Isolation of TIR and non-TIR NBS--LRR resistance gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt).

Toll and interleukin-1 receptor (TIR) and non-TIR nucleotide binding site-leucine rich repeat (NBS-LRR) resistance gene analogues (RGAs) were obtained from chestnut rose (Rosa roxburghii Tratt) by two PCR-based amplification strategies (direct amplification and overlap extension amplification) with degenerate primers designed to the conserved P-loop, kinase-2, and Gly-Leu-Pro-Leu (GLPL) motifs within the NBS domain of plant resistance gene (R gene) products. Thirty-four of 65 cloned PCR fragments contained a continuous open reading frame (ORF) and their predicted protein products showed homology to the NBS-LRR class R proteins in the GenBank database. These 34 predicted protein sequences exhibited a wide range (19.5--99.4%) of sequence identity among them and were classified into two distinct groups by phylogenetic analysis. The first group consisted of 23 sequences and seemed to belong to the non-TIR NBS-LRR RGAs, since they contained group specific motifs (RNBS-A-non-TIR motif) that are often present in the coiled-coil domain of the non-TIR NBS-LRR class R genes. The second group comprised 11 sequences that contained motifs found in the TIR domain of TIR NBS-LRR class R genes. Restriction fragment length polymorphic (RFLP) markers were developed from some of the RGAs and used for mapping powdery mildew resistance genes in chestnut rose. Three markers, RGA 22 C, RGA 4 A, and RGA 7 B, were identified to be linked to a resistance gene locus, designated CRPM 1 for chestnut rose powdery mildew resistance 1, which accounted for 72% of the variation in powdery mildew resistance phenotype in an F1 segregating population. To our knowledge, this is the first report on isolation, phylogenetic analysis and potential utilization as genetic markers of RGAs in chestnut rose.

Xu Q ，Wen X ，Deng X 《THEORETICAL AND APPLIED GENETICS》

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.

Liu JJ ，Ekramoddoullah AK 《-》

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.

Yaish MW ，Sáenz de Miera LE ，Pérez de la Vega M 《GENOME》

Identification and characterization of NBS-LRR class resistance gene analogs in faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.).

A PCR approach with degenerate primers designed from conserved NBS-LRR (nucleotide binding site-leucine-rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from 5 faba bean (Vicia faba) lines and 2 chickpea (Cicer arietinum) accessions. Sixty-nine sequenced clones showed homologies to various R genes deposited in the GenBank database. The presence of internal kinase-2 and kinase-3a motifs in all the sequences isolated confirm that these clones correspond to NBS-containing genes. Using an amino-acid sequence identity of 70% as a threshold value, the clones were grouped into 10 classes of resistance-gene analogs (RGA01 to RGA10). The number of clones per class varied from 1 to 30. RGA classes 1, 6, 8, and 9 were comprised solely of clones isolated from faba bean, whereas classes 2, 3, 4, 5, and 7 included only chickpea clones. RGA10, showing a within-class identity of 99%, was the only class consisting of both faba bean and chickpea clones. A phylogenetic tree, based on the deduced amino-acid sequences of 12 representative clones from the 10 RGA classes and the NBS domains of 6 known R genes (I2 and Prf from tomato, RPP13 from Arabidopsis, Gro1-4 from potato, N from tobacco, L6 from flax), clearly indicated the separation between TIR (Toll/interleukin-1 receptor homology: Gro1-4, L6, N, RGA05 to RGA10)- and non-TIR (I2, Prf, RPP13, RGA01 to RGA04)-type NBS-LRR sequences. The development of suitable polymorphic markers based on cloned RGA sequences to be used in genetic mapping will facilitate the assessment of their potential linkage relationships with disease-resistance genes in faba bean and chickpea. This work is the first to report on faba bean RGAs.

Palomino C ，Satovic Z ，Cubero JI ，Torres AM ... -  《GENOME》

Analysis of TIR- and non-TIR-NBS-LRR disease resistance gene analogous in pepper: characterization, genetic variation, functional divergence and expression patterns.

Pepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide. However, its yield and fruit quality can be severely threatened by several pathogens. The plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family is the largest class of known disease resistance genes (R genes) effective against such pathogens. Therefore, the isolation and identification of such R gene homologues from pepper will provide a critical foundation for improving disease resistance breeding programs. A total of 78 R gene analogues (CaRGAs) were identified in pepper by degenerate PCR amplification and database mining. Phylogenetic tree analysis of the deduced amino acid sequences for 51 of these CaRGAs with typically conserved motifs ( P-loop, kinase-2 and GLPL) along with some known R genes from Arabidopsis and tomato grouped these CaRGAs into the non-Toll interleukin-1 receptor (TIR)-NBS-LRR (CaRGAs I to IV) and TIR-NBS-LRR (CaRGAs V to VII) subfamilies. The presence of consensus motifs (i.e. P-loop, kinase-2 and hydrophobic domain) is typical of the non-TIR- and TIR-NBS-LRR gene subfamilies. This finding further supports the view that both subfamilies are widely distributed in dicot species. Functional divergence analysis provided strong statistical evidence of altered selective constraints during protein evolution between the two subfamilies. Thirteen critical amino acid sites involved in this divergence were also identified using DIVERGE version 2 software. Analyses of non-synonymous and synonymous substitutions per site showed that purifying selection can play a critical role in the evolutionary processes of non-TIR- and TIR-NBS-LRR RGAs in pepper. In addition, four specificity-determining positions were predicted to be responsible for functional specificity. qRT-PCR analysis showed that both salicylic and abscisic acids induce the expression of CaRGA genes, suggesting that they may primarily be involved in defence responses by activating signaling pathways. The identified CaRGAs are a valuable resource for discovering R genes and developing RGA molecular markers for genetic map construction. They will also be useful for improving disease resistance in pepper. The findings of this study provide a better understanding of the evolutionary mechanisms that drive the functional diversification of non-TIR- and TIR-NBS-LRR R genes in pepper.

Wan H ，Yuan W ，Ye Q ，Wang R ，Ruan M ，Li Z ，Zhou G ，Yao Z ，Zhao J ，Liu S ，Yang Y ... -  《BMC GENOMICS》