Plant NBS-LRR proteins: adaptable guards

Plant NBS-LRR proteins: adaptable guards Leah McHale, Xiaoping Tan, Patrice Koehl and Richard W Michelmore Correspondence: Richard W Michelmore. Email: 0 0 Address: The Genome Center, University of California , Davis, CA 95616 , USA The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. This large family is encoded by hundreds of diverse genes per genome and can be subdivided into the functionally distinct TIR-domain-containing (TNL) and CC-domain-containing (CNL) subfamilies. Their precise role in recognition is unknown; however, they are thought to monitor the status of plant proteins that are targeted by pathogen effectors. - Plant NBS-LRR proteins are similar in sequence to members of the mammalian nucleotide-binding oligomerization domain (NOD)-LRR protein family (also called CARD, transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats (CATERPILLER) proteins), which function in inflammatory and immune responses [6]. But although mammalian NOD-LRR proteins have the same tripartite domain organization as plant NBS-LRR proteins, including a nucleotide-binding domain and a LRR domain, the functional similarities between NBS-LRR and mammalian NOD proteins are probably the result of convergent evolution [7]. There are no NOD-related proteins in Caenorhabditis elegans or Drosophila melanogaster and the downstream partners of the two families differ [7,8]. The human NOD protein apoptotic protease activating factor 1 (APAF-1) has an NBS domain with greater protein-sequence similarity to plant NBS-LRR proteins than to other mammalian NOD proteins; however, it shares neither the amino-terminal nor the carboxy-terminal LRR domains characteristic of plant NBS-LRR proteins. Evolution and genome organization Plant NBS-LRR proteins are numerous and ancient in origin. They are encoded by one of the largest gene families known in plants. There are approximately 150 NBS-LRRencoding genes in Arabidopsis thaliana, over 400 in Oryza sativa [3,9,10], and probably considerably more in larger plant genomes that have yet to be fully sequenced. Many NBS-encoding sequences have now been amplified from a diverse array of plant species using PCR with degenerate primers based on conserved sequences within the NBS domain and there are currently over 1,600 NBS sequences in public databases (Additional data file 1). They are found in non-vascular plants and gymnosperms as well as in angiosperms; orthologous relationships are difficult to determine, however, owing to lineage-specific gene duplications and losses [11,12]. In several lineages, NBS-LRR-encoding genes have become amplified, resulting in family-specific subfamilies (Figure 2; Additional data file 2) [13]. Of the 150 Bs4, L6, N protein, C RAC1, RPP5, RPS4 and Y-1 I2, Mi, Mla, Prf, RPP8, RPP13, RPS2, RPS5 and Rx Bs2, RGC2 and RPM1 NBS-LRR sequences in Arabidopsis, 62 have NBS regions more similar to each other than to any other non-Brassica sequences (Figure 2; Additional data file 2). Different subfamilies have been amplified in the legumes (which includes beans), the Solanaceae (which includes tomato and potato), and the Asteraceae (which includes sunflower and lettuce) [13-15]. The spectrum of NBS-LRR proteins present in one species is not therefore characteristic of the diversity of NBSLRR proteins in other plant families. NBS-LRR-encoding genes are frequently clustered in the genome, the result of both segmental and tandem duplications [3,10,16,17]. There can be wide intraspecific variation in copy number because of unequal crossing-over within clusters [18,19]. NBS-LRR-encoding genes have high levels of inter- and intraspecific variation but not high rates of mutation or recombination [19]. Variation is generated by normal genetic mechanisms, including unequal crossingover, sequence exchange, and gene conversion, rather than genetic events particular to NBS-LRR-encoding genes [3,19-21]. The rate of evolution of NBS-LRR-encoding genes can be rapid or slow, even within an individual cluster of similar sequences. For example, the major cluster of NBS-LRRencoding genes in lettuce includes genes with two patterns of evolution [19]: type I genes evolve rapidly with frequent gene conversions between them, whereas type II genes evolve slowly with rare gene conversion events between clades. This heterogeneous rate of evolution is consistent with a birth-anddeath model of R gene evolution, in which gene duplication and unequal crossing-over can be followed by densitydependent purifying selection acting on the haplotype, resulting in varying numbers of semi-independently evolving groups of R genes [19,22]. The impact of selection on the different domains of individual NBS-LRR-encoding genes is also heterogeneous [19]. The NBS domain seems to be subject to purifying selection but not to frequent gene-conversion events, whereas the LRR region tends to be highly variable. Diversifying selection, as indicated by significantly elevated ratios of nonsynonymous to synonymous nucleotide substitutions, has maintained variation in the solvent-exposed residues of the -sheets of the LRR domain (see below) [19,23]. Unequal crossing-over and gene conversion have generated variation in the number and position of LRRs, and in-frame insertions and/or deletions in the regions between the -sheets have probably changed the orientation of individual -sheets. There are, on average, 14 LRRs per protein and often 5 to 10 sequence variants for each repeat; therefore, even within Arabidopsis, there is the potential for well over 9 x 1011 variants, which emphasizes the highly variable nature of the putative binding surface of these proteins. There are two major subfamilies of plant NBS-LRR proteins, defined by the presence of Toll/interleukin-1 receptor (TIR) or coiled-coil (CC) motifs in the amino-terminal domain (Figure 1). Although TIR-NBS-LRR proteins (TNLs) and CCNBS-LRR proteins (CNLs) are both involved in pathogen recognition, the two subfamilies are distinct both in sequence and in signaling pathways (see below) and cluster 9, 12, 5, 2, 1, 1, 1, 1 Amaranthaceae Apiaceae Asteraceae Brassicaceae Caricaceae Cucurbitaceae Cupressaceae Euphorbiaceae Fabaceae Funariaceae Lamiaceae 21 5 7, 2 50 5, 222 Solanaceae Vitaceae Multiple families 77, 16, 10, 8, 7, 3, 1, 1 separately in phylogenetic analyses using their NBS domains (see Additional data file 2) [24,25]. TNLs are completely absent from cereal species, which suggests that the early angiosperm ancestors had few TNLs and that these were lost in the cereal lineage. The presence or absence of TNLs in basal monocots is not currently known. CNLs from monocots and dicots cluster together, indicating that angiosperm ancestors had multiple CNLs (Figure 2) [26]. There are also 58 proteins in Arabidopsis that are related to the TNL or CNL subfamilies but lack the full complement of domains [3,27]. These include (...truncated)