Download NLR functions in plant and animal immune systems: so far and yet

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NLR functions in plant and animal immune
systems: so far and yet so close
© 2011 Nature America, Inc. All rights reserved.
Takaki Maekawa1, Thomas A Kufer2 & Paul Schulze-Lefert1
In plants and animals, the NLR family of receptors perceives non-self and modified-self molecules inside host cells and mediates
innate immune responses to microbial pathogens. Despite their similar biological functions and protein architecture, animal NLRs
are normally activated by conserved microbe- or damage-associated molecular patterns, whereas plant NLRs typically detect strainspecific pathogen effectors. Plant NLRs recognize either the effector structure or effector-mediated modifications of host proteins.
The latter indirect mechanism for the perception of non-self, as well as the within-species diversification of plant NLRs, maximize
the capacity to recognize non-self through the use of a finite number of innate immunoreceptors. We discuss recent insights into
NLR activation, signal initiation through the homotypic association of N-terminal domains and subcellular receptor dynamics in
plants and compare those with NLR functions in animals.
There are fundamental differences between the immune systems of plants
and those of animals. Plants lack an adaptive immune system or specialized cells of the immune response. Instead, plants rely entirely on innate
immune responses and on the ability of each individual cell to recognize and mount resistance responses to pathogenic invaders (viruses,
bacteria, fungi, oomycetes and nematodes). A key feature of the plant
immune system is the presence of two classes of receptors for the perception of non-self that can trigger potent resistance responses: the first
class enables the recognition of pathogen-associated molecular patterns
(also called microbe-associated molecular patterns (MAMPs)) that are
conserved among species of a microbial group1; the second class permits
the detection of polymorphic strain-specific pathogen effectors (effectortriggered immunity)2. The former class consists of membrane-resident
pattern-recognition receptors (PRRs) for the extracellular perception of
MAMPs, whereas the latter comprises proteins, mainly from the NLR family of receptors, that detect either the action or the structure of pathogen
effectors inside host cells. Plant NLRs are often polymorphic between individual plants of a host population, and the sum of genes encoding NLRs in
a host population defines the repertoire for the detection of polymorphic
pathogen effectors. The ability to detect a particular MAMP is usually
conserved within a plant species and between different plant species. In
contrast, most animal NLRs are activated by MAMPs or endogenous substances released after invasion by a pathogen, called damage-associated
molecular patterns (DAMPs). In this context, animal NLRs are functionally analogous to membrane-resident animal PRRs, such as the Toll-like
receptors (TLRs). Despite the similar biochemical modules that act in the
innate immune systems of plants and animals, including NLRs, PRRs,
1Max-Planck
Institute for Plant Breeding Research, Department of PlantMicrobe Interactions, Köln, Germany. 2Institute for Medical Microbiology,
Immunology and Hygiene, University of Cologne, Köln, Germany.
Correspondence should be addressed to P.S.-L. ([email protected]).
Published online 18 August 2011; doi:10.1038/ni.2083
818
mitogen-activated protein kinase signaling cascades and inducible antimicrobial peptides, the plant and animal systems probably evolved by
convergent evolution3. This might be a consequence of the biochemical
constraints of building sensors for non-self and signaling cascades from a
limited number of eukaryotic protein modules in both phyla.
The NLR families in both phyla belong to the STAND (signal-transduction ATPases with numerous domains) P-loop ATPases of the AAA+
superfamily whose functions are unusually complex because regulatory
switch, scaffolding and sensory moieties, as well as signal-generating
moieties, are combined into a single multidomain protein4. The central
nucleotide-binding (NB) domain is typically followed by a C-terminal
LRR domain, but in each phylum there is diversity in the N terminus
of these domains that might have arisen by independent evolutionary
domain fusion events. In plants, coiled-coil (CC) or Toll–interleukin
1 (IL-1) receptor (TIR) domains are used as signaling hubs, similar to
human TLRs. In contrast, caspase-activation and recruitment domains
(CARDs), pyrin domains or baculovirus inhibitor-of-apoptosis repeats
are found only in animal NLRs (Fig. 1a,b). Furthermore, the number of
NLRs encoded in the genomes of plant and animal species varies considerably. Higher plants contain between ~150 and ~460 NLRs (in Arabidopsis
and rice, respectively)5,6, whereas the available vertebrate genomes encode
only ~20 NLRs7. However, early diverging metazoans and some fish have
large NLR repertoires, as exemplified by the sea urchin genome, which
has over 200 NLRs7–11. The Drosophila and Caenorhabditis elegans innate
immune systems seem to function without NLRs and must engage either
no or different receptors for intracellular sensing of non-self7.
The adaptive immune response in vertebrates is orchestrated mainly
by secreted cytokines that are produced by infected cells and/or activated
cells of the immune response. The secretion of cytokines is based on both
transcriptional reprogramming and enzymatic activation of zymogens
and prestored cytokines after activation of NLRs and other classes of PRRs.
The activation of animal NLRs can also trigger primary cell-autonomous
responses. These include, for example, the secretion of antimicrobial peptides that are of particular importance in maintaining the shielding funcvolume 12 number 9 september 2011 nature immunology
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© 2011 Nature America, Inc. All rights reserved.
tion of barrier epithelia and the induction of cell-death responses that can
help clear intracellular pathogens. In plants, events after NLR activation
are linked to the massive generation of reactive oxygen species, a sustained
increase in cytosolic Ca2+ and transcriptional reprogramming, typically
followed by a rapid host-cell death at sites of attempted colonization. The
latter response is widely used as a proxy for plant NLR activation, although
NLR-mediated pathogen resistance has been uncoupled from host-cell
death in several cases12 (Box 1). Although this defense mechanism is still
poorly understood, plants also use phytohormones and secreted peptides
for the systemic orchestration of immune responses, similar to mammalian cytokines and interferons13,14 (Box 2).
Here we outline the present ideas about NLR activation and signaling in plants and discuss functional analogies to the immune system in
animals. We illustrate how combined population and molecular genetics approaches have provided fundamental insight into NLR-mediated
mechanisms for the detection of non-self in plants and how structural
and cell biological approaches are beginning to identify mechanisms of
receptor-specific NLR signal initiation and signal transduction.
Detection of non-self and ‘modified self’ by NLRs
Early studies demonstrated that the LRR domain of the rice NLR Pita
interacts directly with its cognate effector from the rice blast fungus
Magnaporthe grisea15,16. Although direct binding has been confirmed
for a subset of other NLR-effector pairs17–19, combined population
genetic and molecular studies have shown that a conventional receptoreffector ligand-recognition model cannot be the sole mechanism by which
plant NLRs maximize and maintain the repertoire for the perception of
non-self. This is illustrated by allelic diversification of a subset of plant loci
encoding numerous NLR variants in a host population, each recognizing
a distinct strain-specific effector of a pathogen species20–26. Alignment
of such diversified receptor sequences has identified residues that are
under positive selection. Such an approach, combined with domain-swap
experiments, has identified the C-terminal LRR moiety as the main but
not exclusive determinant of effector-recognition specificity22,23,26–33.
Diversifying selection at single NLR-encoding loci with multiple alleles
encoding NLRs with different recognition specificities has been interpreted as evidence of direct binding of cognate effectors, which allows the
prediction of rapidly coevolving host-pathogen interactions and reciprocal
diversifying selection at cognate effector loci29. However, two examples
(Box 3) indicate it is unlikely that functional diversification of allelic NLRencoding loci is solely the product of direct, iterative cycles of receptor and
pathogen effector adaptations (referred to as the ‘coevolutionary arms
race’). Mechanisms other than direct receptor-effector ligand interactions
might drive adaptation changes in NLR repertoires for the detection of
non-self in plant populations (discussed below).
No evidence is available for the direct interaction of microbial structures
with vertebrate NLRs. Although mutational analysis of the NLR Nod1
has suggested direct binding of peptidoglycan subunits to the concave
surface of the LRR domain34, biophysical evidence for such an interaction
has not been reported so far. Only for the NLR-related protein Apaf-1
has biochemical and structural evidence shown direct interaction of its
elicitor, cytochrome-c, with its WD40 domain35,36. In addition, population-genetic evidence for pathogen-driven functional diversification of
sequence encoding the LRR domain in single vertebrate NLR-encoding
loci is sparse. The seemingly less diversification of recognition specificities of animal NLRs might reflect their role in sensing mainly conserved
MAMPs rather than polymorphic effectors, as in plants. Whether the
emergence of the adaptive immune system in vertebrates has replaced
the need for NLR-mediated detection of polymorphic non-self remains
speculative.
Strong experimental evidence supports the idea of indirect detection
of non-self for several plant NLRs. In this model, NLRs sense modified
host proteins (‘modified self ’), which in turn are effector targets. This
requires a preformed complex that contains the NLR and effector targets for the detection of host-protein modification. As the detection of
modified self requires that NLRs respond only to the action rather than
structure of effectors, this mechanism might have a selective advantage
over direct perception of non-self (for example, by antibodies of the vertebrate adaptive immune system) for coping with the limited number of
innate immunoreceptors for rapidly changing and highly diverse pathogen
effector repertoires. This is relevant given the discovery of several hundred
candidate secreted effectors encoded in each genome of a wide range of
plant pathogens37–41. In addition, effector repertoires vary between different strains of a pathogen species, which suggests that the interior of plant
Box 1 NLR-mediated host-cell death and disease resistance
Rapid and localized host cell death is typically seen after activation of CC- or TIR-type NLRs in plants in interactions with diverse
pathogens127–129. Host cells executing Rx NLR–mediated resistance to PVX virus remain alive, but continued transgene-mediated
overexpression of the viral elicitor leads to Rx-dependent cell death130. This could indicate a threshold above which sustained Rx activation
and true signaling leads to cellular collapse. Alternatively, because of the dual localization of Rx in the cytoplasm and nucleus 108,109, the
transgene-delivered effector might be mislocalized and activate Rx in a compartment (or compartments) different from that in which it is
activated in wild-type plants, which could set in motion a qualitatively different cell death–associated resistance pathway.
Two type I metacaspases have been shown to antagonistically control cell death in Arabidopsis129. Conditional expression of one of
these, the ‘anti-death’ metacaspase AtMC2, blocks the cell-death response mediated by activation of the two NLRs tested but does
not affect the restriction of pathogen growth. Conversely, Arabidopsis ndr1 mutants abrogate the function of several CC-type NLRs
by allowing unrestricted pathogen growth but retain NLR-associated cell-death responses 131. These seemingly conflicting data could
be reconciled by the assumption of signal bifurcation at activated NLRs that initiates presumably compartment-specific cell death–
dependent and cell death–independent branch pathways that are (singly or in concert, and dependent on pathogen lifestyle) sufficient to
terminate microbial growth.
Distinct cell-death responses have been described in mammals. Some NLRs, such as NLRC4 and NLRP3, can induce an immunogenic
cell-death response that is initiated by the non-apoptotic caspase caspase-1. This particular type of cell death has been called
‘pyroptosis’ and restricts pathogen propagation in certain cells of the immune response 132 and might be analogous to plant NLR–
mediated cell death. In contrast, apoptosis, another form of cell death, leads to an immunologically silent response, triggered by Shigella
type III effectors, for more propagation of bacteria133. Similarly, Botrytis cinerea, a plant necrotrophic pathogen, triggers a host cell-death
response to facilitate pathogenesis134.
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Plant cell
b
CC
TIR
Other
LRR
LRR
NB-ARC
NACHT
NLR
c
d
Pathogen
NDR1
RIN4
Pathogen
T3SS
P
AvrRpm1
AvrB
Adaptive immune
recognition
AvrRpt2
AvrPto
RPM1
RPS2
Prf
L
MHC
Pto homolog
Rx
EDS1
RanGAP2
AvrL567
TRs
TFs
CIITA
NLRC5
RIP2
Nod1, Nod2
Disease
resistance
and
cell death
Nucleus
Cell wall
ASC
ROS
TFs
NF-kB
AvrMla
NLRC4
NLRP1, NLRP3
NF-kB
MAPK
MLA
Plasma
membrane
Cellular
injury
P Pto
RPS2
SNC1
RPS4
N
PAMPs
DAMPs
RIPK
Feeding RPM1
structure
© 2011 Nature America, Inc. All rights reserved.
NLR
Plasma
membrane
Caspase-1
IL-1b
IL-18
Nucleus
Pyroptosis
(cell death)
Proinflammatory
cytokine response
Katie Vicari
a
Animal cell
CARD
PYD
BIRs
Other
Figure 1 Mode of action of NLRs in plant and animal innate immune systems. (a,b) Simplified tripartite modular structures of plant NLRs (a) and animal NLRs
(b). PYD, pyrin domain; BIRs, baculovirus inhibitor-of-apoptosis repeats. (c) Detection of strain-specific pathogen effectors (‘Avr’) by plant NLRs. Bacterial
pathogens usually secrete effectors via the bacterial type III secretion system (T3SS), whereas filamentous pathogens often export effectors from specialized
feeding structures called ‘haustoria’. The pathogen effectors included here are thought to suppress PRR-triggered resistance responses. A subset of NLRs
shuttle between the nucleus and cytoplasm, whereas others are invariably tethered to the plasma membrane. The lipase-like protein EDS1 shuttles between the
cytoplasm and nucleus and serves as convergence point in the signaling of resistance responses initiated by TIR-type NLRs. NDR1 and RIN4 are membraneassociated proteins that interacting with RPM1. Some NLRs associate with transcriptional repressors (TR), which is thought to amplify ‘defense’ gene
expression activated by transcription factors (TF). (d) Functions of various well-characterized vertebrate NLRs in a human cell. Exposure to bacterial pathogens,
which in some cases can access the host cytoplasm, activates NLR proteins. This results in the expression of proinflammatory cytokines and antimocrobial
peptides. Activation of pyrin domain–containing NLRs such as NLRP1 and NLRP3 can also result in activation of the protease caspase-1, which in turn cleaves
the zymogens of IL-1b and IL-18 for subsequent secretion. Activation of these NLRs by particular bacteria can also induce the caspase-1-dependent cell-death
program of pyroptosis. NLRP3 can also be activated by DAMPs, especially by membrane damage. Nuclear shuttling has been reported for some animal NLRs,
such as CIITA and NLRC5. This results in the activation of genes encoding major histocompatibility complex (MHC) class I and II molecules involved in the
presentation of antigens to T cells of the adaptive immune system. PAMPs, pathogen-associated molecular pattern; MAPK, mitogen-activated protein kinase.
cells encounters a staggeringly large diversity of non-self structures. The
detection of modified self through the use of a finite number of innate
immune sensors also requires that the number of host proteins for which
NLR surveillance is needed is a fraction of the actual host protein complement. Two closely related hypotheses for indirect recognition, referred to
as either the ‘guard’ or ‘decoy’ model, have been proposed42,43. In the guard
model, the NLR is believed to act as a guardian for a host protein, designated the ‘guardee’, which is the target of an effector-mediated modification. In this case, the effector target is thought to have a role in promoting
virulence. In the ‘decoy’ model, some host proteins are believed to have
evolved by duplication of a gene encoding an ancestral ‘guardee’ and are
believed to have lost the original function that made them attractive effector targets in the promotion of pathogenesis. A hybrid model of indirect
and direct recognition of non-self posits that a host protein can serve as a
cofactor for direct association of the effector with the NLR44,45.
An example of indirect recognition involves the tomato NLR Prf and
its associated host protein Pto. The Pseudomonas syringae pathovar (pv.)
tomato effector protein AvrPto binds to the serine-threonine kinase Pto,
thereby inhibiting its kinase activity and activating Prf46–49 (Fig. 1c).
Constitutive gain-of-function Pto mutants, mimicking the action of
820
AvrPto, are sufficient to trigger a Prf-dependent cell-death response48,50.
Because such Pto modifications can activate Prf in the absence of the
effector AvrPto, activation of the receptor does not require direct PrfAvrPto associations. In plants lacking Pto and/or Prf, Pseudomonas AvrPto
dampens immune signaling and promotes virulence by binding to the
cytoplasmic domain of the plasma membrane–resident FLS2-BAK1 PRR
complex46–49. Prf recruits several other Pto-like kinases into Prf immunocomplexes, which might extend its recognition spectrum for other
effectors51. Because a function of Pto in PRR-triggered defense responses
has not been demonstrated and the presence or absence of Pto polymorphisms is widespread in tomato populations, Pto might serve as a ‘decoy’
for restarting immune responses, via Prf, after the delivery of AvrPto into
host cells and interception of PRR-triggered signaling43.
A single plant guardee can serve as convergence point for several
different pathogen effectors and can be guarded by more than one
NLR. This is exemplified by RPM1, an Arabidopsis NLR that mediates
immune responses after detection of the P. syringae pv. maculicola effectors AvrRpm1 and AvrB52 (Fig. 1c). RPM1 is activated by a physical
molecular signature specific to the effector-dependent phosphorylation
of RIN4, a plasma membrane–associated host protein53,54. RIN4 acts as
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Box 2 NLR action and phytohormone-dependent defense signaling
© 2011 Nature America, Inc. All rights reserved.
Salicylic acid, jasmonic acid and ethylene are phytohormones that act in the respective hormone signaling pathways to finely regulate
various types of plant defense responses through a highly interconnected signaling network 13. Because of this network property, their
contribution to NLR-mediated immunity has been examined on a genetic background in which all three hormone signaling pathways are
dysfunctional135. Whereas 80% of RPS2-mediated resistance to P. syringae pv. maculicola expressing the effector AvrRps2 is impaired
in the mutant plants, 80% of RPM1-mediated resistance to the same bacterium expressing the effector AvrRpm1 is retained, which
indicates large differences in the dependence of NLR function on these three phytohormones. The predicted convergence points of NLR
and phytohormone signaling pathways remain so far elusive. Action of these three phytohormones together also accounts for the largest
part of resistance to P. syringae triggered by the PRR FLS2 (ref. 135), which demonstrates overlapping contributions of phytohormones
to PRR- and NLR-mediated resistance responses. Both cell-autonomous and non–cell-autonomous defense responses contribute to
pathogen containment, and this seems to depend on different EDS1 subfunctions mediated by the action of sequence-related PAD4 as
separate protein or by the action of the PAD4-EDS1 heterocomplex136. Whereas local resistance (and associated cell death) mediated
by the RPP1 TIR-type NLR at foci of pathogen infection requires the action of EDS1 and PAD4 together as separate molecular entities,
adjacent defense responses that limit the spread of virulent pathogens require formation of the EDS1-PAD4 heterocomplex for salicylic
acid–dependent transcriptional reprogramming136.
a negative regulator of PRR-triggered defense responses and is essential
for unrestricted growth of the bacterium55. Whether phosphorylation of
RIN4 is directly mediated by AvrB or is mediated by the associated host
receptor-like cytoplasmic kinase RIPK, which itself is activated by AvrB,
remains unclear53,54. Substitutions in RIN4 that mimic the AvrB-mediated
phosphorylation are sufficient to activate RPM1 independently of the bacterial effector. Thus, similar to the Prf activation described above, RPM1
activation does not require direct RPM1-AvrB association. A second NLR
that interacts with RIN4, Arabidopsis RPS2, detects the elimination of
RIN4 caused by the cysteine protease effector AvrRpt2 from P. syringae
pv. tomato56,57. Together these findings show that distinct biochemical
modifications of the same guardee can activate distinct NLRs. If it is
assumed that the diversity of pathogen effector activity is limited by a
finite number of biochemical modifications, this can explain why a finite
number of innate NLRs in plants can indirectly sense the presence of an
overwhelming diversity of polymorphic non-self structures without an
adaptive immune system.
Similar to plant NLRs, many human NLRs respond to surprisingly
diverse microbial structures, indicative of an indirect mode of stimulus
recognition. By analogy to guardees and decoys, this broad recognition
repertoire might be mediated through their association with host proteins.
This is best exemplified by human NLRP3. NLRP3 has been shown to be
activated by a plethora of different elicitors, including particulate substances, such as uric acid crystals (which are associated with the inflammatory condition in gout), fatty acid crystals and mineral crystals, as well as
with particulate adjuvants such as alum. Furthermore, NLRP3 can react to
bacteria-derived toxins, MAMPs, virus-derived nucleic acids and DAMPs.
The immense structural differences among these elicitors indicate direct
recognition is unlikely. Although the molecular details of the interconnection between NLRP3 and its activity cues remain elusive, perturbation of
cellular ion fluxes, the induction of reactive oxygen species and the release
of normally compartmentalized lysosomal proteases such as the cathepsins have been suggested as activation proxies58 (Fig. 1d).
Certain alleles encoding the NLR NAIP (Naip1; also called Birc1) in
mouse strains correlate with resistance to infection with Legionella pneumophila59, which provides an example of potentially NLR allele–specific
disease resistance in animals. Likewise, the interaction between the mouse
and its parasite Toxoplasma gondii60 provides an example of strain-specific
disease resistance comparable to that in plants. Natural polymorphisms
in the virulence of T. gondii strains correlate with the expression of polymorphic ROP kinases that are delivered into host cells. These target a
polymorphic family of mouse interferon-inducible immunity-related
GTPases that are essential for resistance to avirulent strains of T. gondii.
nature immunology volume 12 number 9 september 2011
By analogy to the indirect mode of activation of the NLR Prf in plants
by pathogen effector–mediated modification of host members of the Pto
kinase family46–49, it is conceivable that a mouse NLR indirectly detects
the presence of T. gondii ROP kinases through the modification of host
immunity-related GTPases.
Activation of NLRs by receptor oligomerization
A sequence of intramolecular conformational changes and the replacement of NDP by NTP (in tested cases, of ADP by ATP) are critical for NLR
activation61–63. Plant and mammalian NLRs have a similar architecture
of their central domain, designated the NB-ARC domain and NACHT
domain, respectively. This central domain can be further divided into
subdomains comprising an NB domain, a four-helix bundle (ARC1), a
winged-helix fold (ARC2) and a helical bundle (ARC3). The first three
subdomains are conserved in both phyla, whereas the helical bundle
(ARC3) is absent from plant NLRs62,64. Both loss-of-function and gainof-function mutations are frequently found in the sequence encoding the
central domain of plant and human NLRs64,65. In humans, most prominent are mutations in sequences encoding the NACHT domains of NLRP3
and Nod2 that are associated with hereditary autoinflammatory diseases,
which suggests that these are hypermorphic variants65. However, mutation
of sequences encoding homologous catalytic residues can yield at the same
time hypermorphic and hypomorphic phenotypes in related NLRs, which
suggests subtle differences in NACHT function66.
Studies of Rx, a CC-type plant NLR, have shown that individual
parts of the receptor that are coexpressed physically interact with each
other in planta (for example, CC and NB-ARC–LRR segments) and can
reconstitute a functional receptor67. This is indicative of intramolecular
domain-domain associations in the intact full-length receptor. Notably,
effector-dependent intramolecular dissociation of the NB-ARC and LRR
domains seems to be critical for Rx-triggered resistance67. In this model,
the LRR domain is proposed to act as a negative regulator of Rx activation,
as either substitutions in or absence of the LRR domain cause(s) an ectopic
cell-death response67,68. Consequently, after recognition of a cognate effector, an initial conformational change allows the replacement of ADP by
ATP in the NB domain and a subsequent conformational change initiates
downstream signaling63. Mammalian NLRs are probably activated by a
similar mechanism. Apaf-1 is known to be kept in an inactive (autoinhibitory) state by intramolecular interactions36,69,70, and mutational analysis
of human Nod2 has identified a negative regulatory function of the LRR
domain and the linker between the NACHT and LRR domains71.
It has been proposed that NLRs self-associate through their central
NTPase domain61. Self-association of the NB-ARC domain of plants,
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a
b
© 2011 Nature America, Inc. All rights reserved.
Figure 2 Polymorphic surface patches of
N-terminal TIR and CC domains of plant NLRs
are critical for receptor function. (a) Mapping of
individual residues of flax L6 (ref. 78), tobacco
N77,95 or Arabidopsis RPS439 onto the monomeric
structure of the plant TIR domain from Arabidopsis
thaliana96 (AtTIR; Protein Data Bank accession
code 3JRN); TIR domain surface residues needed
for disease resistance mediated by the NLRs
are in red. The L6 TIR domain functions as
homodimer78. (b) Mapping of CC domain surface
residues needed for disease resistance provided
by MLA10 (ref. 79) and RPM1 (ref. 100) onto the
MLA10 dimer structure79 (red; Protein Data Bank
accession code 3QFL). The RPM1 CC domain is
assumed to adopt a dimer fold similar to the MLA10 CC dimer79. The EDVID motif (blue) is present in many CC-type NLRs. Protomers (structural units of the
oligomeric proteins) are green and brown.
however, has been observed only under nonphysiological conditions72,
which could hint at a unique self-association property of plant NLRs.
This might reflect different evolutionary fusions of N-terminal domains
(CC-TIR versus CARD–pyrin domain–baculovirus inhibitor-of-apoptosis repeat domain) to the shared central domain in both phyla73 and/or
absence of the ARC3 helical bundle from the plant NB-ARC module. The
N-terminal domain of human Nod1, consisting of six antiparallel helices
arranged as a compact bundle (called the ‘death-domain fold’), has been
found to form pH-dependent dimers74,75. That finding and structural
insight into the NLR-related C. elegans CED-4 octamer suggest that the
N-terminal domains of certain animal NLRs engage in the formation of
homomeric complexes76, although the contribution of these interactions
to the biological function of mammalian NLRs awaits further elucidation.
The N-terminal domains of the plant NLRs N, RPS5 and Prf also provide a
self-association interface for homo-oligomerization51,72,77. Direct support
for the proposal of receptor self-association driven by receptor modules
other than the NB central domain has been provided by structural insights
into the CC domain of the barley NLR MLA and the TIR domain of the
flax NLR L6, which have demonstrated homotypic, dimeric associations
for these N-terminal domains78,79.
NLR-mediated signal initiation
Because of the tripartite modular architecture of plant NLRs, in which
the LRR domain serves a role in recognition specificity and the central
NB-ARC domain serves a role in NTP hydrolysis–powered alteration
of receptor conformation, it is tempting to assign a function in signal
initiation to the N-terminal CC or TIR modules. But how good is the
evidence for this? Overexpression of the TIR region from different TIRtype NLRs, including Arabidopsis RPS4 and RPP1, tobacco N and flax L,
triggers an effector-independent cell-death response19,78,80–82. This TIR
domain–triggered autonecrosis depends on EDS1, a lipase-like key regulatory protein with a signaling function in disease resistance mediated by
several TIR-type NLRs82–84. This suggests that the TIR-dependent autoactivity is a genuine immune response rather than the result of nonspecific
perturbation of cellular integrity79. Deletion of the LRR domain from the
Arabidopsis CC-type NLR RPS5 results in autonecrosis, but this cell-death
response is dependent on both CC and NB moieties72. Expression of the
N-terminal CC domain of the barley NLR MLA alone, which forms a
homodimer, is sufficient for the initiation of cell death79. Substitutions in
the MLA CC domain that disrupt homodimerization of the CC domain
abolish both autonecrosis mediated by the CC domain alone and effectordependent immunity in the context of full-length MLA. Thus, it is likely
that only the properly folded CC homodimer with its characteristic surface-charge segregation provides the scaffold needed for signal initiation79.
Reminiscent of those examples in plants, overexpression of the N-terminal
CARD of human Nod1 is sufficient to trigger signaling events in human
cell lines85. Finally, the animal NLR-like proteins Apaf-1 and CED-4 form
oligomers in response to apoptotic stimuli, which brings their N-terminal
CARDs into close proximity for signaling76,86.
Despite accumulating evidence for CC domain– or TIR domain–
dependent initiation of NLR signaling, it remains possible that the overexpression of particular TIR or CC modules leads to nonphysiological
Box 3 Functional diversification of allelic NLRs in plants
Thirteen alleles of the gene encoding L in flax have been identified; each encodes a receptor that mediates resistance to a different strain
of the flax rust fungus Melampsora lini. These alleles encode TIR-type NLRs, of which a subset recognizes rust fungus effectors encoded
by the AvrL567 locus. The crystal structures of two sequence-related AvrL567 effectors are very similar, with polymorphic residues located
on their surface29. Direct interactions have been found in yeast two-hybrid experiments only between the corresponding effector and
receptor pairs17. Notably, however, rust fungus effectors detected by other L-recognition specificities are most likely unrelated to AvrL567
(ref. 137). It is difficult to imagine how L NLRs that detect unrelated fungal effectors evolved in a coevolutionary process of reciprocal
diversifying selection if direct receptor-effector associations were the sole mechanism underlying functional receptor diversification.
Arabidopsis RPP13 is a CC-type NLR that is extraordinarily polymorphic among individual Arabidopsis ‘accessions’ with allelic receptor
variants that detect strain-specific effectors of the oomycete pathogen Hyaloperonospora arabidopsidis22,138. One effector detected by
RPP13 is encoded by the ATR13 locus139. Although this effector gene is polymorphic and is under diversifying selection in the pathogen
population, allelic ATR13 variants with extensive amino acid variation are equally effective in triggering resistance responses mediated by
a molecule encoded by a single RPP13 allele139. Thus, only a small subset of molecules encoded by Arabidopsis RPP13 alleles detect
ATR13 variants, whereas molecules encoded by other RPP13 alleles recognize different H. arabidopsidis effectors140. This makes it
unlikely that diversifying selection at the receptor loci is solely driven by direct receptor-effector ligand interactions. Consistent with that
idea, attempts to demonstrate direct associations between RPP13 and ATR13 have been unsuccessful 140.
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Pre-activation complex
Endogenous
protein
(guardee
or decoy)
LRR
Post-activation complex
Effector
NB-ARC
CC,TIR,
other
?
Signal
initiation
© 2011 Nature America, Inc. All rights reserved.
Figure 3 NLR signal initiation mediated by the N-terminal module. In
healthy plants, the N-terminal NLR module is kept in an inactive state
(orange oval) through intramolecular interactions with other NLR domains
and/or intermolecular interaction(s) with other host proteins (guardee or
decoy). After pathogen effector–mediated modification of the guardee or
decoy, a conformational change in the receptor exposes the active N-terminal
receptor domain (orange hexagon), which enables associations with signal
transducers (gray hexagon) for signal initiation (Post-activation complex).
This might result in the relocation of the receptor inside the cell.
heterotypic associations with full-length NLR members of the same subclass, thereby eliciting spurious receptor activation. This could explain
why the cell-death response seen after overexpression of the Arabidopsis
RPS4 domain is dependent not only on EDS1 but also on the co-chaperone
SGT1 and cytosolic chaperone hsp90, two interacting proteins that are
essential for the effective preactivation accumulation of many full-length
TIR- and CC-type plant NLRs82,87. However, as SGT1 and hsp90 have
also been linked to NLR signaling after activation88, the identification of
molecules that interact with the RPS4 TIR domain is needed to resolve
this ambiguity. Engagement of the evolutionarily conserved SGT1-hsp90
complex has been demonstrated for animal NLRs such as NLRP3 and
Nod1 (refs. 89,90), but details of its contribution to NLR signaling beyond
receptor folding and turnover remain to be established.
The crystal structures of the N-terminal CC and TIR domains of the
plant NLRs MLA and L6 have been solved78,79. Both domains form
homodimers in solution, and homotypic associations of the corresponding full-length receptors seem to be critical for their disease-resistance
function. Whereas the MLA CC structure resembles two springs slammed
together, the global fold of the L6 TIR domain is similar to animal TIR
domains present in TLRs, the adaptor MyD88, the IL-1 receptor and the
effector molecule RapL91–93 (Fig. 2a,b). Surface residues of the L6 TIR
homodimer are polymorphic in other TIR-type plant NLRs. It is possible
that the global fold of the TIR dimer rather than the polymorphic surface
patches are critical for signal initiation, which would entail immediate convergence of signal initiation from 82 predicted members of the Arabidopsis
receptor family94. Remarkably, however, reinspection of published data
has identified residues at the TIR dimer surface and dimer interface that
are critical for plant TIR-type NLR function77,78,82,95,96. This suggests that
unique dimer surface patches determine the specificity of signal initiation
for each receptor (Fig. 2a). Homotypic TIR domain associations are also
key in linking the cytosolic TIR domain of the animal TLRs to intracellular
signal-transduction pathways; for example, via the TIR adapters such as
MyD88 or TRIF. This is thought to induce an asymmetric orientation of
the TIR interaction surfaces, as observed in the plant L6 homodimer78,97.
Other potential homotypic TIR associations in plants could include the
dimerization of TIR domains from different NLRs (predicted for the
NLRs RRS1 and RPS4; discussed below), or pairs in which one partner is a
truncated TIR domain–containing homolog (such as the Arabidopsis proteins TX (TIR domain only) and TN (TIR and NB domain only), which
might have a function analogous to that of the animal adapters MyD88 or
TRIF)6. Although such homotypic TIR associations could define proximal
receptor-specific signaling platforms, almost all TIR-type NLRs tested
so far signal through EDS1 (refs. 83,84), which would define a potential
‘distal’ convergence point for signal transduction initiated by up to 82
TIR-type Arabidopsis NLRs.
The sequences that form the CC domain in plant NLRs are generally diverse, except that they frequently have a pentapeptide motif98,99.
Surface residues of the MLA CC homodimer79 and those predicted on
the RPM1 CC domain100, of which some are critical for receptor function
(Fig. 2b), could define proximal receptor-specific signaling platforms for
the CC-type receptor subclass analogous to those proposed above for the
TIR-type subclass (Fig. 2a). A distinct convergence point for CC-type
NLR–initiated signal transduction analogous to EDS1 has not been established. Given the approximately 150 NLRs encoded by the Arabidopsis
genome6, this indicates the existence of up to 150 unique NLR signalinitiation interfaces. If so, this shifts the conundrum of receptor-specific
signal transduction to the next downstream components. Specific associations between host proteins and the N-terminal CC or TIR domains have
been identified for many plant NLRs (Fig. 3 and Box 4). However, in most
of these cases, these seem to be guardees rather than signal transducers.
Thus, it will be important to determine whether the guardees present in
preactivation NLR complexes are replaced by N terminus–specific signal
transducers after activation (Fig. 3 and Box 4).
The importance of homotypic protein module associations for receptor signal initiation has also been documented for the N-terminal death
domain of mammalian NLRs (Fig. 1b). Genetic evidence suggests that signal-transduction events mediated by the well-characterized NLRs Nod1,
Nod2, NLRC4 (Ipaf), NLRP1 and NLRP3 depend on recruitment of the
specific adaptors RIP2 and ASC in the cytoplasm58,101–103. These interactions are mediated by homotypic interactions involving death-domain
fold domains. Structural data suggest that at least for Nod1 and RIP2,
the association relies on a strong electrostatic component104. However,
Box 4 The role of N-terminal interactors of plant NLRs
The CC region of Arabidopsis RPM1 associates with RIN4 at the plasma membrane as part of a recognition complex 55, but a dedicated
role for RIN4 in effector-triggered RPM1 signaling after activation is unknown. Fluorescence lifetime imaging suggests that the CC
domain of barley MLA interacts in the nucleus with a subfamily of WRKY transcription factors in the presence of the cognate effector.
These WRKY proteins seem to act as repressors of PRR-triggered immune responses, which suggests that MLA interferes with the
repressor function of WRKY, thereby de-repressing PRR-triggered defense141. The TIR domain of Arabidopsis SNC1, a TIR-type NLR of
unknown pathogen-detection function, directly interacts with the transcriptional corepressor TPR1 (ref. 142). The SNC1-TPR1 complex
formation is believed to activate TPR1, thereby repressing the expression of negative regulators of defense responses. Additionally,
the nuclear pore components Nup88 and Nup96, as well as importin a3, are needed for nucleo-cytoplasmic partitioning of SNC1 and
its function112,143,144. NRIP1 is a rhodanese sulfurtransferase that resides inside chloroplasts of naive tobacco plants and becomes
relocalized to the cytoplasm and nucleus after infection with tobacco mosaic virus. The tobacco TIR-type NLR N binds directly by its
TIR region to NRIP1, which in turn associates with the tobacco mosaic virus elicitor p50 (ref. 44). Catalytic activity of NRIP1 is not
needed for the association with N or p50, and a role for NRIP1 in signaling by N after activation is unclear. Whether guardees present in
preactivation NLR complexes are replaced by N terminus–specific post-activation signal transducers remains to be tested (Fig. 3).
nature immunology volume 12 number 9 september 2011
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© 2011 Nature America, Inc. All rights reserved.
structural details of the active signaling complexes, called ‘nodosomes’ or
‘inflammasomes’, remain elusive. Notably, the signaling events triggered
by RIP2 and ASC differ considerably. Once initiated by Nod1 and Nod2,
RIP2 becomes ubiquitinated after activation to recruit and activate the
kinase TAK1 complex that ultimately leads to the activation of mitogenactivated protein kinase and transcription factor NF-kB pathways, which
both converge in extensive transcriptional reprogramming, leading to the
production and release of a variety of proinflammatory mediators105. In
contrast, the recruitment of ASC to NLRC4, NLRP1 or NLRP3 inflammasomes leads to interaction and activation of the pro-caspase-1, which
ultimately feeds into the processing and release of the proinflammatory
cytokines IL-1b and IL-18 (refs. 58,105). In the latter case, transcriptional
reprogramming is involved only in enhancing expression of pro-caspase-1,
pro-IL-1b and IL-18 (ref. 106). Alternatively, activation of NLRC4 can also
trigger a cell-death response mediated by activation of caspase-1 independently of ASC recruitment107.
Compartment-specific receptor subfunctions
Coordinated nucleo-cytoplasmic partitioning of the plant CC-type NLR
Rx, which confers resistance to potato virus X (PVX), seems to be critical
for the function of this receptor108,109. Manipulation of Rx spatial partitioning or its cognate viral elicitor inside plant cells has shown that receptor activation occurs in the cytoplasm109. The N-terminal Rx interactor
RanGAP2 serves as cytoplasmic retention factor for Rx and is needed
for receptor function108 (Fig. 1c). Enforced nuclear accumulation of Rx
results in less receptor-mediated cell death and lower resistance to PVX,
which emphasizes the functional importance of the cytosolic pool108. In
contrast, the nuclear pool of the Arabidopsis TIR-type NLR RPS4 is critical for effector-dependent restriction of pathogen growth and receptor
signaling occurs through the nuclear pool of the defense regulator EDS1,
which enables RPS4-mediated changes in transcriptional outputs110,111
(Fig. 1c). Similar to Rx, however, coordinated shuttling of both RPS4 and
EDS1 between cytoplasmic and nuclear compartments seems to be critical
for full functionality111,112. Collectively, such studies show unexpected
subcellular dynamics of plant NLRs and their associated defense regulators
that hint at coordinated and possibly different subfunctions of the same
NLR in distinct cell compartments. As EDS1 defines a convergence point
for TIR-type NLR signaling in plants and acts in the nucleus downstream
of at least RPS4-triggered bacterial resistance, this indicates that a nucleusderived signal is generally needed for Arabidopsis TIR-type NLR functions independently of their localization. Indirect recognition of pathogen
effectors by NLRs can be used to predict their localization to any cell
compartment that contains effector targets for the formation of receptor
complexes. Therefore, partitioning of NLRs into different compartments
in naive cells could be partly the consequence of the subcellular dynamics
of the corresponding effector targets.
Although most vertebrate NLRs are presumably located in the cytoplasm, in humans, the NLR NLRC5 and the transcriptional coactivator
CIITA can also shuttle to the nucleus (Fig. 1d). Similar to the examples
in plants discussed above, nuclear localization of both NLRC5 and CIITA
is linked to transcriptional reprogramming113,114 (Fig. 1d). Although the
function of NLRC5 is still controversial, it is well established that CIITA
mediates transcriptional activation by serving as a scaffold for the recruitment of transcription factors and histone-modifying enzymes to the promoter region of genes encoding major histocompatibility complex class
II, whose products are pivotal in recognition of non-self by the adaptive
immune system in vertebrates115.
Crosstalk between NLRs
In few cases, different NLRs act together to confer resistance in response
to the identical effector116,117. Remarkably, the pair of TIR-type NLRs
824
RPS4 and RRS1 together condition immunity to at least three different
pathogen species (two bacterial species and a fungus)118. This could hint
at a mechanism by which formation of a heteromeric receptor complex
extends the repertoire of NLR-mediated recognition of non-self119. In
mammals, coimmunoprecipitation experiments have indicated that
NLRs can form hetero-oligomers120. However, distinct evidence for such
interactions has been provided only for NLRP3 and NLRP1, which both
functionally interact with Nod2 (refs. 121,122).
SRFR1 is a tetratricopeptide-repeat protein with weak similarities to
yeast transcriptional repressors; it is conserved between plants and animals123,124. Arabidopsis SRFR1 specifically and negatively regulates TIRtype NLR–mediated resistance responses, including RPS4-mediated
resistance after perception of the P. syringae–derived effector AvrRps4
(refs. 123,124). An additional negative regulatory link has been found
between SRFR1 and the TIR-type NLR homolog SNC1, in that the gene
encoding SNC1 becomes constitutively activated in plants that lack SRFR1
(refs. 124,125). Moreover, the synergistic effect on susceptibility to P. syringae in plants that lack SNC1 and RPS4 indicates that the functions of
these two proteins are interconnected via SRFR1 (ref. 96). This hints at
crosstalk between two TIR-type NLR proteins, which suggests an intriguing scenario in which a TIR-type NLR with a dedicated role in pathogen
detection (RPS4) engages another TIR-type NLR homolog (SNC1) for
resistance signaling. Resolving the issue of whether SNC1 is engaged in
amplification of the signal of other NLRs awaits further studies. NLR
crosstalk for resistance signaling might also explain the aforementioned
SGT1- and hsp90-dependent cell-death response after expression of the
RPS4 TIR domain alone82.
Concluding remarks
Mendelian inheritance of disease resistance in plants was first described
in 1907, and the genetic concept of strain-specific disease resistance was
described in 1947 (refs. 126). The molecular isolation of many genes in
plants and pathogens that underlie this type of plant immunity and access
to full genome sequences of the corresponding organisms has been instrumental in ‘translating’ those results into a molecular genetic framework.
Indirect detection of non-self rather than direct binding of effectors to
NLRs is perhaps the most unexpected aspect of the present molecular
concept. Future understanding of the molecular mechanics of NLR function in plants and animals will ultimately need biochemical reconstitution
of recognition complexes and access to co-crystals containing full-length
receptor, guardee-decoy and/or effector-ligand. This might reveal yet
another unexpected twist in NLR biology.
ACKNOWLEDGMENTS
Supported by German Research Foundation in the collaborative research centre
SFB670 (T.M., T.A.K. and P.S.-L.) and the Max Planck Society (P.S.-L.).
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/natureimmunology/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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volume 12 number 9 september 2011 nature immunology