Resistance Gene Homologues Research Articles

Identification of NBS-Type Resistance Gene Homologs in Tobacco Genome

Tobacco (Nicotiana tabacum) is an important cash crop and an ideal experimental system for studies on plant–pathogen interaction. The sequenced tobacco genome provides an opportunity for examining resistance gene homologs (RGHs) in the tobacco genome. Thirty nucleotide-binding site-type RGHs were annotated from genomic data, and another 281 putative RGHs were identified via PCR amplification from wild and cultivated tobacco. The newly identified RGHs are similar to other known RGHs, and some were categorized into new groups or branches that are different from known Nicotiana R genes or RGHs. Of the 281RGHs, 146 were identified from a single tobacco genome. We did not find any polymorphism at the RGHs in cultivated accessions, implying that strong domestication selection and/or demographic effects might have caused a sharp reduction in nucleotide diversity. Three positive selection sites were found in several RGH groups, while purifying selection is pervasive in the RGH family. Our results provide a primary RGH pool and several positively selected sites for the further functional validation of resistance genes in tobacco.

Evidence that the BONZAI1/COPINE1 protein is a calcium- and pathogen-responsive defense suppressor

Copines are calcium-responsive, phospholipid-binding proteins involved in cellular signaling. The Arabidopsis BONZAI1/COPINE1 (BON1/CPN1) gene is a suppressor of defense responses controlled by the disease resistance (R) gene homolog SNC1. The BON1/CPN1 null mutant cpn1-1 has a recessive, temperature- and humidity-dependent, lesion mimic phenotype that includes activation of Pathogenesis-Related (PR) gene expression. Here, we demonstrated that the accumulation of BON1/CPN1 protein in wild-type plants was up-regulated by bacterial pathogen inoculation and by the activation of defense signaling responses controlled by two R genes, SNC1 and RPS2. Interestingly, however, over-accumulation of BON1/CPN1 in two BON1/CPN1 promoter T-DNA insertion mutants did not affect resistance to a bacterial pathogen. Promoter deletion analysis identified a 280 bp segment of the BON1/CPN1 promoter as being required for pathogen-induced gene expression; the same promoter region was also required for calcium ionophore-induced gene expression. Leaf infiltration with calcium ionophore triggered high-level PR gene expression specifically in cpn1-1 plants grown under permissive conditions, while co-infiltration of the calcium chelator EGTA attenuated this effect. These results explain the conditional nature of the cpn1-1 phenotype and are consistent with BON1/CPN1 being a calcium- and pathogen-responsive plant defense suppressor.

Construction of a potato transcriptome map based on the cDNA–AFLP technique

The cDNA-AFLP technique can be used to monitor differential gene expression, but also for linkage mapping. Extending previous works, we have now constructed an integrated linkage map of the potato transcriptome based on the said technique that has a length of around 800 cM and contains nearly 700 transcriptome derived fragments (TDFs). At the same time, most of these markers are anchored to the bins of a highly saturated reference map in potato, combining in this way the information provided by different marker types. Moreover, we detected and confirmed an elevated degree of allelic fragments with this marker type, which was present in nearly half of all primer combinations and involved around 20% of all fragments. These properties were particularly useful to establish anchor points for integrating the individual parental linkage maps. Comparative expression profiling in different plant materials revealed that only a few additional TDFs were obtained which were specific for mature leaves or tubers compared to the TDFs present in whole in vitro plants. Since TDF markers are derived from coding regions, they generally also represent sequences with a biological function. In four case studies, co-migrating TDFs in different Solanum wild species always represented potential alleles based on elevated homologies among them. Two resistance gene homologs were identified by analysing TDFs, which were co-located with known QTLs.

Genome-Wide and Mla Locus-Specific Characterisation of Latvian Barley Varieties

Genome-Wide and Mla Locus-Specific Characterisation of Latvian Barley Varieties Genetic diversity in locally adapted germplasm forms the basis for crop improvement through breeding. While single loci have been routinely used for studies of genetic diversity, the highthroughput genotyping platforms that have recently become available for large genome crop plants offer an unbiased view on genetic diversity on a genome-wide scale. We assessed genetic diversity in Latvian barley varieties and some progenitors using DArT markers and studied the extent of linkage disequilibrium in Latvian germplasm. Further, genetic diversity at three loci flanking the barley powdery mildew Mla locus conferring race-specific resistance was studied in Latvian barley germplasm. The Mla locus encompasses several closely related resistance gene homologues with a complex evolutionary history, which complicates the design of molecular markers for different Mla genes. We observed significant linkage disequilibrium between the single nucleotide polymorphisms (SNPs) at the three loci, 206i20_T7, ABC15612, and 538P8, flanking the Mla locus. SNP haplotypes were largely in agreement with known phenotypic data and, thus, may be potentially diagnostic for Mla resistance genes in hybrids.

Autoimmune Response as a Mechanism for a Dobzhansky-Muller-Type Incompatibility Syndrome in Plants

Epistatic interactions between genes are a major factor in evolution. Hybrid necrosis is an example of a deleterious phenotype caused by epistatic interactions that is observed in many intra- and interspecific plant hybrids. A large number of hybrid necrosis cases share phenotypic similarities, suggesting a common underlying mechanism across a wide range of plant species. Here, we report that approximately 2% of intraspecific crosses in Arabidopsis thaliana yield F1 progeny that express necrosis when grown under conditions typical of their natural habitats. We show that several independent cases result from epistatic interactions that trigger autoimmune-like responses. In at least one case, an allele of an NB-LRR disease resistance gene homolog is both necessary and sufficient for the induction of hybrid necrosis, when combined with a specific allele at a second locus. The A. thaliana cases provide insights into the molecular causes of hybrid necrosis, and serve as a model for further investigation of intra- and interspecific incompatibilities caused by a simple epistatic interaction. Moreover, our finding that plant immune-system genes are involved in hybrid necrosis suggests that selective pressures related to host–pathogen conflict might cause the evolution of gene flow barriers in plants.

Structure of two melon regions reveals high microsynteny with sequenced plant species

In this study, two melon bacterial artificial chromosome (BAC) clones have been sequenced and annotated. BAC 1-21-10 spans 92 kb and contains the nsv locus conferring resistance to the Melon Necrotic Spot Virus (MNSV) in melon linkage group 11. BAC 13J4 spans 98 kb and belongs to a BAC contig containing resistance gene homologues, extending a previous sequenced region of 117 kb in linkage group 4. Both regions have microsyntenic relationships to the model plant species Arabidopsis thaliana, and to Medicago truncatula and Populus trichocarpa. The network of synteny found between melon and each of the sequenced genomes reflects the polyploid structure of Arabidopsis, Populus, and Medicago genomes due to whole genome duplications (WGD). A detailed analysis revealed that both melon regions have a lower relative syntenic quality with Arabidopsis (eurosid II) than when compared to Populus and Medicago (eurosid I). Although phylogenetically Cucurbitales seem to be closer to Fabales than to Malphigiales, synteny was higher between both melon regions and Populus. Presented data imply that the recently completed Populus genome sequence could preferentially be used to obtain positional information in melon, based on microsynteny.

Identification of Cacao TIR-NBS-LRR Resistance Gene Homologues and Their Use as Genetic Markers

Identifying genetic markers linked to disease resistance in plants is an important goal in marker-assisted selection. Using a candidate-gene approach, we have previously developed genetic markers in cacao (Theobroma cacao L.) for two families of genes involved in disease resistance: non-TIR-NBS-LRR (Toll/Interleukin-1 Receptor-nucleotide binding site-leucine rich repeat) resistance gene homologues and WRKY transcription factor genes; however, we failed to isolate TIR-NBS-LRR genes. Using a novel algorithm to design degenerate primers, we have now isolated TIR-NBS-LRR loci as determined by DNA sequence comparison. These loci have been developed as genetic markers using capillary array electrophoresis (CAE) and single-strand conformational polymorphism (SSCP) analysis. We have mapped three distinct TIR-NBS-LRR loci in an F2 population of cacao and demonstrated that one is located on linkage group 3 and the other two on linkage group 5.

Theoretical Model of the Three-dimensional Structure of a Disease Resistance Gene Homolog Encoding Resistance Protein in Vigna mungo

Plant disease resistance (R) genes, the key players of innate immunity system in plants encode ‘R’ proteins. ‘R’ protein recognizes product of avirulance gene from the pathogen and activate downstream signaling responses leading to disease resistance. No three dimensional (3D) structural information of any ‘R’ proteins is available as yet. We have reported a ‘R’ gene homolog, the 'VMYR1′, encoding ‘R’ protein in Vigna mungo. Here, we describe the homology modeling of the 'VMYR1′ protein. The model was created by using the 3D structure of an ATP-binding cassette transporter protein from Vibrio cholerae as a template. The strategy for homology modeling was based on the high structural conservation in the superfamily of P-loop containing nucleoside triphosphate hydrolase in which target and template proteins belong. This is the first report of theoretical model structure of any ‘R’ proteins.

Isolation, characterization and evolutionary analysis of resistance gene analogs in annual ryegrass, perennial ryegrass and their hybrid

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.

Resistance Gene Mapping for Witches' Broom Disease in Theobroma cacao L. in an F2 Population using SSR Markers and Candidate Genes

A genetic linkage map was created from 146 cacao trees (Theobroma cacao), using an F2 population produced by selfing an F1 progeny of the cross Sca6 and ICS1. Simple sequence repeat (SSR) markers (170) were used principally for this map, with 12 candidate genes [eight resistance gene homologues (RGH) and four stress related WRKY genes], for a total of 182 markers. Joinmap software was used to create the map, and 10 linkage groups were clearly obtained, corresponding to the 10 known chromosomes of cacao. Our map encompassed 671.9 cM, approximately 100 cM less than most previously reported cacao maps, and 213.5 cM less than the one reported high-density map. Approximately 27% of the markers showed significant segregation distortion, mapping together in six genomic areas, four of which also showed distortion in other cacao maps. Two quantitative trait loci (QTL) for resistance to witches' broom disease were found, one producing a major effect and one a minor effect, both showing important dominance effects. One QTL for trunk diameter was found at a point 10.2 cM away from the stronger resistance gene. One RGH flanked the minor QTL for witches' broom resistance, implying possible association. QTLs mapped in F2 populations produce estimates of additive and dominance effects, not obtainable in F1 crosses. As dominance was clearly shown in the QTL found in this study, this population merits further study for evaluation of dominance effects for other traits. This F2 cacao population constitutes a useful link for genomic studies between cacao and cotton, its only widely grown agronomic relative.

Strain-specific and recessive QTLs involved in the control of partial resistance to Fusarium oxysporum f. sp. melonis race 1.2 in a recombinant inbred line population of melon

Fusarium oxysporum f. sp. melonis (FOM) causes serious economic losses in melon (Cucumis melo L.). Two dominant resistance genes have been identified, Fom-1 and Fom-2, which provide resistance to races 0 and 2 and races 0 and 1, respectively, however FOM race 1.2 overcomes these resistance genes. A partial resistance to FOM race 1.2 that has been found in some Far East accessions is under polygenic control. A genetic map of melon was constructed to tag FOM race 1.2 resistance with DNA markers on a recombinant inbred line population derived from a cross between resistant (Isabelle) and susceptible (cv. Védrantais) lines. Artificial root inoculations on plantlets of this population using two strains, one that causes wilting (FOM 1.2w) and one that causes yellowing (FOM 1.2y), resulted in phenotypic and genotypic data that enabled the identification of nine quantitative trait loci (QTLs). These QTLs were detected on five linkage groups by composite interval mapping and explained between 41.9% and 66.4% of the total variation. Four digenic epistatic interactions involving seven loci were detected and increased the total phenotypic variation that was explained. Co-localizations between QTLs and resistance gene homologs or resistance genes, such as Fom-2 and Vat, were observed. A strain-specific QTL was detected, and some QTLs appeared to be recessive.

Analysis of the melon genome in regions encompassing TIR-NBS-LRR resistance genes

Plant genomes contain numerous genes (R-genes) that play a role in initiating defence measures against their particular pathogens. Defence mechanisms controlled by R-genes have been the focus of extensive research over the past several years. The majority of the R-genes described so far belong to a super-family of genes (150-600 members) that encode proteins with a nucleotide binding site (NBS), some leucine-rich repeats (LRR) and an N-terminal domain that shows similarity to the Toll and Interleukin-1 receptors (TIR) or a N-terminal coiled-coil (CC) domain. Analysis of four regions of the melon (Cucumis melo) genome, including two sequenced BACs, identified 14 TIR-NBS-LRR genes. Known disease resistance genes have been mapped in three of these regions. Transcriptional expression was detected for predicted genes that are possibly involved in defence responses to pathogen attack. TIR-NBS-LRR genes appear to be clustered in the melon genome. They contain all the conserved motifs that have previously been described for their counterparts in other species, although differences were also detected. The results presented here may contribute to a better understanding of the genomic distribution and evolution of this group of resistance gene homologues and their variability.

Genome-wide identification of R genes and exploitation of candidate RGA markers in rice

By scanning the whole genomic sequence of japonica rice using 45 known plant disease resistance (R) genes, we identified 2119 resistance gene homologs or analogs (RGAs) and verified that RGAs are not randomly distributed but tend to cluster in the rice genome. The RGAs were classified into 21 families according to their functional domain based on Hidden Markov model (HMM). By comparing the RGAs of japonica rice with the whole genomic sequence of indica rice, we found 702 RGAs allelic between the two subspecies and revealed that 671 (95.6%) of them have length difference (InDels) in their genomic sequences (including coding and non-coding regions) between the two subspecies, suggesting that RGAs are highly polymorphic between the two subspecies in rice. We also exploited 402 PCR-based and co-dominant candidate RGA markers by designing primer pairs on the regions flanking the InDels and validating them via e-PCR. The length differences of the candidate RGA markers between the two subspecies are from 1 to 742 bp, with an average of 10.26 bp. All related information of the RGAs is available from our web site (http://ibi.zju.edu.cn/RGAs/index.html).

Full-genome analysis of resistance gene homologues in rice.

The availability of the rice genome sequence enabled the global characterization of nucleotide-binding site (NBS)-leucine-rich repeat (LRR) genes, the largest class of plant disease resistance genes. The rice genome carries approximately 500 NBS-LRR genes that are very similar to the non-Toll/interleukin-1 receptor homology region (TIR) class (class 2) genes of Arabidopsis but none that are homologous to the TIR class genes. Over 100 of these genes were predicted to be pseudogenes in the rice cultivar Nipponbare, but some of these are functional in other rice lines. Over 80 other NBS-encoding genes were identified that belonged to four different classes, only two of which are present in dicotyledonous plant sequences present in databases. Map positions of the identified genes show that these genes occur in clusters, many of which included members from distantly related groups. Members of phylogenetic subgroups of the class 2 NBS-LRR genes mapped to as many as ten different chromosomes. The patterns of duplication of the NBS-LRR genes indicate that they were duplicated by many independent genetic events that have occurred continuously through the expansion of the NBS-LRR superfamily and the evolution of the modern rice genome. Genetic events, such as inversions, that inhibit the ability of recently duplicated genes to recombine promote the divergence of their sequences by inhibiting concerted evolution.

A homolog of the RPS2 disease resistance gene is constitutively expressed in Brassica oleracea

In this study, we identified disease resistance gene homologs in Brassica oleracea and assessed their expression in lines resistant and susceptible to Xanthomonas campestris pv. campestris (Xcc). Two DNA fragments of approximately 2.5 kb (BI-16/RPS2 and Lc201/RPS2) were amplified by PCR from two Brassica lines using primers based on an RPS2 homologous sequence previously described in the Brassica oleracea ecotype B117. The sequences of these fragments shared high similarity (95-98%) with RPS2 homologs from various Brassica species. The digestion of these fragments with restriction enzymes revealed polymorphisms at the Xba I restriction sites. The length polymorphisms were used as a co-dominant marker in an F2 population developed to segregate for resistance to Xcc, the causal agent of black rot. Linkage analysis showed no significant association between the marker and quantitative trait loci for black rot. RT-PCR with specific primers yielded an expected 453 bp fragment that corresponded to the RPS2 homologs in both resistant and susceptible lines inoculated with the pathogen, as well as in non-inoculated control plants. These results suggest that these homologs are constitutively expressed in B. oleracea.

Linkage map of Cucumis melo including phenotypic traits and sequence-characterized genes.

A new linkage map of Cucumis melo, derived from the F2 progeny of a cross between PI 414723 and C. melo 'TopMark' is presented. The map spans a total of 1421 cM and includes 179 points consisting of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), simple sequence repeats (SSRs), and restriction fragment length polymorphism (RFLP) markers. The map also includes an aphid resistance trait (Vat) and the sex type gene, andromonoecious (a), the two of which are important in resistance breeding and the control of hybrid seed production, as well as a seed-color gene, Wt-2. Most RFLPs represent sequence-characterized cDNA probes from C. melo and Cucumis sativus. These include resistance gene homologues and genes involved in various aspects of plant development and metabolism. A sub-set of our SSR and RFLP markers were also mapped, as part of this study, on additional mapping populations that were published for this species. This provides important reference points ("anchors"), enabling us to identify several linkage groups with respect to other melon maps.

POSITIONAL CLONING OF THE APPLE SCAB RESISTANCE GENE VF

The apple scab disease resistance gene Vf, originating from Malus floribunda 821, is an important gene locus for the development of apple scab disease resistant cultivars. A total of 11 tightly linked sequence-characterized amplified region (SCAR) markers have been tagged to the Vf locus by using the amplified fragment length polymorphism (AFLP) technique followed by successful conversion of AFLP markers into SCAR markers. These 11 SCAR markers along with three previously identified SCAR markers were used to screen two bacterial artificial chromosome (BAC) libraries constructed from M. floribunda 821 and the apple cultivar 'GoldRush' (also containing Vf), respectively. As a result, a BAC contig, consisting of five overlapping BAC clones, was constructed spanning a genetic distance of ∼290 kb of the Vf region. Four receptor-like resistance gene homologues were unveiled in the Vf region after searching for encoding sequences from overlapping BAC clones. These four Vf gene homologues were designated as Vfal, Vfa2, Vfa3, and Vfa4, and co-segregated with the Vf locus in a segregant apple mapping population. The first three homologues, Vfal, Vfa2, and Vfa3, were expressed in young leaves, while the fourth homologue, Vfa4, was expressed in young leaves and strongly expressed in mature leaves. Four cDNA clones corresponding to these four homologues were recovered by using rapid cDNA end amplification (RACE) method. Sequence comparisons between cDNA and genomic DNA were conducted. Deduced amino acid sequences revealed that each of the Vf gene homologues contained both an extracellular leucine-rich repeat (LRR) domain and a transmembrane (TM) domain.

A large scale analysis of resistance gene homologues in Arachis.

Arachis hypogaea L., commonly known as the peanut or groundnut, is an important and widespread food legume. Because the crop has a narrow genetic base, genetic diversity in A. hypogaea is low and it lacks sources of resistance to many pests and diseases. In contrast, wild diploid Arachis species are genetically diverse and are rich sources of disease resistance genes. The majority of known plant disease resistance genes encode proteins with a nucleotide binding site domain (NBS). In this study, degenerate PCR primers designed to bind to DNA regions encoding conserved motifs within this domain were used to amplify NBS-encoding regions from Arachis spp. The Arachis spp. used were A. hypogaea var. Tatu and wild species that are known to be sources of disease resistance: A. cardenasii, A. duranensis, A. stenosperma and A. simpsonii. A total of 78 complete NBS-encoding regions were isolated, of which 63 had uninterrupted ORFs. Phylogenetic analysis of the Arachis NBS sequences derived in this study and other NBS sequences from Arabidopsis thaliana, Medicago trunculata, Glycine max, Lotus japonicus and Phaseolus vulgaris that are available in public databases This analysis indicates that most Arachis NBS sequences fall within legume-specific clades, some of which appear to have undergone extensive copy number expansions in the legumes. In addition, NBS motifs from A. thaliana and legumes were characterized. Differences in the TIR and non-TIR motifs were identified. The likely effect of these differences on the amplification of NBS-encoding sequences by PCR is discussed.

Genetic and physical mapping of homologues of the virus resistance gene Rx1 and the cyst nematode resistance gene Gpa2 in potato.

Nine resistance gene homologues (RGHs) were identified in two diploid potato clones (SH and RH), with a specific primer pair based on conserved motifs in the LRR domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene Rx1. A modified AFLP method was used to facilitate the genetic mapping of the RGHs in the four haplotypes under investigation. All nine RGHs appeared to be located in the Gpa2/ Rx1 cluster on chromosome XII. Construction of a physical map using bacterial artificial chromosome (BAC) clones for both the Solanum tuberosum ssp. tuberosum and the S. tuberosum ssp. andigena haplotype of SH showed that the RGHs are located within a stretch of less than 200 kb. Sequence analysis of the RGHs revealed that they are highly similar (93 to 95%) to Gpa2 and Rx1. The sequence identities among all RGHs range from 85 to 100%. Two pairs of RGHs are identical, or nearly so (100 and 99.9%), with each member located in a different genotype. Southern-blot analysis on genomic DNA revealed no evidence for additional homologues outside the Gpa2/ Rx1 cluster on chromosome XII.

Resistance gene homologues in Theobroma cacao as useful genetic markers

Resistance gene homologue (RGH) sequences have been developed into useful genetic markers for marker-assisted selection (MAS) of disease resistant Theobroma cacao. A plasmid library of amplified fragments was created from seven different cultivars of cacao. Over 600 cloned recombinant amplicons were evaluated. From these, 74 unique RGHs were identified that could be placed into 11 categories based on sequence analysis. Primers specific to each category were designed. The primers specific for a single RGH category amplified fragments of equal length from the seven different cultivars used to create the library. However, these fragments exhibited single-strand conformational polymorphism (SSCP), which allowed us to map six of the RGH categories in an F(2) population of T. cacao. RGHs 1, 4 and 5 were in the same linkage group, with RGH 4 and 5 separated by less than 4 cM. As SSCP can be efficiently performed on our automated sequencer, we have developed a convenient and rapid high throughput assay for RGH alleles.