云杉NBS-LRR基因的克隆与表达分析

为深入研究NBS-LRR基因在川西云杉(Picea balfouriana)抗落针病过程中的分子作用机制,该研究根据GenBank数据库中其他植物NBS-LRR基因保守序列设计引物,利用RT-PCR技术,克隆云杉NBS-LRR基因全长cDNA序列(PbNBS-LRR),分析该基因及其编码蛋白的相关信息并进行基因表达研究。结果表明:(1)成功获得PbNBS-LRR基因的全长2 616 bp(基因登录号:MK044348),且包含一个2 508 bp的完整阅读框(ORF),共编码836个氨基酸,其氨基酸序列具有NBS-LRR类抗病基因典型的NB-ARC结构域和LRR结构域。(2)云杉PbNBS-LRR与北美云杉(Picea sitchensis)NBS-LRR类抗病蛋白相似性最高,达到98%;分子进化分析进一步表明,PbNBS-LRR与北美云杉NBS-LRR亲缘关系最近,其次为糖松(Pinus lambertiana)和火炬松(Pinus taeda)。(3)qRT-PCR分析表明,NBS-LRR基因在川西云杉、粗枝云杉(Picea asperata)和丽江云杉(Picea likiangensis)的根、树干韧皮部、嫩枝及针叶中均有表达,在川西云杉和粗枝云杉的根部以及丽江云杉的树干韧皮部中表达量最高;在落针病病原菌侵染川西云杉和粗枝云杉的初期(5月)以及丽江云杉的后期(9月),NBS-LRR基因的表达量最高,分别为对照的1.73倍、2.11倍和90.49倍,表明NBS-LRR基因参与了云杉落针病的防御反应。     

Characterization of disease resistance gene-like sequences in near-isogenic lines of tomato

 We examined near-isogenic lines (NILs) carrying either of the tomato mosaic virus (ToMV) resistance genes Tm-1 and Tm-2 for sequences homologous to the isolated disease-resistance genes. DNA fragments were amplified from the genomic DNA of the NILs by the polymerase chain reaction (PCR) using primers designed on the basis of sequences of certain domains conserved among some disease-resistance genes. Of ten PCR products cloned, five were identified as having homology to either of the two classes of disease-resistance genes. The first class encoded proteins containing leucine-rich repeats (LRRs) and a nucleotide-binding site (NBS), such as the RPS2 gene in Arabidopsis and the N gene in tobacco. The second class encoded proteins containing a C-terminal membrane anchor but no NBS, such as the Cf 2 and Cf 9 genes in tomato. In Southern hybridization of the genomic DNAs of the NILs carrying either Tm-1 or Tm-2 and their parental NIL carrying neither of these resistance genes, multiple bands could be detected with most of the clones used as probes. This suggests that the genomes of the NILs contain multiple copies of sequences homologous to some of the known disease-resistance genes. No evidence was obtained to show that the Tm-1 and/or Tm-2 loci encode either class of protein, since no polymorphic band patterns between the NILs were detected by Southern hybridization. Received: 15 August 1997 / Accepted: 2 September 1997     

Identification and characterization of NBS-encoding disease resistance genes in Lotus japonicus

Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a range of pathogens and insect pests. Consequently, NBS-encoding genes have been the focus of a number of recent studies in molecular disease resistance breeding programs. However, little is known about NBS-encoding genes in Lotus japonicus. In this study, a full set of disease resistance (R) candidate genes encoding NBS from the complete genome of L. japonicus was identified and characterized using structural diversity, chromosomal locations, conserved protein motifs, gene duplications, and phylogenetic relationships. Distinguished by N-terminal motifs and leucine-rich repeat motifs (LRRs), 92 regular NBS genes of 158 NBS-coding sequences were classified into seven types: CC-NBS-LRR, TIR-NBS-LRR, NBS-LRR, CC-NBS, TIR-NBS, NBS, and NBS-TIR. Phylogenetic reconstruction of NBS-coding sequences revealed many NBS gene lineages, dissimilar from results for Arabidopsis but similar to results from research on rice. Conserved motif structures were also analyzed to clarify their distribution in NBS-encoding gene sequences. Moreover, analysis of the physical locations and duplications of NBS genes showed that gene duplication events of disease resistance genes were lower in L. japonicus than in rice and Arabidopsis, which may contribute to the relatively fewer NBS genes in L. japonicus. Sixty-three NBS-encoding genes with clear conserved domain character were selected to check their gene expression levels by semi-quantitative RT-PCR. The results indicated that 53 of the genes were most likely to be acting as the active genes, and exogenous application of salicylic acid improved expression of most of the R genes.     

A genome‐wide survey reveals abundant rice blast R genes in resistant cultivars

Plant resistance genes (R genes) harbor tremendous allelic diversity, constituting a robust immune system effective against microbial pathogens. Nevertheless, few functional R genes have been identified for even the best‐studied pathosystems. Does this limited repertoire reflect specificity, with most R genes having been defeated by former pests, or do plants harbor a rich diversity of functional R genes, the composite behavior of which is yet to be characterized? Here, we survey 332 NBS‐LRR genes cloned from five resistant Oryza sativa (rice) cultivars for their ability to confer recognition of 12 rice blast isolates when transformed into susceptible cultivars. Our survey reveals that 48.5% of the 132 NBS‐LRR loci tested contain functional rice blast R genes, with most R genes deriving from multi‐copy clades containing especially diversified loci. Each R gene recognized, on average, 2.42 of the 12 isolates screened. The abundant R genes identified in resistant genomes provide extraordinary redundancy in the ability of host genotypes to recognize particular isolates. If the same is true for other pathogens, many extant NBS‐LRR genes retain functionality. Our success at identifying rice blast R genes also validates a highly efficient cloning and screening strategy.     

Motifs specific for the ADR1 NBS–LRR protein family in <Emphasis Type="Italic">Arabidopsis</Emphasis> are conserved among NBS–LRR sequences from both dicotyledonous and monocotyledonous plants

The activated disease resistance (ADR) 1 gene encodes a protein that possesses an N-terminal coiled-coil (CC) motif, nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. ADR1 belongs to a small, atypical Arabidopsis thaliana sub-class containing four CC–NBS–LRR genes. The NBS region of most NBS–LRR proteins possesses numerous conserved motifs. In contrast, the LRR domain, which is subject to positive selection, is highly variable. Surprisingly, sequence analysis revealed that the LRR domain of the ADR1 sub-class was more conserved than the NBS region. Sequence analysis identified two novel conserved motifs, termed TVS and PKAE, specific for this CC–NBS–LRR sub-class. The TVS motif is adjacent to the P-loop, whereas the PKAE motif corresponded to the inter-domain region termed the NBS–LRR linker, which was conserved within the different CC–NBS–LRR classes but varied among classes. These ADR1-specific motifs were employed to identify putative ADR1 homologs in phylogenetically distant and agronomically important plant species. Putative ADR1 homologs were identified in 11 species including rice and in 3 further Poaceae species. The ADR1 sub-class of CC–NBS–LRR proteins is therefore conserved in both monocotyledonous and dicotyledonous plant species.     

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species.

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.     

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily

The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.     

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR_LRRK2) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR_LRRK2 in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR_LRRK2 thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR_LRRK2 multimerization was detected via cross-linking studies. Finally, the LRR_LRRK2 clinical mutations did not influence LRR_LRRK2 secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR_LRRK2 inter- and intramolecular interactions.     

小果野蕉(Musa acuminata)全基因组NBS抗病基因的鉴定与分析

为探讨小果野蕉(Musa acuminata)中NBS基因的功能,基于新近发表的小果野蕉全基因组序列,对NBS基因家族进行鉴定、分类和染色体定位,解析基因的复制特征、系统发育关系及上游启动子调控元件类别,推测这些基因在小果野蕉中可能的功能。结果表明,在小果野蕉全基因组中鉴定出125个NBS基因,包括78个标准和47个非标准NBS基因。多数NBS基因在染色体上以基因簇形式存在,串联复制是NBS基因家族扩张的主要动力。系统发育分析表明标准NBS基因形成两大分支,77个标准NBS基因有EST表达支持。这为群体水平的抗病基因型筛选提供了本底信息,促进栽培香蕉分子抗病育种进程。     

Long-Term Evolution of Nucleotide-Binding Site-Leucine-Rich Repeat Genes: Understanding Gained from and beyond the Legume Family

Proper utilization of plant disease resistance genes requires a good understanding of their short- and long-term evolution. Here we present a comprehensive study of the long-term evolutionary history of nucleotide-binding site (NBS)-leucine-rich repeat (LRR) genes within and beyond the legume family. The small group of NBS-LRR genes with an amino-terminal RESISTANCE TO POWDERY MILDEW8 (RPW8)-like domain (referred to as RNL) was first revealed as a basal clade sister to both coiled-coil-NBS-LRR (CNL) and Toll/Interleukin1 receptor-NBS-LRR (TNL) clades. Using Arabidopsis (Arabidopsis thaliana) as an outgroup, this study explicitly recovered 31 ancestral NBS lineages (two RNL, 21 CNL, and eight TNL) that had existed in the rosid common ancestor and 119 ancestral lineages (nine RNL, 55 CNL, and 55 TNL) that had diverged in the legume common ancestor. It was shown that, during their evolution in the past 54 million years, approximately 94% (112 of 119) of the ancestral legume NBS lineages experienced deletions or significant expansions, while seven original lineages were maintained in a conservative manner. The NBS gene duplication pattern was further examined. The local tandem duplications dominated NBS gene gains in the total number of genes (more than 75%), which was not surprising. However, it was interesting from our study that ectopic duplications had created many novel NBS gene loci in individual legume genomes, which occurred at a significant frequency of 8% to 20% in different legume lineages. Finally, by surveying the legume microRNAs that can potentially regulate NBS genes, we found that the microRNA-NBS gene interaction also exhibited a gain-and-loss pattern during the legume evolution.To combat the constant challenges by pathogens, plants have evolved a sophisticated two-layered defense system, in which proteins encoded by disease RESISTANCE (R) genes serve to sense pathogen invasion signals and to elicit defense responses (Jones and Dangl, 2006; McDowell and Simon, 2006; Bent and Mackey, 2007). Over 140 R genes have been characterized from different flowering plants, which confer resistance to a large array of pathogens, including bacteria, fungi, oomycetes, viruses, and nematodes (Liu et al., 2007; Yang et al., 2013). Among these, about 80% belong to the NBS-LRR class, which encodes a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. Based on whether their N termini are homologous to the Toll/Interleukin1 receptor (TIR), the angiosperm NBS-LRR genes are further divided into the TIR-NBS-LRR (TNL) subclass and the non-TIR-NBS-LRR (nTNL) subclass (Meyers et al., 1999; Bai et al., 2002; Cannon et al., 2002). The latter has been also called CC-NBS-LRR (CNL) subclass, since a coiled-coil (CC) structure is often detected at the N terminus (Meyers et al., 2003). Interestingly, a small group of nTNL genes have an N-terminal RPW8-like domain with a transmembrane region before the CC structure (Xiao et al., 2001). This group of RPW8-NBS-LRR (RNL) genes has been usually viewed as a specific lineage of CNLs (Bonardi et al., 2011; Collier et al., 2011); however, its real phylogenetic relationship with CNLs and TNLs requires further investigation.NBS-LRR genes have been surveyed in many sequenced genomes of flowering plants, including four monocots: rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), and Brachypodium distachyon; one basal eudicot: Nelumbo nucifera; two asterid species: potato (Solanum tuberosum) and tomato (Solanum lycopersicum); and 14 rosids: Vitis vinifera, Populus trichocarpa, Ricinus communis, Medicago truncatula, soybean (Glycine max), Lotus japonicus, Cucumis sativus, Cucumis melo, Citrullus lanatus, Gossypium raimondii, Carica papaya, Arabidopsis (Arabidopsis thaliana), Arabidopsis lyrata, and Brassica rapa (Bai et al., 2002; Meyers et al., 2003; Monosi et al., 2004; Zhou et al., 2004; Yang et al., 2006, 2008b; Ameline-Torregrosa et al., 2008; Mun et al., 2009; Porter et al., 2009; Chen et al., 2010; Li et al., 2010a, 2010b; Guo et al., 2011; Zhang et al., 2011; Jupe et al., 2012; Lozano et al., 2012; Luo et al., 2012; Tan and Wu, 2012; Andolfo et al., 2013; Jia et al., 2013; Lin et al., 2013; Wan et al., 2013; Wei et al., 2013; Wu et al., 2014). Variable numbers (from dozens to hundreds) of NBS-LRR genes were reported from these genomes, making one wonder: how did these genes evolve so variably during flowering plant speciation?Comparative genomic studies conducted in the available genome sequences of closely related species or subspecies revealed that a significant proportion of NBS-LRR genes are not shared. For example, 70 NBS-LRR genes between Arabidopsis and A. lyrata show the presence/absence of polymorphisms (Chen et al., 2010; Guo et al., 2011). Moreover, synteny analysis revealed that, among 363 NBS-LRR gene loci in indica (cv 93-11) and japonica (cv Nipponbare) rice, 124 loci exist in only one genome (Luo et al., 2012). Unequal crossover, homologous repair, and nonhomologous repair are the three ways that NBS-LRR gene deletions are caused in rice genomes (Luo et al., 2012).In many surveyed genomes, the majority of NBS-LRR genes are found in a clustered organization (physically close to each other), with the rest exhibited as singletons. Many clusters are homogenous, with only duplicated members occupying the same phylogenetic lineage, whereas heterogenous clusters comprise members from distantly related clades (Meyers et al., 2003). Leister (2004) defined three types of NBS gene duplications: local tandem, ectopic, and segmental duplications. Although a general agreement on the widespread occurrence of local tandem duplications can be reached by various genome survey studies, the relative importance of ectopic and segmental duplications has been seldom investigated since the earliest surveys of the Arabidopsis genome (Richly et al., 2002; Baumgarten et al., 2003; Meyers et al., 2003; McDowell and Simon, 2006).With more genomic data available in certain angiosperm families, NBS-LRR genes should be further investigated among phylogenetically distant species to fill the gaps in the understanding of their long-term evolutionary patterns. The legume family contains many economically important crop species, such as clover (Trifolium spp.), soybean, peanut (Arachis hypogaea), and common bean (Phaseolus vulgaris). Although these legumes are constantly threatened by various pathogens, only a few functional legume R genes have been characterized, and all of them belong to the NBS-LRR class (Ashfield et al., 2004; Hayes et al., 2004; Gao et al., 2005; Seo et al., 2006; Yang et al., 2008a; Meyer et al., 2009). Therefore, it would be interesting to investigate the NBS-LRR gene repertoire among different legume species. Here, we carried out a comprehensive analysis of NBS-LRR genes in four divergent legume genomes, M. truncatula, pigeon pea (Cajanus cajan), common bean, and soybean, which shared a common ancestor approximately 54 million years ago (MYA; Fig. 1; Lavin et al., 2005). Approximately 1,000 nTNL and 667 TNL subclass NBS-encoding genes were identified in our study. Their genomic distribution, organization modes, phylogenetic relationships, and syntenic patterns were examined to obtain insight into the long-term evolutionary patterns of NBS-LRR genes.Open in a separate windowFigure 1.The phylogenetic tree of four investigated legume species (M. truncatula, pigeon pea, common bean, and soybean). Two WGD events are indicated with triangles: one occurred approximately 59 MYA in the common ancestor of the four investigated legumes, and the other occurred approximately 13 MYA in the Glycine spp. lineage alone (Schmutz et al., 2010). The numbers at the branch nodes indicate divergence times (Lavin et al., 2005; Stefanovic et al., 2009). [See online article for color version of this figure.]     

A large scale analysis of resistance gene homologues in<Emphasis Type="Italic"> Arachis</Emphasis>

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.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Communicated by M.-A. Grandbastien     

Genome-wide identification and resistance expression analysis of the <Emphasis Type="Italic">NBS</Emphasis> gene family in <Emphasis Type="Italic">Triticum urartu</Emphasis>

As the largest class of resistant genes, the nucleotide binding site (NBS) has been studied extensively at a genome-wide level in rice, sorghum, maize, barley and hexaploid wheat. However, no such comprehensive analysis has been conducted of the NBS gene family in Triticum urartu, the donor of the A genome to the common wheat. Using a bioinformatics method, 463 NBS genes were isolated from the whole genome of T. urartu, of which 461 had location information. The expansion pattern and evolution of the 461 NBS candidate proteins were analyzed, and 118 of them were duplicated. By calculating the lengths of the copies, it was inferred that the NBS resistance gene family of T. urartu has experienced at least two duplication events. Expression analysis based on RNA-seq data found that 6 genes were differentially expressed among Tu38, Tu138 and Tu158 in response to Blumeria graminis f.sp.tritici (Bgt). Following Bgt infection, the expression levels of these genes were up-regulated. These results provide critical references for further identification and analysis of NBS family genes with important functions.     

Divergent Evolution of Plant NBS-LRR Resistance Gene Homologues in Dicot and Cereal Genomes

The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well. Received: 17 May 1999 / Accepted: 25 September 1999     

植物NBS类抗病基因的进化

NBS(Nucleotide-binding site)类抗病基因是植物中最重要的一类抗病基因, 其进化模式、结构特点和功能调控一直是抗病基因研究领域的热点。这类基因具有保守的结构域, 广泛存在于植物基因组中, 在不同植物基因组中数目差异较大且具有较低的表达量。此外, 同源NBS类抗病基因之间通过频繁的序列交换产生广泛的序列多样性, 且抗病基因位点具有较差的线性。依据基因之间序列交换的频率, 抗病基因可分为TypeⅠ和TypeⅡ两类。文章从抗病基因的结构、数量、分布、序列多样性、进化模式以及表达调控等方面进行了综述, 旨在为后续NBS类抗病基因的相关研究提供参考。     

Molecular evolution of a family of resistance gene analogs of nucleotide-binding site sequences in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Nucleotide-binding site-leucine-rich repeats (NBS-LRR) gene families are one of the major plant resistance genes. Genomic NBS evolution was studied in many plant species for diverse arrays of NBS gene families. In this study, we focused on one family of NBS sequences in an attempt to understand how closely related NBS sequences evolved in the light of selection in domesticated plant species. A phylogenetic analysis revealed five major clades (A–E) and five subclades (A1–A5) within clade A of cloned NBS sequences. Positive selection was only detected in newly evolved NBS lineages in subclades of clade A. Positively selected codon sites were found among NBS sequences of clade A. A sliding-window analysis revealed that regions with Ka/Ks ratios of >1 were in the inter-motifs when paired clades were compared, but regions with Ka/Ks ratios of >1 were found across NBS sequences when subclades of clade A were compared. Our results based on a family of closely related NBS sequences showed that positive selection was first exerted on specific lineages across all NBS sequences after selective constraints. Subsequently, sequences with mutations in commonly conserved motifs were scrutinized by purifying selection. In the long term, conserved high frequency alleles in commonly conserved motifs and changes in inter-motifs were maintained in the investigated family of NBS sequences. Moreover, codons identified to be under positive selection in the inter-motifs were mainly located in regions involved in functions of ATP binding or hydrolysis.     

Allele-mining of rice blast resistance genes at AC134922 locus

The AC134922 locus is one of the most rapidly evolving nucleotide binding site-leucine-rich repeat (NBS-LRR) gene family in rice genome. Six rice blast resistance (R) genes have been cloned from this locus and other two resistance candidate genes, Pi34 and Pi47, are also mapped to this complex locus. Therefore, it seems that more functional R genes could be identified from this locus. In this study, we cloned 22 genes from 12 cultivars based on allele-mining strategy at this locus and identified 6 rice blast R genes with 4 of them recognizing more than one isolates. Our result suggests that gene stacking might be the evolutionary strategy for complex gene locus to interact with rapidly evolving pathogens, which might provide a potential way for the cloning of durable resistance genes. Moreover, the mosaic structure and ambiguous ortholog/paralog relationships of these homologous genes, caused by frequent recombination and gene conversion, indicate that multiple alleles of this complex locus may serve as a reservoir for the evolutionary novelty of these R genes.     

Genome-Wide Comparative Analysis Reveals Similar Types of NBS Genes in Hybrid Citrus sinensis Genome and Original Citrus clementine Genome and Provides New Insights into Non-TIR NBS Genes

In this study, we identified and compared nucleotide-binding site (NBS) domain-containing genes from three Citrus genomes (C. clementina, C. sinensis from USA and C. sinensis from China). Phylogenetic analysis of all Citrus NBS genes across these three genomes revealed that there are three approximately evenly numbered groups: one group contains the Toll-Interleukin receptor (TIR) domain and two different Non-TIR groups in which most of proteins contain the Coiled Coil (CC) domain. Motif analysis confirmed that the two groups of CC-containing NBS genes are from different evolutionary origins. We partitioned NBS genes into clades using NBS domain sequence distances and found most clades include NBS genes from all three Citrus genomes. This suggests that three Citrus genomes have similar numbers and types of NBS genes. We also mapped the re-sequenced reads of three pomelo and three mandarin genomes onto the C. sinensis genome. We found that most NBS genes of the hybrid C. sinensis genome have corresponding homologous genes in both pomelo and mandarin genomes. The homologous NBS genes in pomelo and mandarin suggest that the parental species of C. sinensis may contain similar types of NBS genes. This explains why the hybrid C. sinensis and original C. clementina have similar types of NBS genes in this study. Furthermore, we found that sequence variation amongst Citrus NBS genes were shaped by multiple independent and shared accelerated mutation accumulation events among different groups of NBS genes and in different Citrus genomes. Our comparative analyses yield valuable insight into the structure, organization and evolution of NBS genes in Citrus genomes. Furthermore, our comprehensive analysis showed that the non-TIR NBS genes can be divided into two groups that come from different evolutionary origins. This provides new insights into non-TIR genes, which have not received much attention.     

Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava ( Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.Communicated by M.-A. Grandbastien