R-gene Analogs Research Articles

Transcriptome-wide identification and characterization of resistant gene analogs (RGAs) of ginger (Zingiber officinale Rosc.) and mango ginger (Curcuma amada Roxb.) under stress induced by pathogen

Resistance genes (R-genes) are responsible for activation of plant defense signaling involving the recognition of pathogen effectors by R-proteins resulting in the onset of the hypersensitive response (HR). R-gene analogs (RGAs) have been characterized in numerous plants and efficiently used for plant breeding purposes. Ginger (Zingiber officinale Rosc.) is an asexual spice crop which is affected by many diseases of which bacterial wilt caused by Ralstonia solanacearum is one of the most important production constraints. Out of the Zingiberaceous species screened for bacterial wilt resistance, mango ginger (Curcuma amada Roxb.) was found to be resistant. With an aim to study the disease resistance mechanism in mango ginger, RGAs have been identified from the ginger transcriptome database (gTDB) and characterized. Screening for the RGAs using bioinformatics-driven methods resulted in the identification of 160 clusters in mango ginger and 212 clusters in ginger with similarity to known R-genes that were classified based on the presence and organization of conserved domains. A large number of the clusters with gene ontology related to disease resistance were identified to be of NBS-LRR type. The gene expression studies of selected NBS-LRR transcripts identified that they got highly expressed in mango ginger in the initial hours post inoculation indicating their possible role in disease resistance. The study paves a way to understand the existing RGAs in ginger and mango ginger. Unfolding the mechanism that confers the high level of resistance in mango ginger may provide new insights for developing bacterial wilt resistance in ginger.

Restriction associated DNA-genotyping at multiple spatial scales in Arabidopsis lyrata reveals signatures of pathogen-mediated selection

BackgroundGenome scans based on outlier analyses have revolutionized detection of genes involved in adaptive processes, but reports of some forms of selection, such as balancing selection, are still limited. It is unclear whether high throughput genotyping approaches for identification of single nucleotide polymorphisms have sufficient power to detect modes of selection expected to result in reduced genetic differentiation among populations. In this study, we used Arabidopsis lyrata to investigate whether signatures of balancing selection can be detected based on genomic smoothing of Restriction Associated DNA sequencing (RAD-seq) data. We compared how different sampling approaches (both within and between subspecies) and different background levels of polymorphism (inbreeding or outcrossing populations) affected the ability to detect genomic regions showing key signatures of balancing selection, specifically elevated polymorphism, reduced differentiation and shifts towards intermediate allele frequencies. We then tested whether candidate genes associated with disease resistance (R-gene analogs) were detected more frequently in these regions compared to other regions of the genome.ResultsWe found that genomic regions showing elevated polymorphism contained a significantly higher density of R-gene analogs predicted to be under pathogen-mediated selection than regions of non-elevated polymorphism, and that many of these also showed evidence for an intermediate site-frequency spectrum based on Tajima’s D. However, we found few genomic regions that showed both elevated polymorphism and reduced FST among populations, despite strong background levels of genetic differentiation among populations. This suggests either insufficient power to detect the reduced population structure predicted for genes under balancing selection using sparsely distributed RAD markers, or that other forms of diversifying selection are more common for the R-gene analogs tested.ConclusionsGenome scans based on a small number of individuals sampled from a wide range of populations were sufficient to confirm the relative scarcity of signatures of balancing selection across the genome, but also identified new potential disease resistance candidates within genomic regions showing signatures of balancing selection that would be strong candidates for further sequencing efforts.

Using RNA Sequencing and In Silico Subtraction to Identify Resistance Gene Analog Markers for Lr16 in Wheat.

Leaf rust, caused by Puccinia triticina Eriks., is one of the most widespread diseases of wheat and breeding for resistance is one of the most effective methods of control. Lr16 is a wheat leaf rust resistance gene (R-gene) that provides resistance at both the seedling and adult stages. Simple-sequence repeat (SSR) markers have been used to map Lr16 to the distal end of chromosome 2B. The objectives of this study were to use RNA sequencing (RNA-seq) and in silico subtraction to identify new R-gene analogs (RGAs) and use them as Lr16 markers. RNA was isolated from the susceptible wheat cultivar Thatcher (Tc) and the resistant Tc isolines TcLr10, TcLr16, TcLr21, and sequenced using Illumina technology. Using in silico subtraction, sequences from the resistant Tc isolines were aligned to a Tc reference expressed sequence tag (EST) set. Sequences not aligning to the Tc reference were assembled into contigs and analyzed using BLASTx to determine putative gene functions. Primer pairs were designed for 181 RGA sequences, of which, 137 amplified in at least one of the parents. A mapping population was developed with 165 F2 lines from a cross between the rust-susceptible cultivar Chinese Spring (CS) and TcLr16. Two RGA markers XTaLr16_RGA266585 and XTaLr16_RGA22128 were identified that mapped proximally 1.2 and 23.8 cM from Lr16, respectively. Three SSR markers Xwmc764, Xwmc661, and Xbarc35 mapped between these two RGA markers at distances of 5.0, 10.9, and 16.1 cM from Lr16, respectively. In silico subtraction is an effective technique for isolating RGAs linked to R-genes of interest.

Characterization of novel wheat NBS domain-containing sequences and their utilization, in silico, for genome-scale R-gene mining

In crop improvement, the isolation, cloning and transfer of disease resistance genes (R-genes) is an ultimate goal usually starting from tentative R-gene analogs (RGAs) that are identified on the basis of their structure. For bread wheat, recent advances in genome sequencing are supporting the efforts of wheat geneticists worldwide. Among wheat R-genes, nucleotide-binding site (NBS)-encoding ones represent a major class. In this study, we have used a polymerase chain reaction-based approach to amplify and clone NBS-type RGAs from a bread wheat cultivar, 'Salambo 80.' Four novel complete ORF sequences showing similarities to previously reported R-genes/RGAs were used for in silico analyses. In a first step, where analyses were focused on the NBS domain, these sequences were phylogenetically assigned to two distinct groups: a first group close to leaf rust Lr21 resistance proteins; and a second one similar to cyst nematode resistance proteins. In a second step, sequences were used as initial seeds to walk up and downstream the NBS domain. This procedure enabled identifying 8 loci ranging in size between 2,115 and 7,653 bp. Ab initio gene prediction identified 8 gene models, among which two had complete ORFs. While GenBank survey confirmed the belonging of sequences to two groups, subsequent characterization using IWGSC genomic and proteomic data showed that the 8 gene models, reported in this study, were unique and their loci matched scaffolds on chromosome arms 1AS, 1BS, 4BS and 1DS. The gene model located on 1DS is a pseudo-Lr21 that was shown to have an NBS-LRR domain structure, while the potential association of the RGAs, here reported, is discussed. This study has produced novel R-gene-like loci and models in the wheat genome and provides the first steps toward further elucidation of their role in wheat disease resistance.

Comparative genomic sequence analysis of strawberry and other rosids reveals significant microsynteny

BackgroundFragaria belongs to the Rosaceae, an economically important family that includes a number of important fruit producing genera such as Malus and Prunus. Using genomic sequences from 50 Fragaria fosmids, we have examined the microsynteny between Fragaria and other plant models.ResultsIn more than half of the strawberry fosmids, we found syntenic regions that are conserved in Populus, Vitis, Medicago and/or Arabidopsis with Populus containing the greatest number of syntenic regions with Fragaria. The longest syntenic region was between LG VIII of the poplar genome and the strawberry fosmid 72E18, where seven out of twelve predicted genes were collinear. We also observed an unexpectedly high level of conserved synteny between Fragaria (rosid I) and Vitis (basal rosid). One of the strawberry fosmids, 34E24, contained a cluster of R gene analogs (RGAs) with NBS and LRR domains. We detected clusters of RGAs with high sequence similarity to those in 34E24 in all the genomes compared. In the phylogenetic tree we have generated, all the NBS-LRR genes grouped together with Arabidopsis CNL-A type NBS-LRR genes. The Fragaria RGA grouped together with those of Vitis and Populus in the phylogenetic tree.ConclusionsOur analysis shows considerable microsynteny between Fragaria and other plant genomes such as Populus, Medicago, Vitis, and Arabidopsis to a lesser degree. We also detected a cluster of NBS-LRR type genes that are conserved in all the genomes compared.

Consequences of Geographical Habitats on Population Structure and Genetic Diversity in Campanula spp.

Characterization of populations by means of DNA techniques provides a tool for precise identification and a quantitative estimate of genetic diversity, crucial in evaluation of genetic fragmentation within and among populations. NBS profiling are PCR-based approaches that sample genetic variation in resistance genes (R-gene), and R gene analogs (RGA). To date, myb patterns have not been used for evaluating genetic diversity in other species. NBS primers are homologous to the conserved sequences in the Nucleotide-Binding-Site of the NBS-LRR class of R-genes. A total of 12 populations from five Campanula species (C. barbata L., C. latifolia L., C. rapunculoides L., C. spicata L. and C. trachelium L.), autochthonous of the West Italian Alps, were genotyped via nucleotide-binding site (NBS) and myb gene profiling. The selected markers produced a total of 361 bands, showing high levels of polymorphism. Genetic diversity among and within species and population structure was evaluated by different statistical analyses performed using TREECON software, Mantel Nonparametric Test, NTSYS package, AMOVA and STRUCTURE. The correlation between genetic variability and geographical location suggests that the five Campanula species have been subjected to long-term evolutionary processes consistent with the natural fragmentation of continuous mountains areas.

Isolation and Characterization of Disease Resistance Gene Analogs from <I>Erianthus arundinaceus</I> cDNA

Plant disease resistance genes (R-genes) encode some conserved motifs. According to the conserved motifs present in the known NBS-LRR R-gene sequences and R gene analogs (RGAs), several degenerate primers were designed and applied in the RGA isolation from Erianthus arundinaceus using PCR approach. In total, 6 RGAs were successfully obtained, with GenBank accession numbers of EU685835, EU685836, EU685837, EU685838, EU685839, and EU685840. Multiple alignments showed that the encoding sequences of the six clones were highly conserved and strikingly similar to the eleven most typical NBS-LRR type R-gene peptide sequences, especially at the four NES motifs of P-loop. kinase-2, kinase-3a, and HD. The identity percentage at the amino-acid level ranged from 8.3% to 93.0% among all 17 sequences tested and from 30.5% to 45.6% among the six RGAs cloned in this study. The results of cluster analysis and the existence of an aspartic acid residue (D) at the final residue position of the kinase-2 motif also indicated that all of E. arundinaceus RGAs might belong to non-TIR group. The Real-time PCR results showed that all of the RGAs were constitutively expressed in roots, stalks and leaves of E. arundinaceus, and their expression could be up-regulated in leaves by the exogenous signal molecules SA and H2O2. Therefore, it suggested that E. arundinaceus RGAs might play important roles in disease resistance in an SA- and H2O2-dependent defense response pathway. Further studies should aim to clone full-length R-genes in E. arundinaceus and characterize their functions in defense responses.

Development of SNP markers linked to the L locus in Capsicum spp. by a comparative genetic analysis

In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence, more BAC clones were identified and a BAC contig spanning 224 kb was constructed. Gene prediction analysis showed that there were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2 cM) to the L 4 gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this study was subjected to linkage analysis in L 4 - and L 3 -segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with L 3 , was located on the opposite side of 087H3T7, about 0.7 cM away from L 4 . This supports the idea that L 3 and L 4 may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking markers in molecular breeding program for introgression of L 4 to elite germplasm against most aggressive tobamoviruses pathotype P1,2,3 is discussed.

Efficient authentic fine mapping of the rice blast resistance gene Pik-h in the Pik cluster, using new Pik-h-differentiating isolates

The Pik-h gene in rice confers resistance to several races of rice blast fungus (Magnaporthe oryzae), and has been classified as a member of the Pik cluster, one of the most resistance (R) gene-dense regions in the rice genome. However, the loss of a key mutant isolate has long made it difficult to differentiate Pik-h from other Pik group genes especially from Pik-m. We identified new natural isolates enabling the differentiation between Pik-h and Pik-m genes, and first confirmed the authenticity of the International Rice Research Institute (IRRI) “monogenic” line IRBLkh-K3, and then fine-mapped the Pik-h gene in the Pik cluster. Using 701 susceptible individuals among 3,060 siblings from a cross of IRBLkh-K3×CO39, the Pik-h region was delimited to 270 kb, the narrowest interval among the Pik group genes reported to date, in the cv. Nipponbare genome. Annotation of this genome region first revealed 6 NBS-LRR type R-gene analogs (RGAs), clustered within the central 120 kb, as possible counterparts of Pik-h and 6 other Pik group R genes. Interestingly, the Pik-h region and the cluster of RGAs were shown to be located 130 kb and 230 kb apart from Xa4 and Xa2 bacterial blight resistance genes, respectively, once classified as belonging to the Pik cluster. The closest recombination events were limited to the margins of the Pik-h region, and recombination was suppressed in the core interval with the RGA cluster. This fine-mapping, performed in a short time using an HEGS system, will facilitate utilization of the cluster’s genetic resources and help to elucidate the mechanism of evolution of R-genes. The presence of natural isolates also confirmed that evolution of Pik-h corresponds to pathogen evolution.

Illegitimate recombination is a major evolutionary mechanism for initiating size variation in plant resistance genes

Current models for the evolution of plant disease resistance (R) genes are based on mechanisms such as unequal crossing-over, gene conversion and point mutations as sources for genetic variability and the generation of new specificities. Size variation in leucine-rich repeat (LRR) domains was previously mainly attributed to unequal crossing-over or template slippage between LRR units. Our analysis of 112 R genes and R gene analogs (RGAs) from 16 different gene lineages from monocots and dicots showed that individual LRR units are mostly too divergent to allow unequal crossing-over. We found that illegitimate recombination (IR) is the major mechanism that generates quasi-random duplications within the LRR domain. These initial duplications are required as seeds for subsequent unequal crossing-over events which cause the observed rapid increase or decrease in LRR repeat numbers. Ten of the 16 gene lineages studied contained such duplications, and in four of them the duplications served as a template for subsequent repeat amplification. Our analysis of Pm3-like genes from rice and three wheat species showed that such events can be traced back more than 50 million years. Thus, IR represents a major new evolutionary mechanism that is essential for the generation of molecular diversity in evolution of RGAs.

Genome-wide isolation of resistance gene analogs in maize (Zea mays L.)

Conserved domains or motifs shared by most known resistance (R) genes have been extensively exploited to identify unknown R-gene analogs (RGAs). In an attempt to isolate all potential RGAs from the maize genome, we adopted the following three methods: modified amplified fragment length polymorphism (AFLP), modified rapid amplification of cDNA ends (RACE), and data mining. The first two methods involved PCR-based isolations of RGAs with degenerate primers designed based on the conserved NBS domain; while the third method involved mining of RGAs from the maize EST database using full-length R-gene sequences. A total of 23 and 12 RGAs were obtained from the modified AFLP and RACE methods, respectively; while, as many as 109 unigenes and 77 singletons with high homology to known R-genes were recovered via data-mining. Moreover, R-gene-like ESTs (or RGAs) identified from the data-mining method could cover all RACE-derived RGAs and nearly half AFLP-derived RGAs. Totally, the three methods resulted in 199 non-redundant RGAs. Of them, at least 186 were derived from putative expressed R-genes. RGA-tagged markers were developed for 55 unique RGAs, including 16 STS and 39 CAPS markers.

The Genetic Architecture of Disease Resistance in Maize: A Synthesis of Published Studies

ABSTRACT Fifty publications on the mapping of maize disease resistance loci were synthesized. These papers reported the locations of 437 quantitative trait loci (QTL) for disease (dQTL), 17 resistance genes (R-genes), and 25 R-gene analogs. A set of rules was devised to enable the placement of these loci on a single consensus map, permitting analysis of the distribution of resistance loci identified across a variety of maize germplasm for a number of different diseases. The confidence intervals of the dQTL were distributed over all 10 chromosomes and covered 89% of the genetic map to which the data were anchored. Visual inspection indicated the presence of clusters of dQTL for multiple diseases. Clustering of dQTL was supported by statistical tests that took into account genome-wide variations in gene density. Several novel clusters of resistance loci were identified. Evidence was also found for the association of dQTL with maturity-related QTL. It was evident from the distinct dQTL distributions for the different diseases that certain breeding schemes may be more suitable for certain diseases. This review provides an up-to-date synthesis of reports on the locations of resistance loci in maize.

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

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

Nucleotide binding site/leucine-rich repeats, Pto-like and receptor-like kinases related to disease resistance in grapevine.

Nucleotide Binding Site/Leucine-Rich Repeat (NBS-LRR) and Serine/Threonine Kinase (STK) genes are two of the known classes of resistance (R-) genes in plants, and occur in large multigene families. Systematic identification of genes for NBS-LRRs and STKs provides a means of access to genomic regions that may be involved in disease resistance. Here we present a picture of these two families of R-gene analogs (RGAs) in grape with the aim of developing a set of resistance-related sequence-tagged-site (STS) markers. One hundred and three NBS-LRR sequences were isolated. They included members of the CC (coiled-coil) and TIR (Toll-interleukin receptor) sub-classes. A comparative analysis with other angiosperm NBSs is provided. Fifty-three genes for receptor-like kinases (RLKs) with serine/threonine specificity were identified. RLK sequences formed a putative monophyletic group within the kinase superfamily. They were similar to both cytoplasmic RLKs, such as Pto, and RLKs with LRR, S-locus, lectin-like and thaumatin-like extracellular binding-domains. The latter resembled the products of the R-related genes Xa21, FLS2, Rlk10, SFR2, and PR5K. Forty-five reference RGAs were converted into STSs by using appropriately designed specific primers. RGA-STSs were present in diverse grape genotypes, and >85% of the primers were capable of amplifying the STSs across the taxa Vitis and Muscadinia. DNA sequence polymorphism among these RGAs was assessed by SSCP (single-strand conformation polymorphism) analysis in over 20 Vitis spp. Finally, 45 universal primers for grape RGAs are proposed that should permit tagging of R-related regions in any grape genome.

Investigation of variations in NBS motifs in alfalfa (Medicago sativa), M. edgeworthii, and M. ruthenica

Medicago edgeworthii Sirjaev and M. ruthenica (L.) Ledebour are allogamous, diploid (2n = 2x = 16) perennials from Asia, which have a remarkable ability to survive stress , but are genetically distant from cultivated alfalfa (M. sativa L., 2n = 4x = 32). In lieu of hybridization, potentially important genes from the Asian species could be transferred to alfalfa using transgenic techniques. Many plant disease resistance genes (R-genes) belong to the nucleotide binding site-leucine rich repeats (NBS-LRR) superfamily and have several highly conserved NBS region motifs, which can be used to identify them. Thus, genomic DNA of alfalfa and the two Asian species was primed with a degenerate forward P-loop primer paired with degenerate reverse primers designed for GLPLA, RNBS-C, or RNBS-D-non-Toll/Interleukin (RNBS-D-non-TIR) motifs. The P-Loop/GLPLA primer combination yielded the largest number of sequences without stop codons, and alfalfa had the greatest variation within the kinase and RNBS-C domains; however several kinase and RNBS-C sequences were unique to M. edgeworthii or M. ruthenica. Priming with the P-loop/RNBS-C primer pair resulted in some open reading frames, which apparently did not belong to the NBS-LRR superfamily. The only open reading frames for the P-loop/RNBS-D-non-TIR primer combination were for M. ruthenica. These non-TIR-type clones, and the only non-TIR-type clone from the P-loop/GLPLA PCR (alfalfa), had sequences that were markedly different from the TIR-type sequences. The RNBS-C region may be useful in identifying non-TIRtype R-gene analogs in future studies. Screening for unique R-gene analogs in genetically distant Medicago species may be a very effective way of isolating potential new R-genes for transfer to alfalfa. Key words: Alfalfa, disease resistance, kinase, Medicago edgeworthii, Medicago ruthenica, NBS-LRR, R-gene analog

Disease resistance gene analogs as candidates for QTLs involved in pepper-pathogen interactions

Whereas resistance genes (R-genes) governing qualitative resistance have been isolated and characterized, the biological roles of genes governing quantitative resistance (quantitative trait loci, QTLs) are still unknown. We hypothesized that genes at QTLs could share homologies with cloned R-genes. We used a PCR-based approach to isolate R-gene analogs (RGAs) with consensus primers corresponding with conserved domains of cloned R-genes: (i) the nucleotide binding site (NBS) and hydrophobic domain, and (ii) the kinase domain. PCR-amplified fragments were sequenced and mapped on a pepper intraspecific map. NBS-containing sequences of pepper, most similar to the N gene of tobacco, were classified into seven families and all mapped in a unique region covering 64 cM on the Noir chromosome. Kinase domain containing sequences and cloned R-gene homologs (Pto, Fen, Cf-2) were mapped on four different linkage groups. A QTL involved in partial resistance to cucumber mosaic virus (CMV) with an additive effect was closely linked or allelic to one NBS-type family. QTLs with epistatic effects were also detected at several RGA loci. The colocalizations between NBS-containing sequences and resistance QTLs suggest that the mechanisms of qualitative and quantitative resistance may be similar in some cases.