Characterization of resistance gene analogs with a nucleotide binding site isolated from a triploid white poplar.

The majority of cloned plant disease resistance genes (R genes) encode a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) domain. In this study, to better understand the R genes in white poplar, 59 resistance gene analogues (RGAs) were identified from a triploid white poplar [(Populus tomentosa x Populus bolleana) x P. tomentosa], based on conserved NBS regions. The 59 RGAs were phylogenetically classified into 10 subfamilies, and 54 RGAs with open-reading frames (ORFs) were further grouped into two classes, toll and interleukin-1 receptor (TIR) and non-TIR. BLAST searches with reference to the genomic sequence of Populus trichocarpa found 96 highly homologous regions distributed in 37 loci, suggesting the abundance and divergence of NBS-encoding genes in the triploid poplar genome. Within subfamilies 1-3, the average non-synonymous/synonymous substitution (omega) rates were < 1, indicating purifying selection on these RGAs, but some sites were clearly under diversifying selection with omega > 1. Many intergenic exchanges were also detected among these RGAs, indicating a probable role in homogenising NBS domains. Quantitative real-time PCR analysis revealed dramatic variations in the transcript level of 18 RGAs in the mature leaves, bark and roots of the triploid poplar, and identified two RGAs that had significantly higher level of transcripts in bark, four RGAs in mature leaves, and 14 in the above-ground portion of poplars, suggesting their probable roles in resistance against diseases attacking the organs. Our results shed light on genetic resources of poplar resistance and will be useful for further resistance gene isolation and exploitation.

Zhang Q ，Zhang ZY ，Lin SZ ，Zheng HQ ，Lin YZ ，An XM ，Li Y ，Li HX ... -  《PLANT BIOLOGY》

Over-expression of the triploid white poplar PtDrl01 gene in tobacco enhances resistance to tobacco mosaic virus.

A full-length cDNA, designated as the Populus tomentosa disease resistance-like 01 (PtDrl01) gene, was isolated from triploid white poplar [(Populus tomentosa × P. bolleana) × P. tomentosa]. The protein thought to be produced by the PtDrl01 gene contains a nuclear localisation sequence (NLS), a toll/interleukin-1 receptor (TIR) homologue region, a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) domain. The protein also exhibits a considerable degree of homology to N-like resistance proteins. Real-time quantitative RT-PCR analysis revealed that expression of the PtDrl01 gene in triploid white poplar leaves could be induced by two defence signalling molecules: methyl jasmonate (MeJA) and salicylic acid (SA). Over-expression of the PtDrl01 gene in transgenic tobacco induced enhanced resistance to tobacco mosaic virus (TMV). Long-term resistance from the PtDrl01 gene to TMV infection was also observed in transgenic tobacco plants. Additionally, over-expression of the PtDrl01 gene resulted in transcriptional changes in genes expressing pathogenesis-related proteins in transgenic tobacco under non-stress conditions. These data strongly suggest that the PtDrl01 gene is involved in plant defence responses to pathogen infection.

Zheng HQ ，Zhang Q ，Li HX ，Lin SZ ，An XM ，Zhang ZY ... -  《-》

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

Glynn NC ，Comstock JC ，Sood SG ，Dang PM ，Chaparro JX ... -  《PEST MANAGEMENT SCIENCE》

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 《-》

Comparative analysis of NBS domain sequences of NBS-LRR disease resistance genes from sunflower, lettuce, and chicory.

Plant resistance to many types of pathogens and pests can be achieved by the presence of disease resistance (R) genes. The nucleotide binding site-leucine rich repeat (NBS-LRR) class of R-genes is the most commonly isolated class of R-genes and makes up a super-family, which is often arranged in the genome as large multi-gene clusters. The NBS domain of these genes can be targeted by polymerase chain reaction (PCR) amplification using degenerate primers. Previous studies have used PCR derived NBS sequences to investigate both ancient R-gene evolution and recent evolution within specific plant families. However, comparative studies with the Asteraceae family have largely been ignored. In this study, we address recent evolution of NBS sequences within the Asteraceae and extend the comparison to the Arabidopsis thaliana genome. Using multiple sets of primers, NBS fragments were amplified from genomic DNA of three species from the family Asteraceae: Helianthus annuus (sunflower), Lactuca sativa (lettuce), and Cichorium intybus (chicory). Analysis suggests that Asteraceae species share distinct families of R-genes, composed of genes related to both coiled-coil (CC) and toll-interleukin-receptor homology (TIR) domain containing NBS-LRR R-genes. Between the most closely related species, (lettuce and chicory) a striking similarity of CC subfamily composition was identified, while sunflower showed less similarity in structure. These sequences were also compared to the A. thaliana genome. Asteraceae NBS gene subfamilies appear to be distinct from Arabidopsis gene clades. These data suggest that NBS families in the Asteraceae family are ancient, but also that gene duplication and gene loss events occur and change the composition of these gene subfamilies over time.

Plocik A ，Layden J ，Kesseli R 《MOLECULAR PHYLOGENETICS AND EVOLUTION》