NBS Domains Research Articles

Genome-Wide Characterization of NLRs in Saccharum spontaneum L. and Their Responses to Leaf Blight in Saccharum

Sugarcane is an important sugar and potential energy crop, and the complexity of its genome has led to stagnant progress in genome decipherment and hindered the genome-wide analysis of the nucleotide binding site leucine-rich repeat (NLR) receptor until the genome of Saccharum spontaneum was published. From the genome of S. spontaneum, 724 allelic and non-allelic NLRs were identified and classified into five types (N, NL, CN, CNL, and P) according to domain architectures and integrity and at least 35 genes encoded non-canonical domains. The phylogenetic analysis indicated NLRs containing the coiled-coil (CC) domain separated from those without CC in six Poaceae species, including S. spontaneum. The motif analysis determined the characteristics and potential functions of the 137 representative non-allelic NLRs, especially the core motifs contained in the NBS and LRR domains, which indicated that motifs were regularly distributed among clades. Through transcription factor binding site (TFBS) profiles, we predicted that the most important transcription regulator of NLRs in sugarcane were ERF, MIKC_MADS, and C2H2. In addition, based on three sets of transcriptome data from two sugarcane hybrids and one S. spontaneum clone infected by the necrotrophic fungal pathogen Stagonospora tainanensis causing sugarcane leaf blight (SLB), the expression dynamics of NLRs responding to the infection in three sugarcane clones were compared. The different genetic background led to the significant difference of NLRs response to SLB in different sugarcane clones, and we got an inference of the potential mechanism of SLB resistance. These results provided a basic reference and new insights to further study and utilize the NLRs.

In silico characterization and expression of disease-resistance-related genes within the collinear region of Brassica napus blackleg resistant locus LepR1′ in B. oleracea

Blackleg is a devastating disease of cabbage (C genome), but so far, only a few resistant cabbage lines have been identified, and the resistance-related genes are yet to be identified. In contrast, numerous R loci have been mapped to the A or B genome. High ancestral synteny among Brassicaceae genomes suggest that homologous regions of R loci in the A or B genome may contain functional R gene(s) in the C genome. Here, we investigated the collinear region of a major Brassica napus R locus, LepR1′, in Brassica oleracea to identify genes of putative disease resistance class via comprehensive in silico analysis. The LepR1′ locus is collinear to a 17.29-Mbp region in chromosome C02 of Brassica oleracea and hosted a total of 1,392 genes, and 36 of these genes contained NBS, LRR, TIR, F-box and RLK domains. The expression of these 36 genes was profiled using qRT-PCR in the cotyledons of resistant (SCNU-03) and susceptible (SCNU-59) lines at 0, 6, 24, and 48 h after inoculation with Leptosphaeria maculans isolates 00–100s and 03–02s having the corresponding avirulent gene, AvrLep1. Among these genes, a cluster of 10 genes were differentially expressed. NBS-LRR gene Bo2g131620 had a 45-bp insertion/deletion (indel) mutation in the 3rd intron between resistant and susceptible lines. The higher expression in resistant lines against both isolates and the indel mutation in Bo2g131620 suggest that these genes may have roles in blackleg resistance in cabbage. Mapping and functional analysis will be necessary to validate such a role. Saturating the region with enough markers is recommended during mapping.

Genome-wide identification and expression analysis of rice NLR genes responsive to the infections of Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae

Nucleotide-binding site, leucine-rich repeat (NLR) genes play a critical role in rice disease resistance. However, the transcriptional activities of rice NLR genes during pathogen invasions are still unclear. To uncover their activities, we identified 430 regular rice NLR genes with both NBS and LRR domains, consisting of 192 CNL and 238 XNL (without a CC motif) members. We performed individual and integrative analyses based on 69 samples from rice microarray after the infections of Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae (Mor). Among the rice NLR genes, 397 were found to be expressed at low/medium level, while 10 NLR genes were observed to show high expression levels. A total of 400 NLR genes were discovered to be differentially expressed in at least one sample. Furthermore, 46 rice NLR genes were identified to be differentially expressed in response to the two pathogens and 38 of them could be validated using RNA-seq data. Six cis-regulatory elements (MYC, STRE, MYB, ABRE, G-box, and AS-1) were observed to occur frequently in the promoter regions of rice NLR genes. Ten NLR genes were selected for qPCR detection, and seven of the NLR genes verified the validity of the microarray and RNA-seq data. Our results are helpful in revealing expressions and regulations of NLR genes in rice resistance to Xoo and Mor.

In Silico Characterization of Nbs-lrr Gene Family in Vitis Vinifera Genome

Introduction: During evolution, plants developed defense mechanisms against pathogen attack, including an infinity of molecular processes that are triggered by pathogen exposure, such as resistance gene synthesis (R). The NBS-LRR gene family is known as one of the most representative families in the R gene class, in which its protein domains are related to one form of plant defense mechanisms. Objective: To identify and characterize candidate sequences of the NBS-LRR gene family in the Vitis vinifera genome. Methodology: Initially, a seed sequence was selected from those curated in the NBS-LRR family and deposited in the UniProt database. This sequence was aligned via tBLASTn against the V. vinifera genome deposited in the NCBI, by adopting a cut-off of e-value ≥ e-10. The sequences were annotated, translated and had its conserved protein domains identified by both ORF Finder and CD-search tools, respectively. Finally, the prediction of the isoelectric point was performed, and the molecular weight was also estimated using the JVirGel 2.0 software. Moreover, the subcellular localization was carried out by the Cell-PLoc 2.0 software. Results and Discussion: A total of 40 candidate sequences were retrieved that are related to the gene of interest. The translated proteins showed a variation from 338 to 944 aa in size. A total of 33 complete NBS domains were found, from which 16 sequences had a whole RX-CC_like domain, while only two sequences have a full-length LRR domain. The candidate proteins had an isoelectric point from 5.25 to 9.46, a molecular weight varying from 38.47 and 108.14 kDa. All proteins showed cytoplasmatic subcellular localization, from which, 21 % also displayed an cellular membrane localization, in agreement with the data described in the literature. Conclusions: The results described here can contribute to a better understanding of the molecular characteristics of the NBS-LRR gene family and their role in defense mechanisms of grapevines to pathogens.

Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae.

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L). Little is known about host-pathogen interactions and host defense mechanisms. Here, individual resistant and susceptible plants were selected from cultivars Maverick and ZG9830 and used for transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate PssALF3. Bioinformatic analysis revealed a number of differentially expressed genes (DEGs) in resistant and susceptible genotypes. Although resistant plants from each cultivar produced a hypersensitive response, transcriptome analyses indicated that they respond differently at the molecular level. The number of DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick, suggesting that ZG9830 plants had a more rapid effector triggered immune response. Unique up-regulated genes in resistant ZG9830 plants included genes encoding putative nematode resistance HSPRO2-like proteins, orthologs for the rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick plants had an over-representation of R genes in the CC-NBS-LRR family including two genes for atypical CCR domains and a putative ortholog of the Arabidopsis RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY family transcription factors and expression of genes involved in protein phosphorylation, regulation of transcription, defense response including synthesis of isoflavonoids, and oxidation-reduction processes. These results will further the identification of mechanisms involved in resistance to facilitate selection of parent populations and development of commercial varieties.

Engineering an auto-activated R protein that is in vivo activated by a viral protease

Autonomous hypersensitive responses (self-HRs) are caused by constitutively active R proteins. In this study, we identified an auto-activated form of the R gene Pvr9 (autoPvr9); the auto-activation results from an amino acid substitution between its NBS and LRR domains. Self-HR was strongly reduced or completely inhibited by fusion of an extra peptide to the autoPvr9 N-terminal or C-terminal, respectively. When an NIa recognition site was placed between autoPvr9 and the extra peptide, the fusion construct could trigger an NIa-dependent HR. Several C-terminal fusions were tested, but only those that maintained detectable protein expression were capable of an NIa-dependent HR. Our results suggest the potential for transforming malfunctioning and auto-activated R proteins into a new construct targeting potyviral NIa proteases.

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest.

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars.

A unique RPW8-encoding class of genes that originated in early land plants and evolved through domain fission, fusion, and duplication.

Duplication, lateral gene transfer, domain fusion/fission and de novo domain creation play a key role in formation of initial common ancestral protein. Abundant protein diversities are produced by domain rearrangements, including fusions, fissions, duplications, and terminal domain losses. In this report, we explored the origin of the RPW8 domain and examined the domain rearrangements that have driven the evolution of RPW8-encoding genes in land plants. The RPW8 domain first emerged in the early land plant, Physcomitrella patens, and it likely originated de novo from a non-coding sequence or domain divergence after duplication. It was then incorporated into the NBS-LRR protein to create a main sub-class of RPW8-encoding genes, the RPW8-NBS-encoding genes. They evolved by a series of genetic events of domain fissions, fusions, and duplications. Many species-specific duplication events and tandemly duplicated clusters clearly demonstrated that species-specific and tandem duplications played important roles in expansion of RPW8-encoding genes, especially in gymnosperms and species of the Rosaceae. RPW8 domains with greater Ka/Ks values than those of the NBS domains indicated that they evolved faster than the NBS domains in RPW8-NBSs.

Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes.

Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

Large-scale bioinformatic analysis of the regulation of the disease resistance NBS gene family by microRNAs in Poaceae

In the present study, we have screened 71, 713, 525, 119 and 241 mature miRNA variants from Hordeum vulgare, Oryza sativa, Brachypodium distachyon, Triticum aestivum, and Sorghum bicolor, respectively, and classified them with respect to their conservation status and expression levels. These Poaceae non-redundant miRNA species (1,669) were distributed over a total of 625 MIR families, among which only 54 were conserved across two or more plant species, confirming the relatively recent evolutionary differentiation of miRNAs in grasses. On the other hand, we have used 257 H. vulgare, 286T. aestivum, 119 B. distachyon, 269 O. sativa, and 139 S. bicolor NBS domains, which were either mined directly from the annotated proteomes, or predicted from whole genome sequence assemblies. The hybridization potential between miRNAs and their putative NBS genes targets was analyzed, revealing that at least 454 NBS genes from all five Poaceae were potentially regulated by 265 distinct miRNA species, most of them expressed in leaves and predominantly co-expressed in additional tissues. Based on gene ontology, we could assign these probable miRNA target genes to 16 functional groups, among which three conferring resistance to bacteria (Rpm1, Xa1 and Rps2), and 13 groups of resistance to fungi (Rpp8,13, Rp3, Tsn1, Lr10, Rps1-k-1, Pm3, Rpg5, and MLA1,6,10,12,13). The results of the present analysis provide a large-scale platform for a better understanding of biological control strategies of disease resistance genes in Poaceae, and will serve as an important starting point for enhancing crop disease resistance improvement by means of transgenic lines with artificial miRNAs.

Functional analysis of alternative transcripts of the soybean Rj2 gene that restricts nodulation with specific rhizobial strains.

The Rj2 gene is a TIR-NBS-LRR-type resistance gene in soybean (Glycine max) that restricts root nodule symbiosis with a group of Bradyrhizobium japonicum strains including USDA122. Rj2 generates two distinct transcript variants in its expression profile through alternative splicing. Alternative splicing of Rj2 is caused by the retention of the 86-bp intron 4. Inclusion of intron 4 in mature mRNA introduces an in-frame stop codon; as such, the alternative transcript is predicted to encode a truncated protein consisting of the entire portion of the TIR, NBS and LRR domains but missing the C-terminal domain of the full-length Rj2 protein encoded by the regular transcript. Since alternative splicing has been shown to be essential for full activity of several plant R genes, we attempted to test whether the alternative splicing is required for Rj2-mediated nodulation restriction. Here we demonstrated that the Rj2-mediated nodulation restriction does not require the combined presence of the regular and alternative transcripts, and the expression of the regular transcript alone is sufficient to confer nodulation restriction.

Analysis of structure and differential expression of Pseudomonas syringae 5-like(RPS5-like) genes in pathogen-infected Vitis flexuosa

Abstract: Nucleotide binding site-leucine-rich repeat (NBS-LRR) genes are the largest gene resistance family in plants. Resistance to Pseudomonas syringae 5 (RPS5) is a member of the NBS-LRR family. RPS5 and its homologs are important disease resistance genes in plants including Arabidopsis. This study identified nine loci of RPS5-like genes in Vitis flexuosa that showed differential expression from transcriptome analysis by next generation sequencing (NGS) of V. flexuosa infected with Elsinoe ampelina based on structural analyses: VfRPS5-like1059 (Vitis flexuosa resistance to Pseudomonas syringae 5-like 1059), VfRPS5-like1833, VfRPS5-like4135, VfRPS5-like4833, VfRPS5-like6172, VfRPS5-like13564, VfRPS5-like20585, VfRPS5-like55532, and VfRPS5-like62178. These genes, 2236-4762 bp in length, exhibited the structure of a coiled coil-nucleotide binding site-leucine-rich repeat (CC-NBS-LRR) resistance gene. The predicted amino acid sequences of all nine genes contained typical NBS domains. Chromosomal localization revealed that the nine genes were located in six chromosomes of the V. flexuosa pseudo-genome. Of the nine loci, only VfRPS5-like55532 showed upregulated expression against all tested pathogens at all tested time points, except 24 h postinoculation (hpi) for E. ampelina. All tested genes also showed differential expression against major grapevine fungal and bacterial pathogens. The results presented herein will also serve as a useful reference dataset for functional analysis and utilization in molecular breeding of resistant grapevines.

Loss/retention and evolution of NBS-encoding genes upon whole genome triplication of Brassica rapa

A genome triplication took place in the ancestor of Brassiceae species after the split of the Arabidopsis lineage. The postfragmentation and shuffling of the genome turned the ancestral hexaploid back to diploids and caused the radiation of Brassiceae species. The course of speciation was accompanied by the loss of duplicate genes and also influenced the evolution of retained genes. Of all the genes, those encoding NBS domains are typical R genes that confer resistance to invading pathogens. In this study, using the genome of Arabidopsis thaliana as a reference, we examined the loss/retention of orthologous NBS-encoding loci in the tripled Brassica rapa genome and discovered differential loss/retention frequencies. Further analysis indicated that loci of different retention ratios showed different evolutionary patterns. The loci of classesII and III (maintaining two and three syntenic loci, respectively, multi-loci) show sharper expansions by tandem duplications, have faster evolutionary rates and have more potential to be associated with novel gene functions. On the other hand, the loci that are retained at the minimal rate (keeping only one locus, class I, single locus) showed opposite patterns. Phylogenetic analysis indicated that recombination and translocation events were common among multi-loci in B. rapa, and differential evolutionary patterns between multi- and single-loci are likely the consequence of recombination. Investigations towards other gene families demonstrated different evolutionary characteristics between different gene families. The evolution of genes is more likely determined by the property of each gene family, and the whole genome triplication provided only a specific condition.

Isolation and characterization of Resistance Gene Analogue (RGA) from Fusarium resistant banana cultivars

Isolation and characterization of resistance genes from local banana cultivars was important in order to support the development of FOC resistant banana cultivars. Resistance gene analogues (RGAs) were isolated and characterized form three fusarium resistant banana cultivars using degenerate primers based on NBS domains. From 91 fragments sequenced, 17 fragments were positively NBS-type sequences and encoded as MNBS1-MNBS17. Phylogenetic analysis of MNBS deduced amino classified into three groups; the first group consisted of 14 members (MNBS1-MNBS14) with 97.4% identity, and the other three groups consisted of one member (MNBS15, MNBS16 and MNBS17, respectively) with 28.5% identity. All MNBS sequences were categorized as non-TIR-NBS-LRR. Comparison and phylogenetic analysis of MNBS with other known RGA and R genes showed that deduced amino acid MNBSs shared 91.7-98.8% identity with Musa NBS-LRR and 19.9-35.5% identity with known R genes. Among them, MNBS17 shared 50.5% identity with RGC2 (ABY75802) that assosiated to FOC race 4 resistant Musa species.

Alternative splicing is required for RCT1-mediated disease resistance in Medicago truncatula

RCT1 is a TIR-NBS-LRR-type resistance (R) gene in Medicago truncatula that confers resistance to multiple races of Colletotrichum trifolii, a hemi-biotrophic fungal pathogen that causes anthracnose disease in Medicago and other closely related legumes. RCT1 undergoes alternative splicing at both coding and 3'-untranslated regions, thereby producing multiple transcript variants in its expression profile. Alternative splicing of RCT1 in the coding region results from the retention of intron 4. Because intron 4 lies downstream of the LRR-encoding exons and contains an in-frame stop codon, the alternative transcript is predicted to encode a truncated protein consisting of the entire portion of the TIR, NBS, and LRR domains but lacks the C-terminal domain of the full-length RCT1 protein encoded by the regular transcript. Here we provide evidence that the RCT1-mediated disease resistance requires the combined presence of the regular and alternative transcripts. Neither the regular nor the alternative RCT1 transcript alone is sufficient to confer resistance against the pathogen. This study, in addition to the reports on the tobacco N and Arabidopsis RPS4 genes, adds another significant example showing the involvement of alternative splicing in R gene-mediated plant immunity.

Regions outside the leucine-rich repeat domain determine the distinct resistance specificities of the rice blast resistance genes Pik and Pik-m

Two alleles of the rice blast resistance (R) Pik locus, Pik-m and Pik, are each composed of a pair of nucleotide-binding site–leucine-rich repeat (NBS–LRR) genes, referred to as the first gene and the second gene. Pik-m and Pik are unique in that many of the amino acid substitutions between them are distributed in or near the N-terminal coiled-coil (CC) domain of the first gene, suggesting that the CC domain of the first gene plays an important role in determinating their R specificity. To examine this hypothesis, I investigated resistance phenotypes of transgenic plants carrying each of two kinds of domain-swapped Pik-m-based recombinant first genes. Replacement of the LRR domain of Pik-m with the equivalent region of Pik did not change the Pik-m-type specificity, indicating that regions outside the LRR domain are responsible for differentiating the R specificity of Pik-m from Pik. In contrast, replacement of both the NBS and LRR domains of Pik-m with the corresponding region of Pik resulted in loss of blast resistance, suggesting that co-adaptation of polymorphisms in the CC and NBS domains is necessary to maintain resistance.

Characterization of the rice blast resistance gene Pik cloned from Kanto51

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

Isolation of an Rx homolog from C. annuum and the evolution of Rx genes in the Solanaceae family

The well-conserved NBS domain of resistance (R) genes cloned from many plants allows the use of a PCR-based approach to isolate resistance gene analogs (RGAs). In this study, we isolated an RGA (CapRGC) from Capsicum annuum “CM334” using a PCR-based approach. This sequence encodes a protein with very high similarity to Rx genes, the Potato Virus X (PVX) R genes from potato. An evolutionary analysis of the CapRGC gene and its homologs retrieved by an extensive search of a Solanaceae database provided evidence that Rx-like genes (eight ESTs or genes that show very high similarity to Rx) appear to have diverged from R1 [an NBS-LRR R gene against late blight (Phytophthora infestans) from potato]-like genes. Structural comparison of the NBS domains of all the homologs in Solanaceae revealed that one novel motif, 14, is specific to the Rx-like genes, and also indicated that several other novel motifs are characteristic of the R1-like genes. Our results suggest that Rx-like genes are ancient but conserved. Furthermore, the novel conserved motifs can provide a basis for biochemical structural–function analysis and be used for degenerate primer design for the isolation of Rx-like sequences in other plant species. Comparative mapping study revealed that the position of CapRGC is syntenic to the locations of Rx and its homolog genes in the potato and tomato, but cosegregation analysis showed that CapRGC may not be the R gene against PVX in pepper. Our results confirm previous observations that the specificity of R genes is not conserved, while the structure and function of R genes are conserved. It appears that CapRGC may function as a resistance gene to another pathogen, such as the nematode to which the structure of CapRGC is most similar.

Insights from molecular modeling and dynamics simulation of pathogen resistance (R) protein from brinjal

Resistance (R) protein recognizes molecular signature of pathogen infection and activates downstream hypersensitive response signalling in plants. R protein works as a molecular switch for pathogen defence signalling and represent one of the largest plant gene family. Hence, understanding molecular structure and function of R proteins has been of paramount importance for plant biologists. The present study is aimed at predicting structure of R proteins signalling domains (CC-NBS) by creating a homology model, refining and optimising the model by molecular dynamics simulation and comparing ADP and ATP binding. Based on sequence similarity with proteins of known structures, CC-NBS domains were initially modelled using CED- 4 (cell death abnormality protein) and APAF-1 (apoptotic protease activating factor) as multiple templates. The final CC-NBS structural model was built and optimized by molecular dynamic simulation for 5 nanoseconds (ns). Docking of ADP and ATP at active site shows that both ligand bind specifically with same residues and with minor difference (1 Kcal/mol) in binding energy. Sharing of binding site by ADP and ATP and low difference in their binding site makes CC-NBS suitable for working as molecular switch. Furthermore, structural superimposition elucidate that CC-NBS and CARD (caspase recruitment domains) domain of CED-4 have low RMSD value of 0.9 A° Availability of 3D structural model for both CC and NBS domains will . help in getting deeper insight in these pathogen defence genes.

Isolation, Characterization and Phylogenetic Analysis of Nucleotide Binding Site-encoding Disease-resistance Gene Analogues from European Aspen (Populus tremula)

Abstract 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/dS) 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.