Plant disease resistance genes (R genes) are important components of the genetic resistance mechanism in plants [1, 2]. Over the past decade, several R genes conferring resistance to a wide spectrum of plant pathogens, including bacteria, fungi, oomycetes, viruses and nematodes, have been cloned from different plant species [2–5]. Sequence analyses revealed that these proteins share a high degree of homology and have a number of conserved motifs. These include a nucleotide-binding site (NBS), a leucine-rich repeat (LRR) region, a motif homologous to the cytoplasmic domains of the Drosophila Toll protein and the mammalian interleukin-1 receptor (TIR), a coiled-coil (CC) or leucine zipper structure, a transmembrane domain (TM) and a protein kinase domain . Although a wide range of pathogens are involved, these R genes encode a limited set of proteins that can be classified into several superfamilies, including NBS-LRR, a receptor-like kinase, LRR-TM and TM-CC [2, 7].
The NBS-LRR class of R genes can be divided into two subfamilies (TIR-NBS-LRR and non-TIR-NBS-LRR) based on the features of their N-terminal structure [2, 8]. These two subfamilies can also be distinguished (95% accuracy) by the last residue, D (Aspartate) or W (Tryptophan), of the conserved kinase-2 motif within the NBS domain . The former corresponds to the TIR-NBS-LRR subfamily, whereas the latter corresponds to the non-TIR-NBS-LRR subfamily of R genes. The ‘NBS’ and ‘LRR’ domains in the NBS-LRR R genes have different roles during host–pathogen recognition. The highly conserved NBS domains can bind and hydrolyze ATP or GTP , whereas the LRR motif is typically involved in protein–protein interactions and is responsible for recognition specificity [2, 11, 12].
To date, eight conserved motifs have been identified in the NBS domain of plant non-TIR- and TIR-NBS-LRR R genes, including P-loop, kinase-2, kinase-3a, GLPL, RNBS-A-TIR, RNBS-D-TIR, RNBS-A-non-TIR and RNBS-D-non-TIR . The first four conserved motifs are common in the TIR and non-TIR-NBS-LRR subfamilies. The RNBS-A-TIR and RNBS-D-TIR motifs are specific to the TIR-NBS-LRR subfamily. The remaining two motifs, RNBS-A-non-TIR and RNBS-D-non-TIR, belong to the non-TIR-NBS-LRR subfamily. These highly conserved motifs within the NBS domain occur across different plant species, making it possible to isolate R gene analogues (RGAs) from other crops using degenerate polymerase chain reaction (PCR) [13–19]. At present, more than 1600 NBS-LRR-type RGAs have been amplified via PCR from a wide range of plant species, and they have been arranged in clusters similar to R genes in plant genomes [5, 20]. Some of these are closely linked to known R gene loci or form a part of the R genes [21–23].
In recent years, the evolutionary patterns of NBS-LRR R genes have been investigated extensively in different plant species. For example, in annual species, such as Arabidopsis and rice, studies have shown that tandem and segmental gene duplication, gene conversion, unequal crossing-over, ectopic recombination and diversifying selection seem to be the primary evolutionary modes of NBS-LRR R genes [3, 24–27]. In woody perennial species (e.g. grapevine and poplar), tandem gene duplication and recombination play major roles in NBS-LRR R gene expansion . Point mutations, small insertions or deletions and gene loss have been proposed as the primary mechanisms by which NBS-LRR R genes evolve [29, 30]. Therefore, the evolution of plant NBS-LRR R genes appears to be a complex process.
Pepper (Capsicum annuum L.), a member of the botanical family Solanaceae, is an important vegetable crop worldwide. However, its production is affected because it is prone to many diseases. At present, three R genes conferring resistance to strains of Xanthomonas campestris pv. vesicatoria and root-knot nematodes have been identified from pepper [31–33]. Of them, two genes (Bs2 and CaMi) encode motifs characteristic of the NBS-LRR class of resistance genes. Moreover, some RGAs in pepper have been identified by modified amplified fragment length polymorphisms, NBS profiling and specific PCR amplification with primers designed from conserved regions of the NBS domain [34–37]. However, no detailed analysis of RGA characteristics is currently available. In this paper, we followed a PCR-based protocol using R gene-specific degenerate primers and data mining to identify and characterize the NBS-LRR CaRGAs and identify putative R genes in pepper. We also analyzed the genetic variations and phylogeny in pepper. Functional divergence analysis provided statistical evidence for altered selective constraints during protein evolution between the two subfamilies and identified some critical amino acid sites involved in this functional divergence. Analyses of non-synonymous (Ka) and synonymous (Ks) substitutions per site revealed a purifying selection in the evolutionary processes of non-TIR- and TIR-NBS-LRR CaRGAs in pepper. Several specificity-determining positions (SDPs) responsible for functional specificity were also predicted. Finally, the expression of representative CaRGAs was analysed in response to hormones and in different organs.