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》

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》

Identification and characterization of novel NBS-LRR resistance gene analogues from the pea.

Pea (Pisum sativum) is one of the most cultivated le-gumes in the world, and its yield and seed quality are affected by a variety of pathogens. In plants, NBS-LRR (nucleotide binding site-leucine-rich repeat) is the main class of disease resistance genes. Using degenerate primers deduced from conserved motifs in the NBS domain of known resistance genes, we identified 10 NBS sequences in three varieties of P. sativum. The deduced amino acid sequences of the iden-tified resistance gene analogues (RGAs) exhibited the typical motifs of the NBS domain (P-loop, kinase-2, kinase-3a, and the hydrophobic domain, GLPL) present in the majority of plant proteins belonging to the NBS-LRR class. Phylogenetic analysis showed that seven RGAs belonged to the non-TIR-NBS-LRR subclass and three to the TIR-NBS-LRR subclass. The results of this study provide insights into the structure of this class of resistance genes in the pea, and their evolution-ary relationships with those of other plant species.

Djebbi S ，Bouktila D ，Makni H ，Makni M ，Mezghani-Khemakhem M ... -  《Genetics and Molecular Research》

Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: isolation, RFLP marker development, and physical mapping.

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence. A computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar Musa acuminata subsp. burmannicoides, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs) were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs), together with RGAs from Arabidopsis thaliana and Oryza sativa, grouped most Musa RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid M. acuminata mapping population. Eighty eight BAC clones were identified in M. acuminata Calcutta 4, M. acuminata Grande Naine, and M. balbisiana Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities. This is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4. Contig sequences were deposited in GenBank and assigned numbers ER935972 - ER936023. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.

Miller RN ，Bertioli DJ ，Baurens FC ，Santos CM ，Alves PC ，Martins NF ，Togawa RC ，Souza MT Jr ，Pappas GJ Jr ... -  《BMC PLANT BIOLOGY》

The Arabidopsis RPS4 bacterial-resistance gene is a member of the TIR-NBS-LRR family of disease-resistance genes.

Plant-disease resistance (R) genes mediate the specific recognition of invading pathogens carrying cognate avirulence (avr) determinants. RPS4 is a disease-resistance locus on chromosome 5 of Arabidopsis thaliana specifying resistance to strains of Pseudomonas syringae pv. tomato expressing avrRps4. We have isolated the RPS4 gene using a map-based cloning approach. RPS4 encodes a predicted protein of 1217 amino acids that contains an N-terminus with homology to the intracellular domains of the Drosophila Toll protein and the mammalian interleukin-1 receptor (TIR domain), a tripartite nucleotide-binding site (NBS), and leucine-rich repeats (LRR). Incomplete splicing of the RPS4 mRNA was observed, which may give rise to truncated protein products consisting mainly of the TIR and NBS domains. These features classify RPS4 as a member of the TIR-NBS-LRR R gene family founded by N, L6 and RPP5, which determine resistance to viral, fungal and oomycete pathogens, respectively. Previous work has shown that RPS4, like other Arabidopsis TIR-NBS-LRR R genes specifying resistance to oomycetes, is dependent on a functional EDS1 allele for disease-resistance signaling. The characterization of RPS4 presented here thus establishes a role for TIR-NBS-LRR R genes in resistance to bacterial pathogens, and provides evidence for the model that dependence of R genes on EDS1 is determined by R protein structure, and not by pathogen type. The cloning of RPS4 and the previous isolation of avrRps4 provide the molecular tools for a genetic and molecular dissection of the TIR-NBS-LRR R gene signaling pathway in Arabidopsis.

Gassmann W ，Hinsch ME ，Staskawicz BJ 《PLANT JOURNAL》