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-LRR-encoding genes in Arabidopsis thaliana, over 400 in Oryza sativa 3910, 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 1112. In several lineages, NBS-LRR-encoding genes have become amplified, resulting in family-specific subfamilies (Figure 2; Additional data file 1) 13. Of the 150 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) 131415. The spectrum of NBS-LRR proteins present in one species is not therefore characteristic of the diversity of NBS-LRR proteins in other plant families.

NBS-LRR-encoding genes are frequently clustered in the genome, the result of both segmental and tandem duplications 3101617. There can be wide intraspecific variation in copy number because of unequal crossing-over within clusters 1819. 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 crossing-over, sequence exchange, and gene conversion, rather than genetic events particular to NBS-LRR-encoding genes 3192021.

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-LRR-encoding 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-and-death model of R gene evolution, in which gene duplication and unequal crossing-over can be followed by density-dependent purifying selection acting on the haplotype, resulting in varying numbers of semi-independently evolving groups of R genes 1922.

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 non-synonymous to synonymous nucleotide substitutions, has maintained variation in the solvent-exposed residues of the β-sheets of the LRR domain (see below) 1923. 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 β-sheets have probably changed the orientation of individual β-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 × 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 CC-NBS-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 separately in phylogenetic analyses using their NBS domains (see Additional data file 2) 2425. 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 327. These include 21 TIR-NBS (TN) and five CC-NBS (CN) proteins that have amino-terminal and NBS domains but lack a LRR domain 27. The function of these proteins is not known, but they have the potential to act as adaptors or regulators of TNL and CNL proteins.

Disease resistance is the only function so far demonstrated for NBS-LRR proteins; however, a role in resistance has yet to be confirmed for most. Functions in other areas of plant biology cannot be excluded, particularly for the more divergent members of the family. The simplest model for NBS-LRR R protein function is as receptors that bind effector molecules secreted by pathogens, but direct interactions between NBS-LRR R proteins and effector proteins have been detected only rarely 4748.

In an alternative model, the 'guard hypothesis', NBS-LRR R proteins monitor the status of plant proteins targeted by pathogen effectors 4950. Such indirect detection of pathogens allows a limited number of NBS-LRR R proteins to detect the activity of multiple pathogen effectors that target points of vulnerability in the plant. This has been best characterized in Arabidopsis: the CNL protein RPM1 detects the phosphorylation of RPM1-Interacting Protein 4 (RIN4) by the pathogen effectors AvrB and AvrRpm1 from Pseudomonas syringae pv. glycinea and pv. maculicola, respectively, and elicits the resistance response (Figure 5) 51. The elicitation of this response can be abrogated by a third effector, AvrRpt2 from P. syringae pv. tomato, a protease that cleaves RIN4 5253. The disappearance of RIN4 is detected, however, by a second CNL, RPS2, that in turn elicits the defense response 5455. There is increasing evidence from several systems that other R proteins similarly act as guards of host targets rather than direct receptors, at least for bacterial effectors 565758.

NBS-LRR proteins function as components of macromolecular complexes 59. Yeast two-hybrid and, more recently, co-immunoprecipitation experiments have identified multiple interacting proteins. All of the constituents and details of the dynamics of these complexes have yet to be determined, however. Oligomerization of animal NOD proteins through the NBS domain or oligomerization of Toll-like receptors through the TIR domain is important for activating the signaling pathway in animal innate immune systems 6061626364, but there are currently few data on the oligomerization of plant NBS-LRR proteins. Effector-induced self-oligomerization of the tobacco N protein (a TNL) has recently been demonstrated in Nicotiana benthamiana; the ability to oligomerize was retained after loss-of-function mutations in the RNBS-A motif and TIR domain, but lost after P-loop mutations 32.

Little is known about the regulation of the plant genes that encode NBS-LRRs. Consistent with the need for a rapid response to pathogen attack, many NBS-LRR-encoding genes are constitutively expressed at low levels in healthy, unchallenged tissue, although some show tissue-specific expression (X.T., unpublished work). They are upregulated, however, in response to bacterial flagellin, which induces basal resistance, suggesting that plants can establish a state of heightened sensitivity to pathogen attack 6566.

Both TNLs and CNLs include members that undergo alternative splicing. Alternative splicing of Toll-like receptors in animals is common and splice variants of the mouse Toll-like receptor TLR4 may be part of a regulatory feedback loop inhibiting excessive responses to bacterial lipopolysaccharide 6768. The induction of splice variants upon pathogen recognition has been observed for plant NBS-LRR proteins, suggesting that alternative splicing may have a regulatory role in the plant defense response 68. Multiple transcripts have been detected for several TNL-encoding genes (RPP5, RPS4, and RAC1 in Arabidopsis, L6 in flax, N in tobacco, Y-1 in potato, and Bs4 in tomato) and fewer CNL-encoding genes 6970717273747576, although their significance to disease resistance is unclear. The ratio of transcripts from the tobacco N gene is critical for resistance to tobacco mosaic virus 71. Both full-length and alternative transcripts are necessary for resistance mediated by RPS4 in Arabidopsis 73.

Triggering of basal resistance and/or cell death associated with specific resistance imposes a heavy cost and is therefore likely to be tightly regulated. There is growing evidence for multiple layers of negative regulation, paralleling that observed in mammals. One layer involves RIN4; the disappearance of RIN4 triggers the basal resistance response (see above) 451. Another level involves the interaction between the LRR and NBS regions; the LRR can act in trans as a negative regulator of the NBS in the CNLs potato Rx and tomato Mi 4243). A third layer involves the conformational change of the NBS following hydrolysis of ATP 31. NBS-LRR R protein activity may also be subject to regulation by heat-shock proteins such as the Hsp90 proteins 4; both CNLs such as Arabidopsis RPM1 and potato Rx and TNLs such as the tobacco N protein require cytosolic HSP90 for their function 777879.

The role of protein degradation in resistance signaling is unclear, but there is increasing evidence for its importance 80. Two proteins, 'Required for Mla12 Resistance 1' (RAR1) and 'Suppressor of G2 Allele of SKP1' (SGT1), are required for the function of several R proteins that signal through different pathways 59. The COP9 signalosome, a multiprotein complex involved in protein degradation, is required for resistance to tobacco mosaic virus mediated by the tobacco TNL N protein 81. The Arabidopsis CNL protein RPM1 is degraded at the onset of the hypersensitive response 82; RING-finger E3 ubiquitin ligases in Arabidopsis are involved in RPM1- and RSP2-mediated elicitation of the hypersensitive response 83. Therefore, either specific or general proteolysis may have roles in controlling the amplitude of the defense response and the extent of cell death associated with the hypersensitive response.

Most NBS-LRR proteins lack a signal peptide or membrane-spanning regions and are therefore assumed to be cytoplasmic. Fractionation studies and interactions in yeast with membrane-associated proteins suggest that several are localized to the inner side of the membrane 51545582. Localization studies are challenging, however, because of the probable dynamic nature of complexes and because of the low endogenous expression levels of NBS-LRR proteins; consequently, data from overexpression studies are difficult to interpret.

Plant NBS-LRR proteins act through a network of signaling pathways and induce a series of plant defense responses, such as activation of an oxidative burst, calcium and ion fluxes, mitogen-associated protein kinase cascade, induction of pathogenesis-related genes, and the hypersensitive response 4848586. At least three independent, genetically defined signaling pathways in Arabidopsis are induced by NBS-LRR proteins 87. TNLs and CNLs tend to signal through different downstream pathways: TNLs signal through the 'Enhanced Disease Susceptibility' protein EDS1 and CNLs through the 'Non-race specific Disease Resistance' protein NDR1, although this correlation is not absolute. A separate pathway independent of EDS1 and NDR1 is activated by the Arabidopsis CNLs RPP8 and RPP13. Several small signaling molecules in the plant defense response, such as salicylic acid, jasmonic acid, ethylene, and nitric oxide, are involved downstream of NBS-LRR proteins and there is complicated cross-talk between the different signaling pathways, involving both synergism and mutual antagonism between pathways 88899091.

The scope and complexity of this protein family provide many opportunities and challenges for both evolutionary and functional studies. An important immediate goal is to obtain crystal structures of NBS-LRR proteins, either in their entirety or as individual domains with and without their ligands. The coevolution of NBS-LRR proteins with their cognate bacterial effectors and their plant targets is of considerable interest, particularly as understanding these genetic changes and selective forces could lead to strategies for generating plants with more durable disease resistance. We also need to address an intriguing conundrum: if the LRR domain is acting as a negative regulator of the NBS domain and NBS-LRR proteins are monitoring the status of conserved host proteins, why is there frequently a strong evolutionary signal of divergent selection acting on solvent-exposed residues on the concave surface of the LRR?

Numerous questions remain at the functional level. Are all NBS-LRR proteins involved in plant defense, or do some have other functions? What are the constituents of the macromolecular complexes involving NBS-LRR proteins and what events occur upon pathogen challenge? Do these complexes often contain multiple NBS-LRR proteins 92? Are pathogen effectors usually detected indirectly, through monitoring their activity on plant targets, or are some effectors, for example from oomycetes or fungi, detected directly by NBS-LRR proteins? Do the proteins with only some of the domains, such as the TN and CN proteins 27, function as regulatory or adaptor molecules?

Other questions include the functions of the variable amino- and carboxy-terminal domains and the multiple layers of positive and negative regulation (transcriptional, alternative splicing, phosphorylation and particularly protein degradation). Also, what is the functional significance of the lack of TNLs in cereals, and does this result in a different spectrum of resistance responses? Finally, what is the molecular basis of 'restricted taxonomic functionality' (resistance function restricted to within a plant family) of NBS-LRR proteins 93 and which additional proteins are required for function in plants other than the source species?

Ultimately, once the evolutionary mechanisms and structure-function relationships are understood in detail, it might be possible to generate NBS-LRR proteins with new recognition specificities that target key pathogen constituents, resulting in new, durable forms of resistance.

The following additional data files are available: Additional data file 1 shows an alignment of 65 amino acids from 1,600 NBS sequences used to generate the neighbor-joining trees shown in Figure 2 and Additional data file 2; in both additional data files, parts (a) show TNL sequences and parts (b) CNL sequences. Additional data file 3 shows an alignment of NBS sequences used to generate the models of the NBS domain of RPS4 and RPS5 shown in Figure 3; PHYRE, a threading service available at 94, identified APAF-1 (PDB code 1z6t) as a reliable template to model the RPS4 and RPS5 NBS domains, with Z-scores of 5 × 10^-23 and 1 × 10^-18, respectively. The PHYRE pairwise sequence alignments of APAF-1 and RPS4 and of APAF-1 and RPS5 were collated into a single alignment without further refinement. Boxes show the positions of the eight motifs identified by Meyers et al. 3 and the position of the anchor region.