云南悬钩子蔷薇NBS LRR类抗病基因同源克隆与分析

为研究云南野生蔷薇属中的NBS类抗病基因,根据已知抗病基因NBS LRR序列中的保守区域设计简并引物,利用RT PCR技术从云南悬钩子蔷薇中进行体外扩增,获得了对应区域的cDNA片段,回收、克隆这些特异片段,测序分析,共得到4个含有NBS LRR保守结构域的抗病基因同源序列(RGAs),分别命名为AC9、AC39、AC50和AC68。它们与已报道的11个NBS类抗病基因相应区段的氨基酸序列相似性为5.4%~79.2%,其中这4个RGAs片段与Mi、RPS2、Pib和RPM1基因聚为一类。表明这4条RGAs序列可进一步用作悬钩子蔷薇抗病候选基因的分子筛选及遗传图谱的构建。     

易变山羊草抗禾谷孢囊线虫基因中核苷酸结合位点区(NBS)的克隆及序列分析

 大部分已克隆的植物抗病基因都包含有核苷酸结合位点区 (NBS)和富含亮氨酸的重复序列区 (LRR) .利用来自节节麦的抗禾谷孢囊线虫基因Cre3位点NBS区保守序列设计特异引物 ,从含有来自易变山羊草的抗禾谷孢囊线虫基因的小麦品系E 10的基因组中PCR扩增得到一条约 5 30bp的单一条带 .将扩增条带克隆和序列分析发现 ,该克隆 (Rccn4 )的编码区长 5 2 8bp ,含一个不完整的开放读码框 ,没有起始密码子、终止密码子和内含子结构 ,它编码一个 176个氨基酸残基的蛋白质 ,分子量为 2 0 4kD .Rccn4含有NBS LRR类抗病基因NBS区共有的保守模体I(V)LDD、T(T S)R、G(L S)PLA(A I L)、RCF(A L)Y ,并且与Cre3基因的NBS编码区核苷酸和氨基酸同源性分别为99 4 %和 98% .它是一个新的含NBS编码区核苷酸的抗禾谷孢囊线虫基因     

云南悬钩子蔷薇NBS-LRR类抗病基因同源克隆与分析

为研究云南野生蔷薇属中的NBS类抗病基因,根据已知抗病基因NBSLRR序列中的保守区域设计简并引物,利用RTPCR技术从云南悬钩子蔷薇中进行体外扩增,获得了对应区域的cDNA片段,回收、克隆这些特异片段,测序分析,共得到4个含有NBSLRR保守结构域的抗病基因同源序列（RGAs）,分别命名为AC9、AC39、AC50和AC68。它们与已报道的11个NBS类抗病基因相应区段的氨基酸序列相似性为5.4%~79.2%,其中这4个RGAs片段与Mi、RPS2、Pib和RPM1基因聚为一类。表明这4条RGAs序列可进一步用作悬钩子蔷薇抗病候选基因的分子筛选及遗传图谱的构建。     

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

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

Expression of two soybean resistance gene candidates shows divergence of paralogous single-copy genes

The cloning of several plant genes directly involved in triggering a disease resistance response has shown that numerous resistance genes in the nucleotide binding site (NBS)/leucine-rich repeat (LRR) class have similar conserved amino acid sequences. In this study, we used a short soybean DNA sequence, previously cloned based on its conserved NBS, as a probe to identify full-length resistance gene candidates. Two homologous, but genetically independent genes were identified. One gene maps to the soybean molecular linkage group (MLG) F and a second is coded on MLG E. The first gene contains a 3,279 nucleotide open reading frame (ORF) sequence and possesses all the functional motifs characteristic of previously cloned NBS/LRR resistance genes. The N-terminal sequence of the deduced gene product is highly characteristic of other resistance genes in the subgroup of NBS/LRR genes which show homology to the Toll/Interleukin-1 receptor genes. The C-terminal region is somewhat more divergent as seen in other cloned disease resistance genes. This region of the F-linked gene contains an LRR region that is characterized by two alternatively spliced products which produce gene products with either a four-repeat or a ten-repeat LRR. The second cloned gene that maps to soybean MLG E contains 1,565 nucleotides of ORF in the N-terminal domain. Despite strong homology, however, the 3′ region of this gene contains several in-frame stop codons and apparent frame shifts compared to the F-linked gene, suggesting that its functionality as a disease resistance gene is questionable. These two disease resistance gene candidates are shown to be closely related to one another and to the members of the NBS/LRR class of disease resistance genes. Received: 29 November 1999 / Accepted: 22 December 1999     

Molecular Marker-Assisted Genotyping of Mungbean Yellow Mosaic India Virus Resistant Germplasms of Mungbean and Urdbean

Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus begomovirus causes the yellow mosaic disease in a number of economically important edible grain legumes including mungbean (Vigna radiata), urdbean (Vigna mungo) and soybean (Glycine max). The disease is severe, critical, open spread and inflicts heavy yield losses annually. The objective of this study is to develop molecular markers linked to MYMIV-resistance to facilitate genotyping of urdbean and mungbean germplasms for MYMIV-reaction. Resistance-linked molecular markers were successfully developed from consensus motifs of other resistance (R) gene or R gene homologue sequences. Applying linked marker-assisted genotyping, plant breeders can carry out repeated genotyping throughout the growing season in absence of any disease incidence. Two MYMIV-resistance marker loci, YR4 and CYR1, were identified and of these two CYR1 is completely linked with MYMIV-resistant germplasms and co-segregating with MYMIV-resistant F₂, F₃ progenies of urdbean. The present study demonstrated that these two markers could be efficiently employed together in a multiplex-PCR-reaction for genotyping both V. mungo and V. radiata germplasms from field grown plants and also directly from the seed stock. This method of genotyping would save time and labour during the introgression of MYMIV-resistance through molecular breeding, as methods of phenotyping against begomoviruses are tedious, labour and time intensive.     

The island cotton NBS‐LRR gene GbaNA1 confers resistance to the non‐race 1 Verticillium dahliae isolate Vd991

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non‐commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS‐LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus‐induced gene silencing of each of the eight NBS‐LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non‐functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full‐length (～1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non‐race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.     

Potato gene Y-1 is an N gene homolog that confers cell death upon infection with potato virus Y

ADG2 is a DNA sequence mapped to a resistance (R) gene-rich region at the distal end of chromosome XI in potato (Solanum tuberosum subsp. andigena). The gene, in which ADG2 represents the predicted nucleotide-binding domain (NBS), was cloned and characterized. The coding region of the gene (designated as Y-1) is 6,187 bp long and structurally similar to gene N that confers hypersensitive resistance to Tobacco mosaic virus in Nicotiana spp. Both belong to the TIR-NBS-LRR class of genes and show 57% identity at the amino acid sequence level. The introns of Y-1 were spliced as predicted from the sequence. Y-1 cosegregated with Ry(adg), a gene for extreme resistance to Potato virus Y (PVY) on chromosome XI, as tested in a potato-mapping population and with independent potato cultivars. Leaves of the transgenic potato plants expressing Y-1 under the control of Cauliflower mosaic virus 35S promoter developed necrotic lesions upon infection with PVY, but no significant resistance was observed, and plants were systemically infected with PVY.     

Proteomic analysis of salicylic acid induced resistance to Mungbean Yellow Mosaic India Virus in Vigna mungo

The role of salicylic acid (SA) in inducing resistance to MYMIV infection in Vigna mungo has been elucidated by proteomics. Twenty-nine proteins identified by MALDI-TOF/TOF, predicted to be involved in stress responses, metabolism, photosynthesis, transport and signal transduction, showed increased abundance upon SA treatment. Susceptible plants showed characteristic yellow mosaic symptoms upon MYMIV infection. A concentration dependent decrease in physiological symptoms associated with MYMIV was observed upon exogenous SA treatment prior to viral inoculation; and no visible symptom was observed at 100 μM SA. SA treatment stimulated SOD and GPX activity and inhibited CAT activity thus preventing ROS mediated damage. Significant increase in chlorophyll, protein, carbohydrate, phenolic content and H(2)O(2) were observed. Involvement of calmodulin for transmission of defense signal by SA is suggested. A metabolic reprogramming leading to enhanced synthesis of proteins involved in primary and secondary metabolisms is necessary for SA mediated resistance to MYMIV. Identification of proteins showing increased abundance, involved in photosynthetic process is a significant finding which restores virus-induced degradation of the photosynthetic apparatus and provides enhanced metabolites required for repartition of resources towards defense.     

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,genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine (<Emphasis Type="Italic">Pinus monticola </Emphasis>Dougl. ex. D. Don.)

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