Diversity and evolutionary relationship of nucleotide binding site-encoding disease-resistance gene analogues in sweet potato (Ipomoea batatas Lam.).

Most plant disease-resistance genes (R-genes) isolated so far encode proteins with a nucleotide binding site (NBS) domain and belong to a superfamily. NBS domains related to R-genes show a highly conserved backbone of an amino acid motif, which makes it possible to isolate resistance gene analogues (RGAs) by degenerate primers. Degenerate primers based on the conserved motif (P-loop and GLPL) of the NBS domain from R -genes were used to isolate RGAs from the genomic DNA of sweet potato cultivar Qingnong no.2. Five distinct clusters of RGAs (22 sequences) with the characteristic NBS representing a highly diverse sample were identified in sweet potato genomic DNA. Sequence identity among the 22 RGA nucleotide sequences ranged from 41.2% to 99.4%, while the deduced amino acid sequence identity from the 22 RGAs ranged from 20.6%to 100%. The analysis of sweet potato RGA sequences suggested mutation as the primary source of diversity. The phylogenetic analyses for RGA nucleotide sequences and deduced amino acids showed that RGAs from sweet potato were classified into two distinct groups--toll and interleukin receptor-1 (TIR)-NBS-LRR and non-TIR-NBS-LRR. The high degree of similarity between sweet potato RGAs and NBS sequences derived from R-genes cloned from tomato, tobacco, flax and potato suggest an ancestral relationship. Further studies showed that the ratio of non-synonymous to synonymous substitution within families was low. These data obtained from sweet potato suggest that the evolution of NBS-encoding sequences in sweet potato occur by the gradual accumulation of mutations leading to purifying selection and slow rates of divergence within distinct R-gene families.

Chen G ，Pan D ，Zhou Y ，Lin S ，Ke X ... -  《JOURNAL OF BIOSCIENCES》

Origin, diversity and evolution of NBS-type disease-resistance gene homologues in coffee trees (Coffea L.).

Noir S ，Combes MC ，Anthony F ，Lashermes P ... -  《-》

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》

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 《-》