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Genome Biology 2006, 7:212
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Plant NBS-LRR proteins: adaptable guards
Leah McHale, Xiaoping Tan, Patrice Koehl and Richard W Michelmore
Address: The Genome Center, University of California, Davis, CA 95616, USA.
Correspondence: Richard W Michelmore. Email: rwmichelmore@ucdavis.edu
Abstract
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.
Published: 26 April 2006
Genome Biology 2006, 7:212 (doi:10.1186/gb-2006-7-4-212)
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2006/7/4/212
© 2006 BioMed Central Ltd
Most of the disease resistance genes (R genes) in plants
cloned to date encode nucleotide-binding site leucine-rich
repeat (NBS-LRR) proteins characterized by nucleotide-
binding site (NBS) and leucine-rich repeat (LRR) domains
as well as variable amino- and carboxy-terminal domains
(Figure 1). These large, abundant, proteins are involved in
the detection of diverse pathogens, including bacteria,
viruses, fungi, nematodes, insects and oomycetes. There
have been numerous extensive reviews since the first NBS-
LRR-encoding genes were cloned from plants in 1994 (for
example [1-5]). This article aims to provide a current
overview of the structure and function of this protein family
as well as to highlight recent advances.
Plant NBS-LRR proteins are similar in sequence to members
of the mammalian nucleotide-binding oligomerization
domain (NOD)-LRR protein family (also called ‘CARD, tran-
scription 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 func-
tional 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-LRR-
encoding 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 duplica-
tions and losses [11,12]. In several lineages, NBS-LRR-encod-
ing genes have become amplified, resulting in family-specific
subfamilies (Figure 2; Additional data file 2) [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 sub-
families 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 NBS-
LRR proteins in other plant families.
NBS-LRR-encoding genes are frequently clustered in the
genome, the result of both segmental and tandem duplica-
tions [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 crossing-
over, 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-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 evolv-
ing groups of R genes [19,22].
The impact of selection on the different domains of individ-
ual 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 selec-
tion, as indicated by significantly elevated ratios of non-
synonymous 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 10
11
vari-
ants, 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
212.2 Genome Biology 2006, Volume 7, Issue 4, Article 212 McHale et al. http://genomebiology.com/2006/7/4/212
Genome Biology 2006, 7:212
Figure 1
The major domains of NBS-LRR proteins. Examples of proteins with each configuration are shown on the right. Bs4, I2, Mi, and Prf are from tomato; L6
from flax; N from tobacco; RAC1, RPP5, RPS4, RRS1, RPP8, RPP13, RPS2, RPS5, and RPM1 from Arabidopsis; Y-1 and Rx from potato; Mla from barley;
RGC2 from lettuce; Bs2 from pepper. N, amino terminus; TIR, Toll/interleukin-1 receptor-like domain; CC, coiled-coil domain; X, domain without
obvious CC motif; NBS, nucleotide binding site; L, linker; LRR, leucine-rich repeat domain; WRKY, zinc-finger transcription factor-related domain
containing the WRKY sequence; C, carboxyl terminus.
C
N
C
N
N
N
L
L
L
L
TIR NBS
NBS
NBS
C
X
TIR
TIR NBS
Bs2, RGC2 and RPM1
I2, Mi, Mla, Prf, RPP8,
RPP13, RPS2, RPS5
and Rx
RRS1
Bs4, L6, N protein,
RAC1, RPP5, RPS4
and Y-1
Examples
CC
WRKY
LRR
LRR
LRR
LRR
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
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Figure 2
Neighbor-joining tree showing the family-specific amplification of NBS sequences. (a) TNLs. (b) CNLs. The complete tree was based on 1,600 sequences
(see Additional data files 1 and 2 for an expanded tree with individual sequences and the alignments used). Clades that contained sequences from
individual plant families were collapsed into single branches and the number of sequences in each branch is indicated. Different taxa are assigned different
colors; clades with representatives from several families are shown in black. The scale bar represents five nucleotide substitutions.
59
7, 6, 1
5
1, 1
14
5, 3, 3
7, 2
4,
1, 1
10
5, 2, 1
9, 12, 5, 2, 1, 1, 1, 1
7
1, 2
11
3, 2
11
1
9, 2, 1
2, 1, 1, 1
4
2, 1
17
15
58
1
1
1
14
21, 1, 1
13
1, 1
1
5
48
18
2, 1
47
11
2, 1
4, 3
6
31
3
2, 1
3
6
1, 1
5
Homo sapiens Apaf1
8, 4, 1
2
35
4, 4, 2
18
5, 3
8, 13, 2, 1, 1
21
48
6
2, 1
7
5
20
19, 1
17
5
18, 3, 2, 1
1
9
3
25
42
4, 4, 1
1
20
1
4, 2
21
5
7, 2
50
2
5,
2
63
4, 2
60
3, 2, 1
3
15, 7, 5, 2, 2, 2
3
26
77, 16, 10, 8, 7, 3, 1, 1
6, 3, 2
9, 9, 4, 2
17
73
8, 6, 1, 1, 1
5
6, 5, 2
13
41
11
Homo sapiens Apaf1
5
13
2
(a) (b)
Amaranthaceae
Apiaceae
Asteraceae
Brassicaceae
Caricaceae
Convolvulaceae
Cucurbitaceae
Cupressaceae
Euphorbiaceae
Fabaceae
Funariaceae
Lamiaceae
Linaceae
Malvaceae
Nymphaceae
Plantaginaceae
Pinaceae
Poaceae
Rosaceae
Rutaceae
Salicaceae
Solanaceae
Vitaceae
Multiple families
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 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.
Characteristic structural features
NBS-LRR proteins are some of the largest proteins known in
plants, ranging from about 860 to about 1,900 amino acids.
They have at least four distinct domains joined by linker
regions: a variable amino-terminal domain, the NBS
domain, the LRR region, and variable carboxy-terminal
domains (Figure 1). Four subfamilies of CNLs and eight sub-
families of TNLs were identified in Arabidopsis from
sequence homology, motifs, intron positions and intron
phase [3]. No crystal structures have been determined for
any part of a plant NBS-LRR protein; crystal structures of
mammalian NBS and LRR domains are, however, available
as templates for homology-modeling approaches.
The amino-terminal domain
There is little experimental information on the function of
the amino-terminal domain. In animals, the TIR domain is
involved in signaling downstream of Toll-like receptors.
Many plant NBS-LRR proteins are thought to monitor the
status of (‘guard’) targets of pathogen virulence effectors (see
below). Given the presence of TIR or CC motifs as well as the
diversity of these domains, the amino termini are thought to
be involved in protein-protein interactions, possibly with the
proteins being guarded or with downstream signaling com-
ponents [4]. Polymorphism in the TIR domain of the flax
TNL protein L6 affects the specificity of pathogen recogni-
tion [28]. An alanine-polyserine motif that may be involved
in protein stability is located immediately adjacent to the
amino-terminal methionine in many TNLs (but not CNLs) in
Arabidopsis [3]. Four conserved TIR motifs span 175 amino
acids within the TIR domain of TNLs [27]. A CC motif is
common but not always present in the 175 amino acids
amino-terminal to the NBS of CNLs [3]. Some CNLs have
large amino-terminal domains; tomato Prf, for example, has
1,117 amino acids amino-terminal of the NBS, much of which
is unique to this protein.
The NBS domain
More is known of the structure and function of the NBS
domain, which is also called the NB-ARC (nucleotide
binding adaptor shared by NOD-LRR proteins, APAF-1, R
proteins and CED4) domain. This domain contains several
defined motifs characteristic of the ‘signal transduction
ATPases with numerous domains’ (STAND) family of
ATPases, which includes the mammalian NOD proteins
[29,30]. STAND proteins function as molecular switches in
disease signaling pathways. Specific binding and hydrolysis
of ATP has been shown for the NBS domains of two tomato
CNLs, I2 and Mi [31]. ATP hydrolysis is thought to result in
conformational changes that regulate downstream signaling.
The first report of NBS-LRR protein oligomerization, a criti-
cal event in signaling from mammalian NOD proteins, is the
oligomerization of tobacco N protein (a TNL) in response to
pathogen elicitors [32]. In Arabidopsis, eight conserved NBS
motifs have been identified through analysis with MEME, a
program for motif identification [3]. NBS domains of TNLs
and CNLs are distinguished by the sequences of three resis-
tance NBS (RNBS) motifs within them (RNBS-A, RNBS-C,
and RNBS-D motifs; see Additional data file 3) [3].
Threading plant NBS domains onto the crystal structure of
human APAF-1 provides informative insights into the spatial
arrangement and function of the motifs conserved in the
plant NBS domains (Figure 3) [30,33]. The nucleotide-
binding domain of APAF-1 consists of three subdomains: a
three-layered ␣/ subdomain (containing the anchor
region), a helical subdomain (containing the kinase-2 motif
and P-loop) and a winged-helix subdomain (containing the
MHDV motif; Figure 3). The specific binding of ADP by
human APAF-1 is achieved by a total of eight direct and four
water-mediated hydrogen bonds; the P-loop portion of the
helical subdomain interacts with the ␣- and -phosphates of
ADP, a histidine and a serine residue on the winged-helix
subdomain interacts with a phosphate and the sugar of ADP,
and a small anchor region in the ␣/ subdomain stabilizes
the adenine base [33].
The binding pocket and patterns of binding to ADP are well
conserved in the threading models of TNLs (exemplified by
the Arabidopsis protein RPS4) and CNLs (exemplified by
the Arabidopsis protein RPS5; Figure 3) ([30] and P.K.,
unpublished work). The NBS domains of TNLs contain
additional loops absent in the NBS domain of CNLs. TNLs
and CNLs have four conserved motifs that are located
around the catalytic cleft: the P-loop, the anchor region,
and the MHDV motif (specifically the histidine residue), all
of which serve to orient the ADP molecule, as well as the
GLPL motif (the MHDV and GLPL motifs are named after
their constituent amino acids in the single-letter code).
While there is no obvious contact between ADP and the
GLPL motif in human APAF-1, the conservation of its posi-
tion on top of the binding site in APAF-1, RPS4 and RPS5
indicates that it may be involved in binding ADP. In addi-
tion, the last two aspartic acids in the kinase-2 motif are
positioned to interact with the third phosphate of ATP, con-
sistent with their role of coordination for the divalent metal
ion required for phosphotransfer reactions, for example the
Mg
2+
of Mg-ATP (Figure 3). The anchor region in the ␣/
subdomain of APAF-1, which consists of the sequence Val-Thr-
Arg, is present as Phe-Gly-Asn in RSP4 and as Val-Gly-Gln in
212.4 Genome Biology 2006, Volume 7, Issue 4, Article 212 McHale et al. http://genomebiology.com/2006/7/4/212
Genome Biology 2006, 7:212
RPS5. This anchor region, consisting of a hydrophobic (Val
or Phe), a small (Gly or Thr) and a polar (Arg, Asn or Gln)
amino acid, was previously unrecognized, but is highly con-
served in plant NBS-LRR proteins (see Additional data file
3). Autoactivating mutations in two CNLs, potato Rx
(Asp460Val) and tomato I2 (Asp495Val), map next to the
histidine in the MHDV motif; these mutations may perturb
the binding of the -phosphate of ADP and result in a more
open structure [30].
The LRR domain
The LRR domain is a common motif found in more than
2,000 proteins, from viruses to eukaryotes, and it is involved
in protein-protein interactions and ligand binding [1]. The
crystal structures of more than 20 LRR proteins have
revealed that LRR domains characteristically contain a
series of -sheets that form the concave face shaped like a
horseshoe or banana [34]. Less is known, however, about the
quaternary arrangements of LRR proteins. At least three
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Figure 3
Predicted structures of NBS domains. Structural models for the NBS domain of TNL RPS4 and CNL RPS5 of Arabidopsis were generated using a self-
consistent mean-field homology modeling technique [95], in the absence of ADP. ADP was added to the two NBS models by inference from the APAF-1-
ADP complex without further refinement of the models to illustrate the position of the nucleotide relative to the conserved motifs. (a) The structures
of the NBS domains of RPS4 and RPS5, showing the positions of the conserved motifs. The protein structures are shown as ribbon diagrams and ADP is
shown as a stick model. TIR-type and CC-type NBS domains are made up of motifs, in order from the amino terminus [3]: P-loop (or Walker A site,
blue); RNBS-A (green); kinase-2 (or Walker B site, magenta); RNBS-B (green); RNBS-C (green); GLPL (yellow); RNBS-D (green); MHDV (orange).
(b) The binding sites of human APAF-1 (PDB code 1z6tA), Arabidopsis RPS4, and RPS5, showing the residues interacting with ADP and ATP. The
coordination of ADP in the three proteins involves three different conserved motifs. A small anchor region at the amino terminus of the NBS domain
coordinates the adenine of ADP or ATP, the P-loop coordinates the ␣- and -phosphates, and the MHDV motif (in the winged-helix subdomain in
APAF-1) coordinates either the sugar or the -phosphate of ADP. The two terminal aspartic acids from the kinase-2 motif are located in the pocket in
which the ␥-phosphate of ATP would sit. Images were generated using PyMol [96].
P-loop
Anchor
Kinase-2
Winged helix domain
ADP
D243
D244
K160
H438
S422
V127
R129
APAF-1
P-loop
Anchor
Kinase-2
ADP
D108
D109
K36
H302
F290
F2
N4
MHDV
RPS4
P-loop
Anchor
Kinase-2
ADP
D110
D111
K34
C308
V3
Q5
MHDV
H323
RPS5
ADP
P-loop
Kinase-2
RNBS-A
RNBS-B
RNBS-C
RNBS-D
MHDV
RNBS-B
RNBS-A
Kinase-2
P-loop
RNBS-C
RNBS-D
MHDV
ADP
RPS5
RPS4
Anchor
Anchor
GLPL
GLPL
(b)
(a)
different types of dimers have been observed, involving
interactions of either their concave surfaces [35] or their
convex surfaces [36,37], or by concatenation involving an
antiparallel -sheet at the interface [38]. Threading of the
LRR domain of Arabidopsis RPS5 onto the crystal structure
of the bovine decorin protein, a member of the small LRR
proteoglycans (SLRP) protein family with a protein core
composed of LRRs [35], provided a model consistent with a
curved horseshoe-like surface of -sheets (Figure 4; P.K.,
unpublished work). The number of repeats in the LRR
domains in TNLs and CNLs of Arabidopsis is similar (mean
14, range 8 to 25), but this number can be considerably
higher in other species. In the lettuce CNL Resistance Gene
Candidate 2 (RGC2) proteins, an example of which is Dm3,
the LRR domain appears to be duplicated and there can be
as many as 47 LRRs in total [19]. Each LRR comprises a core
of about 26 amino acids containing the Leu-xx-Leu-xx-Leu-
x-Leu-xx-Cys/Asn-xx motif (where x is any amino acid),
which forms a -sheet; each core region is separated by a
section of variable length that varies from zero to 30 amino
acids. In many NBS-LRR proteins, the putative solvent-
exposed residues (shown as x in the consensus sequence
above) show significantly elevated ratios of nonsynonymous
to synonymous substitutions, indicating that diversifying
selection has maintained variation at these positions. The
LRR domain is involved in determining the recognition
specificity of several R proteins (for example [18,39-42]);
direct interaction with pathogen proteins has rarely been
shown, however.
The LRR domain may be involved predominantly in regula-
tory intramolecular interactions. The LRR domain of the
potato CNL Rx interacts with the NBS domain even when
expressed in trans; this interaction is disrupted by the
potato virus X elicitor, a viral coat protein that can induce a
host defense response [43]. Also, the inner, concave surface
of the -sheets may not be the only binding surface. The
LRR domain of TLR3, a human Toll-like receptor, is pre-
dicted to form a heterodimer and to bind double-stranded
RNA from pathogens against its looped surface, on the oppo-
site side from the -sheets [37].
Analysis using MEME identified few motifs in common
between the LRR domains of TNLs and CNLs in Arabidopsis
[3]. The third LRR was one of the few that contained a con-
served motif. Mutation in this LRR of the CNL RPS5 results in
epistatic inhibitory effects on multiple NBS-LRR proteins,
suggesting that the LRR may interact with downstream signal-
ing components [5,44]; also, a mutation within this LRR in the
CNL Rx of potato results in a constitutively active form [45].
The carboxyl termini
CNLs and TNLs differ markedly in the size and composition
of their carboxy-terminal domains. Those of TNLs are larger
and more variable than those of CNLs. CNLs typically have
only 40-80 amino acids carboxy-terminal to the LRR
domain, whereas the carboxyl termini of TNLs often have an
additional 200-300 amino acids, equaling the size of the
LRR domain. Several TNLs have extensions with similarity
to other proteins [3]. One of the larger TNLs in Arabidopsis,
RRS1, which becomes localized to the nucleus in response to
infection, encodes a 1,388 amino-acid protein with a nuclear
localization signal and a WRKY motif (a motif also found in
zinc-finger transcription factors and containing the
sequence Trp-Arg-Lys-Tyr) at the carboxyl terminus [46].
Function, localization and regulation
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
212.6 Genome Biology 2006, Volume 7, Issue 4, Article 212 McHale et al. http://genomebiology.com/2006/7/4/212
Genome Biology 2006, 7:212
Figure 4
The predicted structure of an LRR domain resulting from the threading of
the LRR domain of Arabidopsis RPS5 onto bovine decorin (PDB code
1xku). (a) A cartoon representation of the predicted structure of the
RPS5 LRR domain generated using PyMol [96]. The -sheets forming the
concave face of the ‘horseshoe’ are represented as arrows. The
conserved aliphatic residues are shown in blue. N, amino terminus; C,
carboxyl terminus. (b) Alignment of the 12 leucine-rich repeats in
decorin and the 13 repeats in RPS5 as well as the amino terminal nine
amino acids. The conserved aliphatic residues are shown in blue.
N
C
(a)
(b)
Decorin
LRR1
LRR2
LRR3
LRR4
LRR5
LRR6
LRR7
LRR8
LRR9
LRR10
LRR11
LRR12
RPS5
LRR1
LRR2
LRR3
LRR4
LRR5
LRR6
LRR7
LRR8
LRR9
LRR10
LRR11
LRR12
LRR13
V
L
T
L
L
L
M
L
L
L
L
I
Y
V
V
L
L
L
L
L
L
I
L
L
L
L
L
C
R
A
H
E
Q
I
S
T
A
R
Q
S
A
P
R
T
V
R
I
R
E
K
R
S
T
E
K
P
V
L
T
R
E
V
Y
E
K
E
V
G
A
K
K
T
V
Y
H
T
V
E
K
R
F
T
V
F
V
L
L
L
L
V
I
L
L
L
V
V
V
V
I
L
L
F
L
L
I
V
L
V
L
L
I
R
Q
D
I
Y
R
E
R
H
G
H
Y
S
Q
K
S
F
D
N
N
G
T
D
G
F
E
H
H
C
C
L
L
L
V
L
I
L
L
L
L
L
L
D
L
L
L
L
L
L
L
F
I
I
V
L
V
Q
S
Q
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H
G
A
D
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N
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W
M
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K
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A
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N
N
S
N
N
K
E
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H
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I
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H
L
N
N
N
N
N
T
N
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N
N
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C
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G
K
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P
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comment
divergent members of the family. The simplest model for
NBS-LRR R protein function is as receptors that bind effec-
tor molecules secreted by pathogens, but direct interactions
between NBS-LRR R proteins and effector proteins have
been detected only rarely [47,48].
In an alternative model, the ‘guard hypothesis’, NBS-LRR R
proteins monitor the status of plant proteins targeted by
pathogen effectors [49,50]. 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 pro-
tease that cleaves RIN4 [52,53]. The disappearance of RIN4
is detected, however, by a second CNL, RPS2, that in turn
elicits the defense response [54,55]. There is increasing evi-
dence from several systems that other R proteins similarly
act as guards of host targets rather than direct receptors, at
least for bacterial effectors [56-58].
NBS-LRR proteins function as components of macromol-
ecular complexes [59]. Yeast two-hybrid and, more
recently, co-immunoprecipitation experiments have identi-
fied 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 [60-64], 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 [65,66].
Both TNLs and CNLs include members that undergo alterna-
tive 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
[67,68]. 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
[69-76], 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 disap-
pearance of RIN4 triggers the basal resistance response (see
above) [4,51]. Another level involves the interaction between
the LRR and NBS regions; the LRR can act in trans as a neg-
ative regulator of the NBS in the CNLs potato Rx and tomato
Mi [42,43]). 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 [77-79].
The role of protein degradation in resistance signaling is un-
clear, but there is increasing evidence for its importance [80].
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Genome Biology 2006, 7:212
Figure 5
The regulatory interactions between two Arabidopsis CNL proteins, RPM1
and RPS2, RPM1-Interacting Protein 4 (RIN4) and three pathogen
virulence effectors, AvrB, AvrRpm1 and AvrRpt2 [51-55]. The protein
RPM1 detects the phosphorylation of RIN4 by AvrB and AvrRpm1 and
elicits the resistance response. This outcome can be blocked by AvrRpt2,
a protease that cleaves RIN4. The disappearance of RIN4 is detected by
RPS2, resulting in elicitation of the defense response.
RIN4
RPM1 RPS2
AvrRpm1
AvrB
AvrRpt2
Resistance response
–
+
+
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 ampli-
tude 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 cytoplas-
mic. Fractionation studies and interactions in yeast with
membrane-associated proteins suggest that several are local-
ized to the inner side of the membrane [51,54,55,82]. Local-
ization 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 [4,84-86]. 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 acti-
vated 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 signal-
ing pathways, involving both synergism and mutual antago-
nism between pathways [88-91].
Frontiers
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 effec-
tors, 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 degrada-
tion). 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.
Additional data files
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 addi-
tional 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 x 10
-23
and 1 x 10
-18
,
respectively. The PHYRE pairwise sequence alignments of
APAF-1 and RPS4 and of APAF-1 and RPS5 were collated
212.8 Genome Biology 2006, Volume 7, Issue 4, Article 212 McHale et al. http://genomebiology.com/2006/7/4/212
Genome Biology 2006, 7:212
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.
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