Nucleotide Binding Site Domains Research Articles

Characterizing nucleotide binding site domain (NBD) of ZzR1 resistance gene from Zingiber zerumbet: in silico ligand docking and optimizing heterologous expression

The Nucleotide-binding site domain (NBD) of plant resistance (R) genes plays a vital role during plant defense signaling. The functional significance of CC-NBS-LRR (Coiled coil-NBS-Leucine Rich Repeat) class of R gene designated ZzR1, characterized from Zingiber zerumbet in earlier studies, was determined by molecular modeling and docking studies. Docked complex showed the ligand GTP interacts with amino acid residues in the cleft made by GLPL and P-loop motifs of the NBD. Heterologous expression of ZzR1 NBS protein was optimized using expression vectors, pEcoli-Nterm-6xHN and pET Directional TOPO and transformed in five Escherichia coli strains namely DH5α, TOP10, BL21DE3, BL21DE3 star and BL21plysS cells. The NBS protein of 36 kDa molecular size was expressed in E. coli BL21DE3 strain using pET TOPO vector. Optimum induction was detected at 30 °C using isopropyl-1-thio-β-D-galactopyranoside (IPTG) (1 mM). The present study provides valuable information on ligand interactions and heterologous expression of ZzR1 NBD protein.

Cloning of a BDV-NS3 Conserved Fragment for the Preparation of a Reporter Cell Line

: Border disease virus (BDV) is a serious pathogen of sheep and goats worldwide. The virus causes considerable economic losses in animal husbandry. Therefore, developing effective and genetic-based therapies to control viral infections, such as BDV, has been remarkable over recent years. A common way to evaluate such therapeutic strategies is by cloning the desired fragment into appropriate vectors to develop cell lines expressing it. Due to the required duties of the NS3 gene for BDV proliferation, this gene's HELICc and nucleotide binding site domain was synthesized and cloned in a lentiviral vector upstream of the GFP gene. The cloning accuracy was verified by digestion with restriction enzymes and sequencing. Thus, the BDV-NS3 HELICc and nucleotide binding site carrying plasmid was prepared successfully and will be applied in a second or third lentiviral packaging system for stable expression of the desired gene.

Bioinformatics analysis and function prediction of NBS-LRR gene family in Broussonetia papyrifera.

Most of the currently available disease resistance (R) genes have NBS (nucleotide-binding site) and LRR (leucine-rich-repeat) domain which belongs to the NBS-LRR gene family. The whole genome sequencing of Broussonetia papyrifera provides an important bioinformatics database for the study of the NBS-LRR gene family. In this study, 328 NBS-LRR family genes were identified and classified in B. papyrifera according to different classification schemes, where there are 92N types, 47 CN type, 54 CNL type, 29 NL types, 55 TN type, and 51 TNL type. Subsequently, we conducted bioinformatics analysis of the NBS-LRR gene family. Classification, motif analysis of protein sequences, and phylogenetic tree studies of the NBS-LRR genes in B. papyrifera provide important basis for the functional study of NBS-LRR family genes. Additionally, we performed structural analysis of the chromosomal location, physicochemical properties, and sequences identified by genetic characterization. In addition, through the analysis of GO enrichment, it was found that NBS-LRR genes were involved in defense responses and were significantly enriched in biological stimulation, immune response, and abiotic stress. In addition, we found that Bp06g0955 was the most sensitive to low temperature and encoded the RPM1 protein by analyzing the low temperature transcriptome data of B. papyrifera. Quantitative results of gene expression after 48h of Fusarium infection showed that Bp01g3293 increased 14 times after infection, which encodes RPM1 protein. The potential of NBS-LRR gene responsive to biotic and abiotic stresses can be exploited to improve the resistance of B. papyrifera.

The TIR-NBS protein TN13 associates with the CC-NBS-LRR resistance protein RPS5 and contributes to RPS5-triggered immunity in Arabidopsis.

Nucleotide-binding site (NBS)-leucine-rich repeat (LRR) domain receptor (NLR) proteins play important roles in plant innate immunity by recognizing pathogen effectors. The Toll/interleukin-1 receptor (TIR)-NBS (TN) proteins belong to a subtype of the atypical NLRs, but their function in plant immunity is poorly understood. The well-characterized Arabidopsis thaliana typical coiled-coil (CC)-NBS-LRR (CNL) protein Resistance to Pseudomonas syringae 5 (RPS5) is activated after recognizing the Pseudomonas syringae type III effector AvrPphB. To explore whether the truncated TN proteins function in CNL-mediated immune signaling, we examined the interactions between the Arabidopsis TN proteins and RPS5, and found that TN13 and TN21 interacted with RPS5. However, only TN13, but not TN21, was involved in the resistance to P. syringae pv. tomato (Pto) strain DC3000 carrying avrPphB, encoding the cognate effector recognized by RPS5. Moreover, the regulation of Pto DC3000 avrPphB resistance by TN13 appeared to be specific, as loss of function of TN13 did not compromise resistance to Pto DC3000 hrcC- or Pto DC3000 avrRpt2. In addition, we demonstrated that the CC and NBS domains of RPS5 play essential roles in the interaction between TN13 and RPS5. Taken together, our results uncover a direct functional link between TN13 and RPS5, suggesting that TN13 acts as a partner in modulating RPS5-activated immune signaling, which constitutes a previously unknown mechanism for TN-mediated regulation of plant immunity.

Molecular and functional analysis of a brown planthopper resistance protein with two nucleotide-binding site domains.

The brown planthopper (Nilaparvata lugens Stål, BPH) resistance gene BPH9 encodes an unusual coiled-coil (CC) nucleotide-binding leucine-rich repeat (LRR) protein with two nucleotide-binding site (NBS) domains. To understand how this CC-NBS-NBS-LRR (CNNL) protein regulates defense signaling and BPH resistance, we dissected each domain's functions. The CC domain of BPH9 self-associated and was sufficient to induce cell death. The region of 97-115 residues in the CC domain is crucial for self-association and activation. NBS2, which contains a complete set of NBS function motifs and inhibits CC domain activation, rather than NBS1, acts as a molecular switch to regulate the activity of BPH9. We demonstrated that the CC domain, the NBS domain, and the LRR domain of BPH9 associate with each other and themselves in planta. Further domain swapping experiments revealed that the CC domains of BPH9 and susceptible alleles were similarly competent to induce resistance and the hypersensitive response, while the LRR domain of BPH9 confers resistance specificity to BPH. These findings provide new insights into the regulatory mechanisms governing the activity of CNNL proteins.

A compendium of genome-wide sequence reads from NBS (nucleotide binding site) domains of resistance genes in the common potato

SolariX is a compendium of DNA sequence tags from the nucleotide binding site (NBS) domain of disease resistance genes of the common potato, Solanum tuberosum Group Tuberosum. The sequences, which we call NBS tags, for nearly all NBS domains from 91 genomes—representing a wide range of historical and contemporary potato cultivars, 24 breeding programs and 200 years—were generated using just 16 amplification primers and high-throughput sequencing. The NBS tags were mapped to 587 NBS domains on the draft potato genome DM, where we detected an average, over all the samples, of 26 nucleotide polymorphisms on each locus. The total number of NBS domains observed, differed between potato cultivars. However, both modern and old cultivars possessed comparable levels of variability, and neither the individual breeder or country nor the generation or time appeared to correlate with the NBS domain frequencies. Our attempts to detect haplotypes (i.e., sets of linked nucleotide polymorphisms) frequently yielded more than the possible 4 alleles per domain indicating potential locus intermixing during the mapping of NBS tags to the DM reference genome. Mapping inaccuracies were likely a consequence of the differences of each cultivar to the reference genome used, coupled with high levels of NBS domain sequence similarity. We illustrate that the SolariX database is useful to search for polymorphism linked with NBS-LRR R gene alleles conferring specific disease resistance and to develop molecular markers for selection.

Identification of NBS-encoding genes linked to black rot resistance in cabbage (Brassica oleracea var. capitata).

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.

Divergence and Conservative Evolution of XTNX Genes in Land Plants.

The Toll-interleukin-1 receptor (TIR) and Nucleotide-binding site (NBS) domains are two major components of the TIR-NBS-leucine-rich repeat family plant disease resistance genes. Extensive functional and evolutionary studies have been performed on these genes; however, the characterization of a small group of genes that are composed of atypical TIR and NBS domains, namely XTNX genes, is limited. The present study investigated this specific gene family by conducting genome-wide analyses of 59 green plant genomes. A total of 143 XTNX genes were identified in 51 of the 52 land plant genomes, whereas no XTNX gene was detected in any green algae genomes, which indicated that XTNX genes originated upon emergence of land plants. Phylogenetic analysis revealed that the ancestral XTNX gene underwent two rounds of ancient duplications in land plants, which resulted in the formation of clades I/II and clades IIa/IIb successively. Although clades I and IIb have evolved conservatively in angiosperms, the motif composition difference and sequence divergence at the amino acid level suggest that functional divergence may have occurred since the separation of the two clades. In contrast, several features of the clade IIa genes, including the absence in the majority of dicots, the long branches in the tree, the frequent loss of ancestral motifs, and the loss of expression in all detected tissues of Zea mays, all suggest that the genes in this lineage might have undergone pseudogenization. This study highlights that XTNX genes are a gene family originated anciently in land plants and underwent specific conservative pattern in evolution.

Transcriptome profiling and characterization of gene families with zinc finger and nucleotide binding site (NBS) domains in Larix kaempferi

Larix kaempferi is widely cultivated because of its significant ecological and economic values. However, the molecular mechanism underlying the growth and development of Larix kaempferi remains largely unknown because of its long growth cycle and highly complex genetic background. In this study, global transcriptome sequencing of Larix kaempferi was conducted via Solexa sequencing. In total, 15.49 million clean reads were assembled into 20,395 unigenes and then annotated. The study found hundreds of highly expressed unigenes involved in growth and development (e.g. plant hormone regulation pathways or starch and sucrose metabolic pathways), of which 25 unigenes with full-length coding regions interested us the most. In addition, two gene families containing the zinc finger and nucleotide-binding site (NBS) domains were further explored. A total of 48 C3H-type zinc finger genes, most containing a conversed C-X7–10-C-X5-C-X3-H motif, were identified and divided into eight distinct subfamilies through phylogenetic analysis. Twenty-one genes containing the NBS domain represented four subfamilies. These findings provided new insights into the transcriptome characteristics of Larix kaempferi and abundant sequence information to elucidate the roles of the functional genes in Larix kaempferi.

Cloning of novel rice blast resistance genes from two rapidly evolving NBS-LRR gene families in rice.

Most rice blast resistance genes (R-genes) encode proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. Our previous study has shown that more rice blast R-genes can be cloned in rapidly evolving NBS-LRR gene families. In the present study, two rapidly evolving R-gene families in rice were selected for cloning a subset of genes from their paralogs in three resistant rice lines. A total of eight functional blast R-genes were identified among nine NBS-LRR genes, and some of these showed resistance to three or more blast strains. Evolutionary analysis indicated that high nucleotide diversity of coding regions served as important parameters in the determination of gene resistance. We also observed that amino-acid variants (nonsynonymous mutations, insertions, or deletions) in essential motifs of the NBS domain contribute to the blast resistance capacity of NBS-LRR genes. These results suggested that the NBS regions might also play an important role in resistance specificity determination. On the other hand, different splicing patterns of introns were commonly observed in R-genes. The results of the present study contribute to improving the effectiveness of R-gene identification by using evolutionary analysis method and acquisition of novel blast resistance genes.

Mapping of clubroot (Plasmodiophora brassicae) resistance in canola (Brassica napus)

Clubroot disease, caused by Plasmodiophora brassicae, has become a major problem in the production of cruciferous crops worldwide. In this study, a population of 121 doubled haploid (DH) lines derived from a crossing between a resistant and a susceptible canola (Brassica napus) genotype was subjected to phenotypic and genotypic studies to determine the inheritance and location of the resistance gene(s). After inoculation with pathotype 3 of P. brassicae, the lines showed a 1:1 segregation ratio for resistance, indicating that resistance in this population is controlled by a single gene. Fifteen PCR‐based markers that were known to be linked to clubroot resistance (CR) genes were screened against genomic DNA from parents and resistant and susceptible bulks. Marker GC1680, linked to the CR gene CRa, exhibited polymorphism between the parents and between the resistant and susceptible bulks. CRa target primers were used to amplify fragments from the two parents and the resultant sequences were compared. A high degree of sequence similarity was found between the parents in the nucleotide binding site domain of CRa. In contrast, sequence polymorphisms were detected in the leucine‐rich repeat (LRR) domain. One pair of primers that amplify a band from the LRR region of the resistant parent but not the susceptible parent was used to screen the DH population. Amplicons were obtained from 60 of the 61 resistant lines and two of the 60 susceptible lines; thus, three recombinants were found. Based on these results, a resistance locus linked to CRa was found.

Identification and distribution of the NBS-LRR gene family in the Cassava genome.

BackgroundPlant resistance genes (R genes) exist in large families and usually contain both a nucleotide-binding site domain and a leucine-rich repeat domain, denoted NBS-LRR. The genome sequence of cassava (Manihot esculenta) is a valuable resource for analysing the genomic organization of resistance genes in this crop.ResultsWith searches for Pfam domains and manual curation of the cassava gene annotations, we identified 228 NBS-LRR type genes and 99 partial NBS genes. These represent almost 1% of the total predicted genes and show high sequence similarity to proteins from other plant species. Furthermore, 34 contained an N-terminal toll/interleukin (TIR)-like domain, and 128 contained an N-terminal coiled-coil (CC) domain. 63% of the 327 R genes occurred in 39 clusters on the chromosomes. These clusters are mostly homogeneous, containing NBS-LRRs derived from a recent common ancestor.ConclusionsThis study provides insight into the evolution of NBS-LRR genes in the cassava genome; the phylogenetic and mapping information may aid efforts to further characterize the function of these predicted R genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1554-9) contains supplementary material, which is available to authorized users.

Cloning and characterization of gene-resistant analogs (RGAs) involved in rust (Puccinia psidii) resistance in Eucalyptus grandis

Disease-resistant genes play an important role in defending against a variety of pathogens and insect pests in plants. Most of the disease-resistant genes encode proteins with conserved leucine rich repeat and nucleotide binding site domains. In this study, we cloned and characterized gene-resistant analogs (RGAs) from Eucalyptus grandis using degenerate PCR, with primers specifically targeting these two domains. The amplified fragments were cloned into the pGEM-T vector and transformed into Escherichia coli. Among the 90 clones obtained, 13 were sequenced and compared with each other and with previously identified gene-resistant diseases. A BLASTX search in GenBank revealed high similarities among the conserved domains of these cloned genes with RGA genes. Some clones, however, showed no significant similarity with DNA sequences in GenBank. Southern blotting analysis identified several polymorphic RFLP loci between distinct genotypes. However, none of them co-segregated with the Puccinia psidii Winter resistance gene 1 (Ppr1) in a population study.

Isolation and characterization of NBS–LRR resistance gene analogues from mango

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.

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.

Substitutions of Two Amino Acids in the Nucleotide-Binding Site Domain of a Resistance Protein Enhance the Hypersensitive Response and Enlarge the PM3F Resistance Spectrum in Wheat

Proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains are major components of the plant immune system. They usually mediate resistance against a subgroup of races of a specific pathogen. For the allelic series of the wheat powdery mildew resistance gene Pm3, alleles with a broad and a narrow resistance spectrum have been described. Here, we show that a broad Pm3 spectrum range correlates with a fast and intense hypersensitive response (HR) in a Nicotiana transient-expression system and this activity can be attributed to two particular amino acids in the ARC2 subdomain of the NBS. The combined substitution of these amino acids in narrow-spectrum PM3 proteins enhances their capacity to induce an HR in Nicotiana benthamiana, and we demonstrate that these substitutions also enlarge the resistance spectrum of the Pm3f allele in wheat. Finally, using Bph14, we show that the region carrying the relevant amino acids also plays a role in the HR regulation of another coiled-coil NBS-LRR resistance protein. These results highlight the importance of an optimized NBS-'molecular switch' for the conversion of initial pathogen perception by the LRR into resistance-protein activation, and we describe a possible approach to extend the effectiveness of resistance genes via minimal targeted modifications in the NBS domain.

Overexpression of the TIR-X gene results in a dwarf phenotype and activation of defense-related gene expression in Arabidopsis thaliana

The Arabidopsis genome encodes various proteins with a Toll/interleukin-1 receptor (TIR) domain. Many of these proteins also contain nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains and function as resistance (R) proteins. However, the protein encoded by At2g32140 (a TIR-X gene) contains a TIR domain but lacks NBS and LRR domains. We found that transgenic plants overexpressing At2g32140 displayed a dwarf phenotype and showed increased expression of defense-related genes. In general, the growth defect caused by activation of defense responses is suppressed under high-temperature conditions. However, transgenic plants overexpressing At2g32140 displayed a much stronger dwarf phenotype at 28°C than at 22°C. This dwarf phenotype was suppressed under the combination with known salicylic-acid pathway mutants. These findings suggest that At2g32140 encodes a protein involved in the plant defense response.

Characterization of Resistance Gene Analogues (RGAs) in Apple (Malus × domestica Borkh.) and Their Evolutionary History of the Rosaceae Family

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.

Cloning and characterization of an NBS-LRR resistance gene from peanuts (Arachis hypogaea L.)

The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family accounts for the largest number of known disease resistance genes and is one of the largest gene families in plant genomes. In this study, resistance gene analogs (RGAs) were isolated from peanuts based on the NBS domain. A full-length cDNA, PnAG3, was obtained by rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the length of PnAG3 was 1882 bp, which included a complete open reading frame of 1335 bp that encoded for the PnAG3 protein composed of 444 amino acids. Multiple analyses showed that this protein had homology with known resistance proteins, the highest being 48.01% with a resistance protein from Arachis cardenasii. The polypeptide has a typical non-TIR-NBS-LRR gene structure. Real-time fluorescence quantitative PCR analysis showed that after Aspergillus flavus infection, expression of the PnAG3 gene in J11 (A. flavus-resistant species) increased by 16.68, 11.16 and 25.96 in the seed coat, kernel and pericarp, respectively. However, it only increased 2–3 times in JH1012 (A. flavus-sensitive species). Cloning of the putative resistance gene from peanut provides a basis for studying the structure and function of peanut disease resistance-related genes and disease resistance genetic breeding in peanuts.

Genome-Wide Analysis of Nucleotide-Binding Site (NBS) Disease Resistance (R) Genes in Sacred Lotus (Nelumbo nucifera Gaertn.) Reveals Their Transition Role During Early Evolution of Land Plants

Nucleotide-binding site (NBS) containing genes comprise the largest class in identified plant resistance genes. A total of 137 NBS class resistance genes were identified from the newly sequenced sacred lotus genome (Nelumbo nucifera Gaertn.) through a reiterative computational sequence analysis. Three distinct groups of NBS-encoding genes were classified: 5 with Toll/interleukin-1 receptor homology (TIR) domain at N-terminal (TIR-NBS [-LRR (leucine-rich repeat)]), 37 with CC (coiled coil) domain (CC-NBS [-LRR]), and 95 with neither TIR nor CC at N-terminal (NBS [-LRR]). Sequence analysis revealed high divergence of NBS-LRR genes in sacred lotus. The result of cluster and syntenic analysis of NBS genes suggested a duplication and recombination event, which is consistent with the correspondent result of whole genome analysis. In addition, we also identified 52 NBS genes which have a putative NACHT domain embedded in the NBS domains. This characteristic has only been reported in animals, fungi and bacteria, suggesting that NACHT and NBS domains shared a similar ancient origin; and sacred lotus NBS (NACHT) genes may represent a transition role during the early evolution of disease resistance in land plants.