), Arabidopsis (Meyers et al., 2003), cotton (He et al., 2004) and sunflower (Radwan et al., 2008 ). The number of subfamilies, which identified in this work, is greater than these identified by Yuksel et al. (2005) and equal the subfamily numbers identified by Bertioli et al. (2003). ...
The nucleotide-binding-site-leucine-rich-repeat (NBS-LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. Here, we describe a collection of peanut NBS-LRR resistance gene candidates (RGCs) isolated from peanut (Arachis) species by mining Gene Bank data base. NBS-LRR sequences assembled into TIR-NBS-LRR (75.4%) and non-TIR-NBS-LRR (24.6%) subfamilies. Total of 20 distinct clades were identified and showed a high level of sequence divergence within TIR-NBS and non-TIR-NBS subfamilies. Thirty-four primer pairs were designed from these RGC sequences and used for screening different genotypes belonging to wild and cultivated peanuts. Therefore, peanut RGC identified in this study will provide useful tools for developing DNA markers and cloning the genes for resistance to different pathogens in peanut.
... With this technique, other investigators were successful in cloning the paralogous genes -member of the multigene family, for which at least one cloned member was available (Kanazin et al. 1996;Yu et al. 1996;Mago et al. 1999;Penuela et al. 2002). RGAs have been isolated from many plant species such as soybean (Yu et al. 1996;Penuela et al. 2002), potato (Leister et al. 1996), tomato (Ohmori et al. 1998), common bean (Rivkin et al. 1999), coffee (Noir et al. 2001), Arabidopsis (Aarts et al. 1998), sunflower (Gentzbittel et al. 1998), lettuce , grapevine (Donald et al. 2002), chickpea (Huettel et al. 2002), apple (Lee et al. 2003), chicory (Plocik et al. 2004) and grain crops including rice, wheat, barley and maize Seah et al. 1998). ...
Leaf rust caused by Puccinia hordei is an important disease of barley (Hordeum
vulgare) in many regions of the world. Yield losses up to 62% have been reported in
susceptible cultivars. The Rph5 gene confers resistance to the most prevalent races (8 and
30) of barley leaf rust in the United States. Therefore, the molecular mapping of Rph5 is
of great interest. Genetic studies were performed by analysis of 93 and 91 F2 plants
derived from the crosses ‘Bowman’ (rph5) × ‘Magnif 102’ (Rph5) and ‘Moore’ (rph5) ×
Virginia 92-42-46 (Rph5), respectively. Linkage analysis positioned the Rph5 locus to the
extreme telomeric region of the short arm of barley chromosome 3H at 0.2 cM proximal
to RFLP marker VT1 and 0.5 cM distal from RFLP marker C970 in the Bowman ×
Magnif 102 population. Synteny between rice chromosome 1 and barley chromosome 3
was employed to saturate the region within the sub-centimorgan region around Rph5
using sequence-tagged site (STS) markers that were developed based on barley expressed
sequence tags (ESTs) syntenic to the phage (P1)-derived artificial chromosome (PAC)
clones comprising distal region of the rice chromosome 1S. Five rice PAC clones were
used as queries to blastn 370,258 barley ESTs. Ninety four non-redundant EST sequences
were identified from the EST database and used as templates to design 174 pairs of
primer combinations. As a result, 10 EST-based STS markers were incorporated into the
‘Bowman’ × ‘Magnif 102’ high-resolution map of the Rph5 region. More importantly, six
markers, including five EST-derived STS sequences, co-segregate with Rph5. Genes,
represented by these markers, are putative candidates for Rph5. Results of this study
demonstrate the usefulness of rice genomic resources for efficient deployment of barley
EST resources for marker saturation of targeted barley genomic region.
... Some of the genes have been successfully used in blast resistance breeding. When a map-based cloning strategy is used in the identification of R-genes, resistance gene analogue (RGA) markers that are designed according to the conserved NBS domain could be used to identify the corresponding candidate NBS-LRR resistance gene (CHAUHAN et al. 2002;CHEN et al. 2004;LEE et al. 2003;MAGO et al. 1999;SALLAUD et al. 2003;ZHUANG et al. 2002 In fact, we also tried to identify the blast R gene other than Pid3 revealed in the genetic analysis of the F2 population by using the same strategy, but we could not find any other pseudogene marker associated with the susceptible individuals. This implies 19 that not all of the susceptible alleles of blast R genes are pseudogenes. ...
... The NBS domain is believed to participate in signal transduction, while the LRR domain is thought to be involved in ligand binding and pathogen recognition (Young, 2000). P-loop, RNBS-A, kinase 2, RNBS-B, RNBS-C, GLPL, and RNBS-D are also highly conserved motifs generally present in the NBS domain of the R genes (Lee et al., 2003). A new PCR-based strategy, using degenerated primers designed from these conserved motifs, has resulted in the isolation of numerous resistance gene analogs (RGAs) from a variety of plant species such as potato (Leister et al., 1996), bean (Ferrier-Cana et al. 2003), rice (Leister & Katagiri, 2000) and several others (for review see Chelkowski & Koczyk, 2003). ...
A maioria dos genes de resistência (R) clonados e caracterizados até o momento contém domínios NBS (nucleotide binding site) e LRR (leucine-rich repeat). Dentro destes domínios, encontram-se "motifs" altamente conservados. Análogos de genes de resistência (RGAs) são marcadores genéticos obtidos por uma estratégia, baseada em PCR, que usa primers degenerados desenhados a partir desses "motifs" altamente conservados dos genes R. Esta estratégia possui a vantagem do elevado grau de conservação da estrutura e seqüência dos aminoácidos observados nos genes R. O objetivo do presente estudo foi realizar uma busca por RGAs em Carica papaya L. e Vasconcellea cauliflora Jacq. A. DC. De três combinações de primers avaliadas, somente uma obteve sucesso na amplificação. O produto da amplificação foi então clonado em pCR2.1TOPO e seqüenciado utilizando os primers universais M13 forward e reverse. Quarenta e oito clones foram seqüenciados de cada espécie vegetal. Das 96 seqüências geradas para cada espécie, retiraram-se as seqüências do vetor e, em seguida, as mesmas foram agrupadas utilizando o programa "CAP3 assembler". A partir do GENEBANK, foi identificado um RGA em C. papaya apresentando um BlastX e-value de 2x10-61 com o "gb|AAP45165.1| putative disease resistant protein RGA3 (Solanum bulbocastanum)". Na extensão do nosso conhecimento, este é o primeiro relato de um RGA na família Caricaceae Dumort. Estudos preliminares de estrutura foram realizados visando à maior caracterização deste potencial "NBS-LRR type protein". Esforços para encontrar novos análogos de genes de resistência devem continuar, principalmente para fornecer bases para o desenvolvimento de plantas de mamão transgênicas com resistência a doenças.
... Structural specificity of the isolated R-genes has catalyzed the isolation of homologous sequences by candidate gene approach using R-gene specific degenerate primers, especially from those plants with a relatively large genome size (Leister et al., 1996) and/or wherein genetic studies have not been initiated so far. This approach has been successfully applied for the isolation and characterization of RGCs from many plant species (Aarts et al., 1998;Cordero and Skinner, 2002;Deng et al., 2000;Gedil et al., 2001;Hinchliffe et al., 2005;Lee et al., 2003;Li et al., 2006;Martinez-Zamora et al., 2004;Rivkin et al., 1999;Song et al., 1995). Some of these RGCs have been demonstrated to be linked to known R-genes (Leister et al., 1996;Shen et al., 1998). ...
Soft rot disease caused by the oomycete Pythium aphanidermatum (Edson) Fitzp. is the most economically significant disease of ginger (Zingiber officinale Rosc.) in tropical countries. All available ginger cultivars are susceptible to this pathogen. However a wild ginger relative viz., Z. zerumbet L. Smith, was identified as a putative soft rot resistance donor. In the present study, a putative resistance (R) gene designated, ZzR1 was isolated and characterized from Z. zerumbet using sequence information from Zingiber RGCs identified in our earlier experiments. Analysis of the 2280bp segment revealed a 2157bp open reading frame (ORF) encoding a putative cytoplasmically localized protein. The deduced ZzR1 protein shared high homology with other known R-genes belonging to the CC-NBS-LRR (Coiled coil-Nucleotide Binding Site-Leucine rich repeat) class and had a calculated molecular weight of 84.61kDa. Real time PCR analysis of ZzR1 transcription in Z. zerumbet following pathogen infection demonstrated activation at 3hpi thus suggesting involvement of ZzR1in Z. zerumbet defense mechanism. Although many R-genes have been characterized from different taxa, none of them will help in development of resistant ginger cultivars owing to the phenomenon of "Restricted Taxonomic Functionality" (RTF). Thus ZzR1 gene characterized from the resistant wild Zingiber accession represents a valuable genomic resource for ginger improvement programs. This first report on R-gene isolation from the Zingiber secondary gene pool is pivotal in designing strategies for engineering resistance in ginger, which is otherwise not amenable to conventional improvement programs owing to sexual reproduction barriers.
... The NBS domain is believed to participate in signal transduction, while the LRR domain is thought to be involved in ligand binding and pathogen recognition (Young, 2000). P-loop, RNBS-A, kinase 2, RNBS-B, RNBS-C, GLPL, and RNBS-D are also highly conserved motifs generally present in the NBS domain of the R genes (Lee et al., 2003). The use of resistance genes is an effective, economical and ecological method to control epidemics of leaf rust disease. ...
*Agricultural Genetic Engineering Research Institute (AGERI), ARC
**Department of Genetics, Faculty of Agriculture, Cairo University
Leaf (brown) rust caused by Puccinia triticina is a fungal disease of wheat (Triticum aestivum L.) that causes significant yield losses annually in many wheat-growing regions of the world. Host plant resistance is the most economically viable and environmentally responsible method for controlling Puccinia triticina, the causal agent of leaf rust in wheat. The identification and utilization of new resistance sources is critical to continue the development of improved cultivars. The objective of this work was to identify defense-related genes against rust in the Egyptian rust resistant cultivar Giza168. Specific primers were designed on the basis of converse motifs of cloned resistance genes of the resistance gene analog (RGA) and leaf rust resistance gene (Lr21) in wheat (Triticum aestivum L.). The designed PCR primers were subsequently used for RT-PCR using RNA isolated from a resistant variety to amplify fragments of 445 bp and 235 bp for RGA and Lr21 genes, respectively. The amplified products were cloned, sequenced and submitted to the GenBank. The nucleotide sequences of the amplified fragments were aligned with their corresponding genes using the BLAST. The expressions of the two genes in the infected and healthy plants were studied using RT-PCR. The RGA expression was induced and detected by RT-PCR, which is up-regulated by fungal infection. The Lr21 expression was detected on both healthy and infected plants, although the expression was higher in infected plants. Key words: RGA gene, Lr21 gene, wheat cv. Giza168.
... With the universal primers designed to target the P-loop and the GLPL motifs, which are supposed to occur in both groups of NBS-LRR-RGAs, predominantly TIR-RGAs were isolated. The same bias towards the amplification of TIR-RGAs with P-loop/GLPL-primers was observed in many other RGA studies (Di Gaspero and Cipriani 2003, Kanazin et al. 1996, Lee et al. 2003. Bertioli et al. (2003) suggest competition during the PCR as a reason for this phenomenon because in plants, TIR-RGAs generally outnumber LZ-RGAs by a factor of 2-3. ...
To isolate resistance gene analogues (RGAs) from roses we used various degenerate oligonucleotide primers targeting conserved motifs within the NBS region of nucleotide binding site (NBS)-leucine-rich repeat (LRR) resistance genes. A large RGA sublibrary consisting of 7000 clones was constructed. This sublibrary contains at least 40 unique RGA families of the TIR (toll-/interleukin-1 receptor) and the LZ (leucine zipper) type, which were further analysed. Phylogenetic studies revealed close relationships of some rose RGAs to R genes and RGAs from other plants and gave rise to the assumption that rose R genes evolved from different starting points, prior to and subsequent to speciation. Southern blot analyses showed that the RGAs were organized as single, low and multicopy loci in the rose genome. None of the analysed sequences detected any hybridization signal in Prunus cérasus indicating that the analysed RGAs are not conserved across genera. The efficiency and selectivity of the different degenerate primers used for the RGA isolation is discussed in detail.
... With this technique other investigators were successful in cloning the paralogous genes -members of the multigene families, for which at least one cloned member was available (Kanazin et al. 1996, Yu et al. 1996, Leister et al. 1998, Mago et al. 1999, Penuela et al. 2002. RGAs have been isolated from many plant species such as soybean (Yu et al. 1996, Penuela et al. 2002, potato (Leister et al. 1996), tomato (Ohmori et al. 1998), common bean (Rivkin et al. 1999), coffee (Noir et al. 2001), Arabidopsis (Aarts et al. 1998), sunflower (Gentzbittel et al. 1998), lettuce (Shen et al. 1998), grapewine (Donald et al. 2002), chickpea (Huettel et al. 2002), apple (Lee et al. 2003), chicory (Plocik et al. 2004) and grain crops including rice, wheat, barley and maize (Leister et al. 1998, Seah et al. 1998, Collins et al. 2001. ...
The majority of verified plant disease resistance genes isolated to date belong to the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. Using degenerate primers, designed from the conserved motifs of the NBS region in tobacco N and Arabidopsis RPS2 genes, we isolated 190 resistance gene analogs (RGA) clones from barley genomic DNA. A total of 13 single- and low-copy RGAs were genetically mapped onto chromosomes 1H–7H (except 5H) using three barley double haploid (DH) mapping populations: Steptoe × Morex, Harrington × TR306 and LUGC × Bowman. Sequence analysis of the RGAs showed that they are members of a diverse group. As a result of BLAST searches, one RGA proved unique as it did not detect any significant hit. Another RGA is putatively functional, because it detected several barley expressed sequence tag (EST) matches. To physically map the RGAs, 13 sequences were used to screen a 6.3 × cv. ‘Morex’ bacterial artificial chromosome (BAC) library. After fingerprint analysis, eight contigs were constructed incorporating 62 BAC clones. These BAC contigs are of great value for positional cloning of disease resistance genes, because they span the regions where various barley R genes have been genetically mapped.
... Because plant-resistance genes are known to be clustered in the genome (Hulbert et al. 2001, Leister 2004) they can serve as tightly linked molecular markers for another gene of the same cluster, or even as a starting point for a map-based cloning approach. More than 100 resistance gene analogues (RGA) of the NBS-LRR (nucleotide binding site/leucin rich repeat) class of plant resistance genes have been identified by PCR-based approaches and mapped for the most part in the Malus genome (Thiermann 2002, Lee et al. 2003, Baldi et al. 2004, Calenge et al. 2005. Although the first fungal resistance genes isolated from apple, conferring resistance to apple scab (Venturia inaequalis), do not belong to this major resistance gene class but to the receptor-like protein (RLP) class (Vinatzer et al. 2001, Xu andKorban 2002), it is possible that other fungal pathogens are controlled by NBS-LRR genes. ...
Molecular markers for the major apple powdery mildew resistance gene Pl1 were identified and are presently used in marker-assisted selection in apple breeding. However, the precise map position of the Pl1 gene in the apple genome was not known. The objectives of this investigation were the identification of the Malus linkage group (LG) carrying the Pl1 locus, mapping of the resistance gene by simple sequence repeat (SSR) markers, and the analysis of genetic associations between the Pl1 gene and the numerous NBS-LRR resistance gene candidates already mapped in the apple genome. A two-step linkage mapping was used, based on two different apple families. The identification of LG 12 carrying Pl1 was performed indirectly by mapping the SCAR marker AT20 in an apple progeny for which there was a core genetic map but no mildew data available. Then, the position of Pl1 on LG 12 was determined by SSR markers in a second population which has been scored for mildew over 6 years in a greenhouse and in the field. The SSR Hi07f01, previously mapped on LG 12 [Tree Genet. Genomes, 2 (2006), 202] cosegregated with AT20 and was closely linked (∼1 cM) to the Pl1 gene. The TIR-NBS-LRR resistance gene analogue 15G11 mapped by the SSCP technique was also closely linked to the Pl1 resistance locus and might be a candidate for Pl1 itself, a second powdery mildew major resistance gene (Pld, [Theor. Appl. Genet., 110 (2004), 175]), or two scab resistance genes (Vg, [IOBC/WPRS Bull., 23 (2000), 245]; Vb, [Genome, 49 (2006), 1238]) which all seem to be located in a common R gene cluster at the distal end of apple LG 12.
... Many studies have been performed in which fragments of R genes have been isolated, mainly by the use of degenerate primers. Thus, R gene candidates have been identified in pea (Timmerman-Vaughan et al. 2000), barley Mammadov et al. 2006), apple (Lee et al. 2003;Calenge et al. 2005), potato (van der Linden et al. 2004), wheat (Dilbirligi et al. 2004), apricot (Soriano et al. 2005), Avena species (Irigoyen et al. 2006), poplar ) and ryegrass ). ...
Severe yield loss in plant crops caused by pathogens constantly demands plant breeders to improve disease resistance in crop
species. To date, more than 90 plant resistance (R) genes have been isolated, many of them belonging to the NBSLRR class of
R genes. These genes are often found in clusters along the chromosomes and frequently lack genetic synteny between species,
which makes them difficult to identify based on sequence homology to previously isolated R genes. However, once identified,
R genes provide the basis for the application of functional markers (FMs), which efficiently assist phenotypic selection in
all phases of resistance breeding. FMs are derived from polymorphic sites within genes causally involved in phenotypic trait
variation. The major advantage of FMs is that they are in complete linkage disequilibrium with causative genes. Consequently,
they can be used in a number of different genetic backgrounds. In fact, only a few FMs for R genes have been published in
crop species up to now, although several alleles have been identified for a number of cloned R genes. But the rapidly increasing
number of cloned R genes, decreasing costs for allele sequencing and the increasing availability of TILLING populations (targeting-induced local lesions in genomes) for most crop species will significantly contribute to the establishment of FMs for most major disease R genes within
the next decade. Thus, this review summarises the current status of FM development in R genes and discusses implications of
the availability of FMs for resistance breeding.
... PCR amplification with degenerate primers targeted to the short conserved regions in the NBS has proven to be an efficient method for identifying R gene analogues (RGAs). This method has been successfully used for isolation of NBS-LRR RGAs from a wide variety of plant species [7][8][9][10][11][12][13][14][15][16][17]. Genetic analysis showed that RGAs often mapped to known disease resistance loci where they tended to occur in clusters [7]. ...
According to the conservative regions of the nucleotide-binding site and the leucine-rich repeat (NBS-LRR) in resistance genes (R genes), the polymerase chain reaction (PCR) with degenerate primers was employed to isolate R gene analogues (RGAs) from five species of banana (Musa spp.), i.e. wild species of Gongjiao (AA) and Xinyiyejiao (BB), cultivated species of Zhongshandajiao (ABB), Fenjiao (AAB) and Williams (AAA), respectively. As a result, a total of 91 sequences with uninterrupted open reading frame (ORFs) were typical of RGAs out of 208 clones sequenced, of which 33 sequences with identity of deduced amino acid sequence below 97% were further analyzed. Based on the phylogenetic analysis by using MEGA software, the 33 sequences could be divided into 12 distinct Musa RGAs. All of them belong to the non-TIR-NBS type. No representatives of the TIR-type RGAs were detected by PCR amplification using TIR-type specific primers, suggesting the TIR-NBS-type of R genes were selectively lost in banana during evolution process. Comparison and phylogenetic analysis of the Musa RGAs with the known R genes from other plant species revealed their evolutionary relationship. Despite the high diversity of the RGAs found in banana, the 12 Musa RGAs were detected in all banana species tested, with the exception of two Musa RGAs: Musa RGA-I only presented and expressed in Zhongshandajiao (ABB) and Gongjiao (AA), while Musa RGA-K only appeared and expressed in Zhongshandajiao, suggesting there was minor differentiation between wild and cultivated forms in general. To the best of our knowledge, the isolation, characterization and phylogenetic analysis of RGAs in banana has not been reported in the literature to date.
... Comparative analysis in the Asteraceae showed that gene duplication and loss events have changed the composition of these gene subfamilies (Plocik et al. 2004). Comparative analysis was also carried out within specific species, mostly focused on comparison between representative genotypes (Geffroy et al. 2009;Lee et al. 2003;Martinez Zamora et al. 2004). However, to date, little is known about NBS gene evolution in the Rutaceae family. ...
The nucleotide-binding site leucine-rich repeat (NBS-LRR) genes are the largest class of disease resistance genes in plants. However, our understanding of the evolution of NBS-LRR genes in Rutaceae fruit crops is rather limited. We report an evolutionary study of 103 NBS-encoding genes isolated from Poncirus trifoliata (trifoliate orange), Citrus reticulata (tangerine) and their F(1) progeny. In all, 58 of the sequences contained a continuous open reading frame. Phylogenetic analysis classified the 58 NBS genes into nine clades, eight of which were genus specific. This was taken to imply that most of the ancestors of these NBS genes evolved after the genus split. The motif pattern of the 58 NBS-encoding genes was consistent with their phylogenetic profile. An extended phylogenetic analysis, incorporating citrus NBS genes from the public database, classified 95 citrus NBS genes into six clades, half of which were genus specific. RFLP analysis showed that citrus NBS-encoding genes have been evolving rapidly, and that they are unstable when passed through an intergeneric cross. Of 32 NBS-encoding genes tracked by gene-specific PCR, 24 showed segregation distortion among a set of 94 F(1) individuals. This study provides new insight into the evolution of Rutaceae NBS genes and their behaviour following an intergeneric cross.
... The NBS domain is believed to participate in signal transduction, while the LRR domain is thought to be involved in ligand binding and pathogen recognition (Young, 2000). P-loop, RNBS-A, kinase 2, RNBS-B, RNBS-C, GLPL, and RNBS-D are also highly conserved motifs generally present in the NBS domain of the R genes (Lee et al., 2003). A new PCR-based strategy, using degenerated primers designed from these conserved motifs, has resulted in the isolation of numerous resistance gene analogs (RGAs) from a variety of plant species such as potato (Leister et al., 1996), bean (Ferrier-Cana et al. 2003), rice (Leister & Katagiri, 2000) and several others (for review see Chelkowski & Koczyk, 2003). ...
The majority of cloned resistance (R) genes characterized so far contain a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain, where highly conserved motifs are found. Resistance genes analogs (RGAs) are genetic markers obtained by a PCR-based strategy using degenerated oligonucleotide primers drawn from these highly conserved "motifs". This strategy has the advantage of the high degree of structural and amino acid sequence conservation that is observed in R genes. The objective of the present study was to search for RGAs in Carica papaya L. and Vasconcellea cauliflora Jacq. A. DC. Out of three combinations of primers tested, only one resulted in amplification. The amplified product was cloned in pCR2.1TOPO and than sequenced using M13 forward and reverse primers. Forty-eight clones were sequenced from each species. The 96 sequences generated for each species were cleaned of vector sequences and clustered using CAP3 assembler. From the GENEBANK, one RGA was identified in C. papaya showing a BlastX e-value of 2x10-61 to the gb|AAP45165.1| putative disease resistant protein RGA3 (Solanum bulbocastanum). To the extent of our knowledge this is the first report of a RGA in the Caricaceae Dumort family. Preliminary structural studies were performed to further characterize this putative NBS-LRR type protein. Efforts to search for other RGAs in papaya should continue, mostly to provide basis for the development of transgenic papaya with resistance to diseases
... Some of the genes have been successfully used in blast resistance breeding. When a map-based cloning strategy is used in the identification of R-genes, resistance gene analogue (RGA) markers that are designed according to the conserved NBS domain could be used to identify the corresponding candidate NBS-LRR resistance gene (CHAUHAN et al. 2002;CHEN et al. 2004;LEE et al. 2003;MAGO et al. 1999;SALLAUD et al. 2003;ZHUANG et al. 2002 In fact, we also tried to identify the blast R gene other than Pid3 revealed in the genetic analysis of the F2 population by using the same strategy, but we could not find any other pseudogene marker associated with the susceptible individuals. This implies 19 that not all of the susceptible alleles of blast R genes are pseudogenes. ...
Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. The two major subspecies of Asian cultivated rice (Oryza sativa L.), indica and japonica, have shown obvious differences in rice blast resistance, but the genomic basis that underlies the difference is not clear. We performed a genomewide comparison of the major class of resistant gene family, the nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene family, between 93-11 (indica) and Nipponbare (japonica) with a focus on their pseudogene members. We found great differences in either constitution or distribution of pseudogenes between the two genomes. According to this comparison, we designed the PCR-based molecular markers specific to the Nipponbare NBS-LRR pseudogene alleles and used them as cosegregation markers for blast susceptibility in a segregation population from a cross between a rice blast-resistant indica variety and a susceptible japonica variety. Through this approach, we identified a new blast resistance gene, Pid3, in the indica variety, Digu. The allelic Pid3 loci in most of the tested japonica varieties were identified as pseudogenes due to a nonsense mutation at the nucleotide position 2208 starting from the translation initiation site. However, this mutation was not found in any of the tested indica varieties, African cultivated rice varieties, or AA genome-containing wild rice species. These results suggest that the pseudogenization of Pid3 in japonica occurred after the divergence of indica and japonica.
... However, weak similarities, and even no similarity at all with any peptide sequence, does not necessarily indicate that these sequences are not RGAs. The NBS primers we used amplified DNA outside of the NBS domain, whereas the majority of the RGA sequences populating the sequence databases, including a number of apple RGAs (Lee et al. 2003), correspond to the NBS domain itself. Our NBS sequences therefore do not overlap with this population of RGAs. ...
We used a new method called nucleotide-binding site (NBS) profiling to identify and map resistance gene analogues (RGAs) in apple. This method simultaneously allows the amplification and the mapping of genetic markers anchored in the conserved NBS-encoding domain of plant disease resistance genes. Ninety-four individuals belonging to an F1 progeny derived from a cross between the apple cultivars 'Discovery' and 'TN10-8' were studied. Two degenerate primers designed from the highly conserved P-loop motif within the NBS domain were used together with adapter primers. Forty-three markers generated with NBS profiling could be mapped in this progeny. After sequencing, 23 markers were identified as RGAs, based on their homologies with known resistance genes or NBS/leucine-rich-repeat-like genes. Markers were mapped on 10 of the 17 linkage groups of the apple genetic map used. Most of these markers were organized in clusters. Twenty-five markers mapped close to major genes or quantitative trait loci for resistance to scab and mildew previously identified in different apple progenies. Several markers could become efficient tools for marker-assisted selection once converted into breeder-friendly markers. This study demonstrates the efficiency of the NBS-profiling method for generating RGA markers for resistance loci in apple.
The cultivated apple (Malus domestica Borkh.) is a cross-pollinated perennial fruit tree of great economic importance. Previous versions of apple reference genomes were unphased, fragmented, and lacked comprehensive insights into the highly heterozygous genome, which impeded genetic studies and breeding programs in apple. In this study, we assembled a haplotype-resolved telomere-to-telomere reference genome for the diploid apple cultivar Golden Delicious. Subsequently, we constructed a pangenome based on twelve assemblies from wild and cultivated apples to investigate different types of resistance gene analogs (RGAs). Our results revealed the dynamics of the gene gain and loss events during apple domestication. Compared with cultivated species, more gene families in wild species were significantly enriched in oxidative phosphorylation, pentose metabolic process, responses to salt, and abscisic acid biosynthesis process. Interestingly, our analyses demonstrated a higher prevalence of RGAs in cultivated apples than their wild relatives, partially attributed to segmental and tandem duplication events in certain RGAs classes. Other types of structural variations, mainly deletions and insertions, have affected the presence and absence of TIR-NB-ARC-LRR (TNL), NB-ARC-LRR (NL), and CC-NB-ARC-LRR (CNL) genes. Additionally, hybridization/introgression from wild species has also contributed to the expansion of resistance genes in domesticated apples. Our haplotype-resolved T2T genome and pangenome provide important resources for genetic studies of apples, emphasizing the need to study the evolutionary mechanisms of resistance genes in apple breeding programs.
Background Bacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli , results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance ( R )-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R -genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB.
Results We identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R -genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli via qRT-PCR. Six R -genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession.
Conclusion We identified six putative candidate R -genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.
Genomic analysis in Juglans (walnuts) is expected to transform the breeding and agricultural production of both nuts and lumber. To that end, we report here the determination of reference sequences for six additional relatives of Juglans regia: Juglanssigillata (also from section Dioscaryon), Juglans nigra, Juglans microcarpa, Juglanshindsii (from section Rhysocaryon),Juglans cathayensis (from section Cardiocaryon), and the closely related Pterocarya stenoptera. While these are 'draft' genomes, ranging in size between 640Mbp and 990Mbp, their contiguities and accuracies can support powerful annotations of genomic variation that are often the foundation of new avenues of research and breeding. We annotated nucleotide divergence and synteny by creating complete pairwise alignments of each reference genome to the remaining six. In addition, we have re-sequenced a sample of accessions from four Juglans species (including regia). The variation discovered in these surveys comprises a critical resource for experimentation and breeding, as well as a solid complementary annotation. To demonstrate the potential of these resources the structural and sequence variation in and around the polyphenol oxidase loci, PPO1 and PPO2 were investigated. As reported for other seed crops variation in this gene is implicated in the domestication of walnuts. The apparently Juglandaceae specific PPO1 duplicate shows accelerated divergence and an excess of amino acid replacement on the lineage leading to accessions of the domesticated nut crop species, Juglans regia and sigillata.
Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from Pongamia glabra, Adenanthera pavonina, Clitoria ternatea and Solanum trilobatum using PCR based approach with primers designed from conserved regions of NBS domain. The presence of consensus motifs viz., kinase 1a, kinase 2, kinase 3a and hydrophobic domain provided evidence that the cloned sequences may belong to the NBS-LRR gene family. Conservation of tryptophan as the last residue of kinase-2 motif further confirms their position in non-TIR NBS-LRR family of resistance genes. The Resistance Gene Analogs (RGAs) cloned from P. glabra, A. pavonina, C. ternatea and S. trilobatum clustered together with well- characterized non-TIR-NBS-LRR genes leaving the TIR-NBS-LRR genes as a separate cluster in the average distance tree constructed based on BLOSUM62. All the four RGAs had high level of identity with NBS-LRR family of RGAs deposited in the GenBank. The extent of identity between the sequences at NBS region varied from 29% (P. glabra and S. trilobatum) to 78% (A. pavonina and C. ternatea), which indicates the diversity among the RGAs.
The current literature describes recovery from virus–induced symptoms as a RNA silencing defense, but immunity-related genes (IRGs), including the structurally specific resistance gene analogs (RGAs) that may play a key role in tolerance and recovery is not yet reported. In this study, the transcriptome data of tolerant cassava TME3 (which exhibits a recovery phenotype) and susceptible cassava T200 infected with South African cassava mosaic virus (SACMV) were explored for RGAs. Putative resistance protein analogs (RPAs) with amide-like indole-3-accetic acid–Ile-Leu-Arg (IAA-ILR) and leucine-rich repeat (LRR)-kinase conserved domains were unique to TME3. Common responsive RPAs in TME3 and T200 were the dirigent-like protein (DIR), coil-coil (CC) nucleotide-binding site (NBS) and toll-interleukin-resistance (TIR), disease resistance zinc finger chromosome condensation-like protein (DZC) and NBS-apoptosis repressor with caspase recruitment (ARC)–LRR domains. Mutation in RPAs in the MHD motif of the NBS-ARC2 subdomain associated with the recovery phase in TME3 was observed. Additionally, a cohort of 25 RGAs mined solely during the recovery process in TME3 was identified. Phylogenetic and expression analyses support that diverse RGAs are differentially expressed during tolerance and recovery. This study reveal that in cassava a perennial crop, RGAs participate in tolerance and differentially accumulate during recovery as a complementary defense mechanism to natural occurring RNA silencing to impair viral replication.
Based on the conservative regions of the nucleotide-binding site and the leucine-rich repeat (NBS-LRR) in cloned wilt resistance genes, the polymerase chain reaction with degenerate primers was employed to isolate resistance gene analogues (RGAs) from the genomic DNA of wilt resistance germplasm ‘Goldfinger’ (AAAB) banana. As a result, twenty fragments of RGAs were isolated, which were of expected size (about 530 bp). Analysis of the deduced amino acids of these RGAs show that they share the NB-ARC domain and belong to the non-TIR-NBS class resistance gene candidates, containing 4 conservative amino acid domains, i.e. P-loop (GMGGVGKTT), Kinase-2 (LLVLDDIW), RNBS-B (CKVLFTTRS), and hydrophobic amino acids GLPL (GLPLALKVL). Other results reveal that sequence identity among the 20 RGAs rang from 41.1% to 99.3%, while identity of the deduced amino acid sequences range from 33.2% to 96.3%. The phylogenetic analysis of the RGA nucleotide sequences and the deduced amino acids showed that the 20 sequences could be divided into 5 distinct types. All of the amino acids deduced from the RGAs share a homology of 28%~54% with those deduced from the known wilt resistance genes such as Fom-2, I2C-1, I2C-2 and I2. This result to some degree indicates the conservation of disease resistance gene evolution. Technically, these RGAs isolated in the present study would lay a base for the further cloning of wilt resistance genes in banana, which could also be used as molecular markers for screening candidate wilt resistance genes in banana.
The NBS-LRR class resistance-gene candidate sequences (Pt8a and Pt9a) were used to develop new specific markers to the citrus nematode resistance gene locus Tyr1. By high-density colony screening, over 200 positive clones were pulled out from the BAC library. A few of the clones were found to be closely linked with the Tyr1 region, because the primers from these clones insert sequence produced polymorphism which matched up with the phenotype after bulked segregant analysis. By primer walking approach, three integrate NBS-LRR class resistance-gene sequences were tagged and identified separately in three clones (7A4, 4L17 and 29F20). More specific markers were developed from these tagged sequences and relatively high-density genetic maps were constructed by incorporating the newly developed markers and previously developed markers in the '9145 family'. New markers were applied in '9401 family' trying to estimate roughly the genetic distance between the Ctv and Tyr1 region.
Degenerate primers designed from the conserved regions of nucleotide-binding site domains of known plant resistance gene products were used to scan a complete set of wheat–Lophopyrum elongatum addition lines. Forty-six clones were isolated and they were grouped into seven families of resistance gene analogues (RGA). All families were characterised as the Toll–Interleukin receptor group of R-genes (plant disease resistance genes). The putative chromosome-specific SNP (single-nucleotide polymorphism) loci were identified by multiple sequence alignments. Furthermore, 20 E chromosome-specific RGA markers were used to identify polymorphic fragments in wheat–Lo. elongatum addition lines and amphidiploid. The results suggested that these chromosome-specific markers could be not only useful for marker-assisted selection and map-based cloning of R-genes in Lo. elongatum, but also feasible for investigating the evaluation process of the E genome.
Most of the disease resistance genes (R-genes) discovered in plants have conserved functional domains, predominantly among them are nucleotide binding sites (NBS) and leucine rich repeats (LRR). The sequence information of the conserved domains can be invariably used to mine similar sequences from other plant species, using degenerate and specific primers for their amplification in a polymerase chain reaction. Such derived sequences, known as Resistance Gene Analogues (RGAs), can serve as molecular markers for rapid identification and isolation of R-genes. Besides, they can also provide clues about the evolutionary mechanism of resistance genes and the interaction involved in pathogen recognition. In the recent years, this sequence-homology based approach has been used extensively for the cloning and mapping of RGAs in cereals, pulses, oilseeds, coffee, spices, forest trees and horticultural crops. In this article, the current status of cloning of RGAs from different crops has been reviewed. A general method of RGA cloning and its modifications like NBS-profiling and AFLP-NBS have also been discussed along with examples. Further, it has been suggested that the RGAs cloned in various crops would be a useful genomic resource for developing cultivars with durable resistance to diseases in different crop breeding programmes.
According to the conservative regions of the nucleotide-binding site and leucine-rich repeat domain (NBS–LRR) in resistance genes (R-gene), a homology-based cloning method was used to isolate disease resistance gene candidates (RGCs) from six species of pear, including Pyrus betulaefolia, P. bretschneideri, P. pyrifolia, P. ussriensis, P. sinkiangensis, P. communis and their interspecific hybrids. Approximately 100 disease resistance gene candidates from 39 cultivars in these pear species were identified. Among these RGC, 98 of genomic sequences which could be translated into polypeptides without stop codons, while the other two sequences presented multiple stop codons. The deduced amino acid sequences of the 98 RGC displayed high diversity, ranging from 20 to 100%, and could be divided into 17 distinct RGC families (RGC 1–RGC 17) based on the phylogenetic analysis with the threshold of 68% identity. The 98 RGC contained both the toll interleukin receptor (TIR) group and non-toll interleukin receptor (non-TIR) group. RGC families RGC 16 and RGC 17 belonged to non-TIR group, the other 15 RGC families were classified to TIR group. Sequences analysis indicated that there was a strong identity of these RGCs to the known R-gene and RGC from other plants. In each RGC family, one representative RGC was selected to test the expression profile at transcriptional level, and all the 17 selected RGCs could be detected at mRNA level. In response to Venturia nashicola inoculation or salicylic acid (SA) treatment, the expression level of RGC Pb-Zs1 (RGC 1) from pear scab-resistant cultivar ‘Zaosu’ pear changed while the expression level of RGC Pb-Dshs2 (RGC 8) from pear scab-susceptible pear cultivar ‘Dangshansu’ seemed less affected. These RGCs isolated in this study could help understand the R-gene in pear species and will serve as a potential resource for future improvement of disease resistance in pear.
Based on the conservative regions of the nucleotide-binding site and the leucine-rich repeat (NBS-LRR) in cloned wilt resistance genes, the polymerase chain reaction with degenerate primers was employed to isolate resistance gene analogues (RGAs) from the genomic DNA of wilt resistance germplasm 'Goldfinger' (AAAB) banana. As a result, twenty fragments of RGAs were isolated, which were of expected size (about 530 bp). Analysis of the deduced amino acids of these RGAs show that they share the NB-ARC domain and belong to the non-TIR-NBS class resistance gene candidates, containing 4 conservative amino acid domains, i.e. P-loop (GMGGVGKTT), Kinase-2 (LLVLDDIW), RNBS-B (CKVLFTTRS), and hydrophobic amino acids GLPL (GLPLALKVL). Other results reveal that sequence identity among the 20 RGAs rang from 41.1% to 99.3%, while identity of the deduced amino acid sequences range from 33.2% to 96.3%. The phylogenetic analysis of the RGA nucleotide sequences and the deduced amino acids showed that the 20 sequences could be divided into 5 distinct types. All of the amino acids deduced from the RGAs share a homology of 28%~54% with those deduced from the known wilt resistance genes such as Fom-2， I2C-1， I2C-2 and I2. This result to some degree indicates the conservation of disease resistance gene evolution. Technically, these RGAs isolated in the present study would lay a base for the further cloning of wilt resistance genes in banana, which could also be used as molecular markers for screening candidate wilt resistance genes in banana.
Plant R genes confer resistance to pathogens in a gene-for-gene mode. Seventy-five putative resistance gene analogs (RGAs) containing
conserved domains were cloned from Rubus idaeus L. cv. ‘Latham’ using degenerate primers based on RGAs identified in Rosaceae species. The sequences were compared to 195
RGA sequences identified from five Rosaceae family genera. Multiple sequence alignments showed high similarity at multiple
nucleotide-binding site (NBS) motifs with homology to Drosophila Toll and mammalian interleukin-1 receptor (TIR) and non-TIR
RNBSA-A motifs. The TIR sequences clustered separately from the non-TIR sequences with a bootstrap value of 76%. There were
11 clusters each of TIR and non-TIR type sequences of multiple genera with bootstrap values of more than 50%, including nine
with values of more than 75% and seven of more than 90%. Polymorphic sequence characterized amplified region and cleaved amplified
polymorphic sequence markers were developed for nine Rubus RGA sequences with eight placed on a red raspberry genetic linkage map. Phylogenetic analysis indicated four of the mapped
sequences share sequence similarity to groupTIR I, while three others were spread in non-TIR groups. Of the 75 Rubus RGA sequences analyzed, members were placed in five TIR groups and six non-TIR groups. These group classifications closely
matched those in 12 of 13 studies from which these sequences were derived. The analysis of related DNA sequences within plant
families elucidates the evolutionary relationship and process involved in pest resistance development in plants. This information
will aid in the understanding of R genes and their proliferation within plant genomes.
Phylogenetic relationships of the nucleotide binding site (NBS)-encoding resistance gene homologues (RGHs) among 12 species in five genera of Rosaceae fruit crops were evaluated. A total of 228 Rosaceous RGHs were deeply separated into two distinct clades, designated as TIR (sequences within this clade containing a Toll Interleukin-1 Receptor domain) and NonTIR (sequences lacking a TIR domain). Most Rosaceous RGH genes were phylogenetically distinct from Arabidopsis, Rice or Pine genes, except for a few Rosaceous members which grouped closely with Arabidopsis genes. Within Rosaceae, sequences from multiple species were often phylogenetically clustered together, forming heterogenous groups, however, apple- and chestnut rose-specific groups really exist. Gene duplication followed by sequence divergence were proposed as the mode for the evolution of a large number of distantly or closely related RGH genes in Rosaceae, and this mode may play a role in the generation of new resistance specificity. Positively selected sites within NBS-coding region were detected and thus nucleotide variation within NBS domain may function in determining disease resistance specificity. This study also discusses the synteny of a genomic region that encompass powdery mildew resistance locus among Malus, Prunus and Rosa, which may have potential use for fruit tree disease breeding and important gene cloning.
Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05(504,) NBS-09(711), NBS-07(688), NBS-03(509) and EST-SSR-04(241)) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.
Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from Pongamia glabra , Adenanthera pavonina , Clitoria ternatea and Solanum trilobatum using PCR based approach with primers designed from conserved regions of NBS domain. The presence of consensus motifs viz., kinase 1a, kinase 2, kinase 3a and hydrophobic domain provided evidence that the cloned sequences may belong to the NBS-LRR gene family. Conservation of tryptophan as the last residue of kinase-2 motif further confirms their position in non-TIR NBS-LRR family of resistance genes. The Resistance Gene Analogs (RGAs) cloned from P. glabra, A. pavonina, C. ternatea and S. trilobatum clustered together with well- characterized non-TIR-NBS-LRR genes leaving the TIR-NBS-LRR genes as a separate cluster in the average distance tree constructed based on BLOSUM62. All the four RGAs had high level of identity with NBS-LRR family of RGAs deposited in the GenBank. The extent of identity between the sequences at NBS region varied from 29% (P. glabra and S. trilobatum) to 78% (A. pavonina and C. ternatea), which indicates the diversity among the RGAs.
In Arabidopsis ecotype Landsberg erecta (L er), 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 L er RPP5 haplotype with the corresponding 90 kb of Arabidopsis ecotype Columbia (Col-0). Relative to the remainder of the genome, the L er 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 fungus Synchytrium endobioticum, the causal agent of potato wart disease, is subject to world-wide quarKantine regulations due to the production of persistent
resting spores and lack of effective chemical control measures. The selection of Synchytrium-resistant potato cultivars may be facilitated by using markers closely linked with a resistance gene or by transferring a
cloned gene for resistance into susceptible cultivars. Sen1, a gene for resistance to Synchytrium endobioticum race 1, was localized on potato chromosome XI in a genomic region which is related to the tobacco genome segment harbouring
the N gene for resistance to TMV. Using N as probe, we isolated homologous cDNA clones from a Synchytrium-resistant potato line. The N-homologous sequences of potato identified by RFLP mapping a family of resistance gene-like sequences closely linked with
the Sen1 locus. Sequence analysis of two full-length N-homologous cDNA clones revealed the presence of structural domains associated with resistance gene function. One clone (Nl-25)
encodes a polypeptide of 61 kDa and harbours a Toll-interleukin like region (TIR) and a putative nucleotide binding site (NBS).
The other clone (Nl-27) encodes a polypeptide of 95 kDa and harbours besides the TIR and NBS domains five imperfect leucine-rich
repeats (LRRs). Both clones have at their amino terminus a conserved stretch of serine residues that was also found in the
N gene, the RPP5 gene from Arabidopsis thaliana and several other resistance gene homologues, suggesting a function in the resistance response. Cloning of the disease resistance
locus based on map position and the establishment of PCR-based marker assays to assist selection of wart resistant potato
genotypes are discussed.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
Statistical tests for detecting gene conversion are described for a sample of homologous DNA sequences. The tests are based on imbalances in the distribution of segments on which some pair of sequences agrees. The methods automatically control for variable mutation rates along the genome and do not depend on a priori choices of potentially monophyletic subsets of the sample. The tests show strong evidence for multiple intragenic conversion events at two loci in Escherichia coli. The gnd locus in E. coli shows a highly significant excess of maximal segments of length 70-200 bp, which suggests conversion events of that size. The data also indicate that the rate of these short conversion events might be of the order of neutral mutation rate. There is also evidence for correlated mutation in adjacent codon positions. The same tests applied to a locus in an RNA virus were negative.
The products of plant disease resistance genes are postulated to recognize invading pathogens and rapidly trigger host defense responses. Here we describe isolation of the resistance gene N of tobacco that mediates resistance to the viral pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator transposon. A genomic DNA fragment containing the N gene conferred TMV resistance to TMV susceptible tobacco. Sequence analysis of the N gene shows that it encodes a protein of 131.4 kDa with an amino-terminal domain similar to that of the cytoplasmic domain of the Drosophila Toll protein and the interleukin-1 receptor (IL-1R) in mammals, a nucleotide-binding site (NBS), and 14 [corrected] imperfect leucine-rich repeats (LRR). The sequence similarity of N, Toll, and IL-1R suggests that N mediates rapid gene induction and TMV resistance through a Toll-IL-1-like pathway.
The tobacco N and Arabidopsis RPS2 genes, among several recently cloned disease-resistance genes, share highly conserved structure, a nucleotide-binding site (NBS). Using degenerate oligonucleotide primers for the NBS region of N and RPS2, we have amplified and cloned the NBS sequences from soybean. Each of these PCR-derived NBS clones detected low-or moderate-copy soybean DNA sequences and belongs to 1 of 11 different classes. Sequence analysis showed that all PCR clones encode three motifs (P-loop, kinase-2, and kinase-3a) of NBS nearly identical to those in N and RPS2. The intervening region between P-loop and kinase-3a of the 11 classes has high (26% average) amino acid sequence similarity to the N gene although not as high (19% average) to RPS2. These 11 classes represent a superfamily of NBS-containing soybean genes that are homologous to N and RPS2. Each class or subfamily was assessed for its positional association with known soybean disease-resistance genes through near-isogenic line assays, followed by linkage analysis in F2 populations using restriction fragment length polymorphisms. Five of the 11 subfamilies have thus far been mapped to the vicinity of known soybean genes for resistance to potyviruses (Rsv1 and Rpv), Phytophthora root rot (Rps1, Rps2, and Rps3), and powdery mildew (rmd). The conserved N- or RPS2-homologous NBS sequences and their positional associations with mapped soybean-resistance genes suggest that a number of the soybean disease-resistance genes may belong to this superfamily. The candidate subfamilies of NBS-containing genes identified by genetic mapping should greatly facilitate the molecular cloning of disease-resistance genes.
Sequences of cloned resistance genes from a wide range of plant taxa reveal significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. In this study, oligonucleotide primers designed for conserved sequences from coding regions of disease resistance genes N (tobacco), RPS2 (Arabidopsis) and L6 (flax) were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing of amplification products indicated that at least nine classes of resistance gene analogs (RGAs) were detected. Genetic mapping of members of these classes located them to eight different linkage groups. Several RGA loci mapped near known resistance genes. A bacterial artificial chromosome library of soybean DNA was screened using primers and probes specific for eight RGA classes and clones were identified containing sequences unique to seven classes. Individual bacterial artificial chromosomes contained 2-10 members of single RGA classes. Clustering and sequence similarity of members of RGA classes suggests a common process in their evolution. Our data indicate that it may be possible to use sequence homologies from conserved motifs of cloned resistance genes to identify candidate resistance loci from widely diverse plant taxa.
Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (avr) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (R) gene products. Members of the NBS-LRR class of R genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBS-LRR protein from Arabidopsis thaliana that confers resistance to Pseudomonas syringae expressing either avrRpm1 or avrB. RPM1 protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.
DnaSP is a Windows integrated software package for the analysis of the DNA polymorphism from nucleotide sequence
data. DnaSP version 3 incorporates several methods for estimating the amount and pattern of DNA polymorphism and divergence,
and for conducting neutrality tests. AVAILABILITY: For academic uses, DnaSP is available free of charge from: http://www.bio.ub.es/julio/DnaSP.html
CONTACT: julio@porthos.bio.ub.es
The tolerance of plants to herbivory reflects the degree to which a plant can regrow and reproduce after damage from herbivores. Autoecological factors, as well as the influence of competitors and mutualists, affect the level of plant tolerance. Recent work indicates that there is a heritable basis for tolerance and that it can evolve in natural plant populations. Although tolerance is probably not a strict alternative to plant resistance, there could be inter- and intraspecific tradeoffs between these defensive strategies.
Turnip crinkle virus (TCV) inoculation onto TCV-resistant Arabidopsis leads to a hypersensitive response (HR) controlled by the dominant gene HRT. HRT is a member of the class of resistance (R) genes that contain a leucine zipper, a nucleotide binding site, and leucine-rich repeats. The chromosomal position of HRT and its homology to resistance gene RPP8 and two RPP8 homologs indicate that unequal crossing over and gene conversion may have contributed to HRT evolution. RPP8 confers resistance to an oomycete pathogen, Peronospora parasitica. Despite very strong similarities within the HRT/RPP8 family, HRT and RPP8 are specific for the respective pathogens they detect. Hence, the HRT/RPP8 family provides molecular evidence that sequence changes between closely related members of multigene families can generate novel specificities for radically different pathogens. Transgenic plants expressing HRT developed an HR but generally remained susceptible to TCV because of a second gene, RRT, that regulates resistance to TCV. However, several transgenic plants that overexpressed HRT produced micro-HRs or no HR when inoculated with TCV and were resistant to infection. Expression of the TCV coat protein gene in seedlings containing HRT resulted in massive necrosis and death, indicating that the avirulence factor detected by the HRT-encoded protein is the TCV coat protein.
The performance of 14 different recombination detection methods was evaluated by analyzing several empirical data sets where
the presence of recombination has been suggested or where recombination is assumed to be absent. In general, recombination
methods seem to be more powerful with increasing levels of divergence, but different methods showed distinct performance.
Substitution methods using summary statistics gave more accurate inferences than most phylogenetic methods. However, definitive
conclusions about the presence of recombination should not be derived on the basis of a single method. Performance patterns
observed from the analysis of real data sets coincided very well with previous computer simulation results. Previous recombination
inferences from some of the data sets analyzed here should be reconsidered. In particular, recombination in HIV-1 seems to
be much more widespread than previously thought. This finding might have serious implications on vaccine development and on
the reliability of previous inferences of HIV-1 evolutionary history and dynamics.
Pathogens can be an important selective agent in plant evolution because they can severely reduce plant fitness and growth. However, the role of pathogen selection on plant evolution depends on the extent of genetic variation for resistance traits and their covariance with host fitness. Although it is usually assumed that resistance traits will covary with plant fitness, this assumption has not been tested rigorously in plant-pathogen interactions. Many plant species are tolerant to herbivores, decoupling the relationship between resistance and fitness. Tolerance to pathogens can reduce selection for resistance and alter the effect of pathogens on plant evolution. In this study, we measured three components of Arabidopsis thaliana resistance (pathogen growth, disease symptoms, and host fitness) to the bacteria Pseudomonas syringae and investigated their covariation to determine the relative importance of resistance and tolerance. We observed extensive quantitative variation in the severity of disease symptoms, the bacterial population size, and the effect of infection on host fitness among 19 accessions of A. thaliana infected with P. syringae. The severity of disease symptoms was strongly and positively correlated with bacterial population size. Although the average fitness of infected plants was smaller than noninfected plants, we found no correlation between the bacterial growth or symptoms expressed by different accessions of A. thaliana and their relative fitness after infection. These results indicate that the accessions studied vary in tolerance to P. syringae, reducing the strength of selection on resistance traits, and that symptoms and bacterial growth are not good predictors of host fitness.
In "gene-for-gene" interactions between plants and their pathogens, incompatibility (no disease) requires a dominant or semidominant resistance (R) gene in the plant, and a corresponding avirulence (Avr) gene in the pathogen. Many plant/pathogen interactions are of this type. R genes are presumed to (a) enable plants to detect Avr-gene-specified pathogen molecules, (b) initiate signal transduction to activate defenses, and (c) have the capacity to evolve new R gene specificities rapidly. Isolation of R genes has revealed four main classes of R gene sequences whose products appear to activate a similar range of defense mechanisms. Discovery of the structure of R genes and R gene loci provides insight into R gene function and evolution, and should lead to novel strategies for disease control.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
Turnip crinkle virus (TCV) inoculation onto TCV-resistant Arabidopsis leads to a hypersensitive response (HR) controlled by the dominant gene HRT. HRT is a member of the class of resistance (R) genes that contain a leucine zipper, a nucleotide binding site, and leucine-rich repeats. The chromosomal position of HRT and its homology to resistance gene RPP8 and two RPP8 homologs indicate that unequal crossing over and gene conversion may have contributed to HRT evolution. RPP8 confers resistance to an oomycete pathogen, Peronospora parasitica. Despite very strong similarities within the HRT/RPP8 family, HRT and RPP8 are specific for the respective pathogens they detect. Hence, the HRT/RPP8 family provides molecular evidence that sequence changes between closely related members of multigene families can generate novel specificities for radically different pathogens. Transgenic plants expressing HRT developed an HR but generally remained susceptible to TCV because of a second gene, RRT, that regulates resistance to TCV. However, several transgenic plants that overexpressed HRT produced micro-HRs or no HR when inoculated with TCV and were resistant to infection. Expression of the TCV coat protein gene in seedlings containing HRT resulted in massive necrosis and death, indicating that the avirulence factor detected by the HRT-encoded protein is the TCV coat protein.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
A total of 17 Pl and TAC clones each representing an assigned region of chromosome 5 were isolated from P1 and TAC genomic
libraries of Arabidopsis thaliana Columbia, and their nucleotide sequences were determined. The length of the clones sequenced in this study summed up to 1,081,958
bp. As we have previously reported the sequence of 9,072,622 bp by analysis of 125 P1 and TAC clones, the total length of
the sequences of chromosome 5 determined so far is now 10,154,580 bp. The sequences were subjected to similarity search against
protein and EST databases and analysis with computer programs for gene modeling. As a consequence, a total of 253 potential
protein-coding genes with known or predicted functions were identified. The positions of exons which do not show apparent
similarity to known genes were also assigned using computer programs for exon prediction. The average density of the genes
identified in this study was 1 gene per 4277 bp. Introns were observed in 74% of the potential protein genes, and the average
number per gene and the average length of the introns were 4.3 and 168 bp, respectively. The sequence data and gene information
are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.
An algorithm is presented for the multiple alignment of sequences, either proteins or nucleic acids, that is both accurate
and easy to use on microcomputers. The approach is based on the conventional dynamic-programming method of pairwise alignment.
Initially, a hierarchical clustering of the sequences is performed using the matrix of the pairwise alignment scores. The
closest sequences are aligned creating groups of aligned sequences. Then close groups are aligned until all sequences are
aligned in one group. The pairwise alignments included in the multiple alignment form a new matrix that is used to produce
a hierarchical clustering. If it is different from the first one, iteration of the process can be performed. The method is
illustrated by an example : a global alignment of 39 sequences of cytochrome c.
Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and, also, the evolutionary rates when the divergence times are known). In comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but both are purines or both pyrimidines, the difference is called type I (or "transition" type), while, if one of the two is a purine and the other is a pyrimidine, the difference is called type II (or "transversion" type). Letting P and Q be respectively the fractions of nucleotide sites showing type I and type II differences between two sequences compared, then the evolutionary distance per site is K = -(1/2) ln [(1-2P-Q) square root of 1-2Q]. The evolutionary rate per year is then given by k = K/(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions per site is estimated by K'S = -(1/2) ln (1-2P-Q). Also, formulae for standard errors were obtained. Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.
Plant genes for pathogen resistance can be used to engineer disease resistant crops. Oligonucleotides were designed from sequence motifs conserved between resistance genes of tobacco and Arabidopsis thaliana and used as PCR primers in potato DNA. Amplification products were obtained that were homologous to known resistance genes and linked without recombination with the nematode resistance locus Gro1 and the Phytophthora infestans resistance locus R7 of potato. Map positions of PCR-derived potato gene fragments were also correlated with resistance loci of the related tomato and tobacco genomes. Our results indicate that plant resistance genes that are effective against nematodes, fungi, viruses and bacteria may be isolated based on common sequence motifs and PCR methodology.
The recent cloning of genes for resistance against diverse pathogens from a variety of plants has revealed that many share conserved sequence motifs. This provides the possibility of isolating numerous additional resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We amplified resistance gene candidates (RGCs) from lettuce with multiple combinations of primers with low degeneracy designed from motifs in the nucleotide binding sites (NBSs) of RPS2 of Arabidopsis thaliana and N of tobacco. Genomic DNA, cDNA, and bacterial artificial chromosome (BAC) clones were successfully used as templates. Four families of sequences were identified that had the same similarity to each other as to resistance genes from other species. The relationship of the amplified products to resistance genes was evaluated by several sequence and genetic criteria. The amplified products contained open reading frames with additional sequences characteristic of NBSs. Hybridization of RGCs to genomic DNA and to BAC clones revealed large numbers of related sequences. Genetic analysis demonstrated the existence of clustered multigene families for each of the four RGC sequences. This parallels classical genetic data on clustering of disease resistance genes. Two of the four families mapped to known clusters of resistance genes; these two families were therefore studied in greater detail. Additional evidence that these RGCs could be resistance genes was gained by the identification of leucine-rich repeat (LRR) regions in sequences adjoining the NBS similar to those in RPM1 and RPS2 of A. thaliana. Fluorescent in situ hybridization confirmed the clustered genomic distribution of these sequences. The use of PCR with degenerate oligonucleotide primers is therefore an efficient method to identify numerous RGCs in plants.
Classical genetic and molecular data show that genes determining disease resistance in plants are frequently clustered in the genome. Genes for resistance (R genes) to diverse pathogens cloned from several species encode proteins that have motifs in common. These motifs indicate that R genes are part of signal-transduction systems. Most of these R genes encode a leucine-rich repeat (LRR) region. Sequences encoding putative solvent-exposed residues in this region are hypervariable and have elevated ratios of nonsynonymous to synonymous substitutions; this suggests that they have evolved to detect variation in pathogen-derived ligands. Generation of new resistance specificities previously had been thought to involve frequent unequal crossing-over and gene conversions. However, comparisons between resistance haplotypes reveal that orthologs are more similar than paralogs implying a low rate of sequence homogenization from unequal crossing-over and gene conversion. We propose a new model adapted and expanded from one proposed for the evolution of vertebrate major histocompatibility complex and immunoglobulin gene families. Our model emphasizes divergent selection acting on arrays of solvent-exposed residues in the LRR resulting in evolution of individual R genes within a haplotype. Intergenic unequal crossing-over and gene conversions are important but are not the primary mechanisms generating variation.
Sixteen P1 and TAC clones assigned to Arabidopsis thaliana chromosome 5 were sequenced, and their sequence features were analyzed using various computer programs. The total length of the sequences determined was 1,013,767 bp. Together with the nucleotide sequences of 109 clones previously reported, the regions of chromosome 5 sequenced so far now total 9,072,622 bp, which presumably covers approximately one-third of the chromosome. A similarity search against the reported gene sequences predicted the presence of a total of 225 protein-coding genes and/or gene segments in the newly sequenced regions, indicating an average gene density of one gene per 4.5 kb. Introns were identified in 72.4% of the potential protein genes for which the entire gene structure was predicted, and the average number per gene and the average length of the introns were 3.3 and 163 bp, respectively. These sequence features are essentially identical to those in the previously reported sequences. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.
The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.
The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.
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.
Several common themes have shaped the evolution of plant disease resistance genes. These include duplication events of progenitor resistance genes and further expansion to create clustered gene families. Variation can arise from both intragenic and intergenic recombination and gene conversion. Recombination has also been implicated in the generation of novel resistance specificities. Resistance gene clusters appear to evolve more rapidly than other regions of the genome. In addition, domains believed to be involved in recognitional specificity, such as the leucine-rich repeat (LRR), are subject to adaptive selection. Transposable elements have been associated with some resistance gene clusters, and may generate further variation at these complexes.
The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.
Scab caused by the fungal pathogen Venturia inaequalis is the most common disease of cultivated apple (Malus x domestica Borkh.). Monogenic resistance against scab is found in some small-fruited wild Malus species and has been used in apple breeding for scab resistance. Vf resistance of Malus floribunda 821 is the most widely used scab resistance source. Because breeding a high-quality cultivar in perennial fruit trees takes dozens of years, cloning disease resistance genes and using them in the transformation of high-quality apple varieties would be advantageous. We report the identification of a cluster of receptor-like genes with homology to the Cladosporium fulvum (Cf) resistance gene family of tomato on bacterial artificial chromosome clones derived from the Vf scab resistance locus. Three members of the cluster were sequenced completely. Similar to the Cf gene family of tomato, the deduced amino acid sequences coded by these genes contain an extracellular leucine-rich repeat domain and a transmembrane domain. The transcription of three members of the cluster was determined by reverse transcriptionpolymerase chain reaction to be constitutive, and the transcription and translation start of one member was verified by 5' rapid amplification of cDNA ends. We discuss the parallels between Cf resistance of tomato and Vf resistance of apple and the possibility that one of the members of the gene cluster is the Vf gene. Cf homologs from other regions of the apple genome also were identified and are likely to present other scab resistance genes.
The majority of plant disease-resistance genes (R-genes) isolated so far encode a predicted nucleotide-binding site (NBS) domain. NBS domains related to R-genes show a highly conserved backbone of amino acid motifs, which makes it possible to isolate resistance gene analogues (RGAs) by PCR with degenerate primers. Multiple combinations of primers with low degeneracy, designed from two conserved motifs in the NBS regions of R-genes of various plants, were used on genomic DNA from coffee trees, an important perennial tropical crop. Nine distinct classes of RGAs of the NBS-like type, representing a highly diverse sample, were isolated from Coffea arabica and C. canephora species. The analysis of one coffee RGA family suggested point mutations as the primary source of diversity. With one exception, coffee RGA families appeared to be closely related in sequence to at least one cloned R-gene. In addition, deduced amino acid sequences of coffee RGAs were identified that showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)-type R-genes. The high degree of similarity between particular coffee RGAs and R-genes isolated from other angiosperm species, such as Arabidopsis, tomato and rice, indicates an ancestral relationship and the existence of common ancestors. The data obtained from coffee species suggests that the evolution of NBS-encoding sequences involves the gradual accumulation of mutations and slow rates of divergence within distinct R-gene families, rather than being a rapid process. Functional inferences drawn from the suggested pattern of evolution of NBS-type R-genes is also discussed.
In Arabidopsis, RPP4 confers resistance to Peronospora parasitica (P.p.) races Emoy2 and Emwa1 (downy mildew). We identified RPP4 in Col-0 as a member of the clustered RPP5 multigene family encoding nucleotide-binding leucine-rich repeat proteins with Toll/interleukin-1 receptor domains. RPP4 is the orthologue of RPP5 which, in addition to recognizing P.p. race Noco2, also mediates resistance to Emoy2 and Emwa1. Most differences between RPP4 and RPP5 occur in residues that constitute the TIR domain and in LRR residues that are predicted to confer recognition specificity. RPP4 requires the action of at least 12 defence components, including DTH9, EDS1, PAD4, PAL, PBS2, PBS3, SID1, SID2 and salicylic acid. The ndr1, npr1 and rps5-1 mutations partially compromise RPP4 function in cotyledons but not in true leaves. The identification of RPP4 as a TIR-NB-LRR protein, coupled with its dependence on certain signalling components in true leaves, is consistent with the hypothesis that distinct NB-LRR protein classes differentially signal through EDS1 and NDR1. Our results suggest that RPP4-mediated resistance is developmentally regulated and that in cotyledons there is cross-talk between EDS1 and NDR1 signalling and processes regulating systemic acquired resistance.
MEGA: Mo-lecular Evolutionary Genetics Analysis (Version 2.1). Pennsyl-vania State University A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plants
- S K Tamura
- Jakobsen Ib
- Nei
S, Tamura K, Jakobsen IB, Nei M (2001) MEGA: Mo-lecular Evolutionary Genetics Analysis (Version 2.1). Pennsyl-vania State University, University Park Leister D, Ballvora A, Salamini F, Gebhardt C (1996) A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plants. Nat Genet 14:421