818 volume 12 number 9 september 2011 nature immunology

NLR functions in plant and animal immune systems: so far and yet so closeTakaki 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 strain-specific 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 special-ized cells of the immune response. Instead, plants rely entirely on innate immune responses and on the ability of each individual cell to recog-nize 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 per-ception 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 (effector-triggered 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 fam-ily of receptors, that detect either the action or the structure of pathogen effectors inside host cells. Plant NLRs are often polymorphic between indi-vidual 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 sub-stances released after invasion by a pathogen, called damage-associated molecular patterns (DAMPs). In this context, animal NLRs are function-ally 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,

mitogen-activated protein kinase signaling cascades and inducible anti-microbial 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-trans-duction 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 consider-ably. 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 pep-tides that are of particular importance in maintaining the shielding func-

1Max-Planck Institute for Plant Breeding Research, Department of Plant-

Microbe Interactions, Köln, Germany. 2Institute for Medical Microbiology,

Immunology and Hygiene, University of Cologne, Köln, Germany.

Correspondence should be addressed to P.S.-L. (schlef@mpiz.mpg.de).

Published online 18 August 2011; doi:10.1038/ni.2083

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nature immunology volume 12 number 9 september 2011 819

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 NLR-encoding 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, popula-tion-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 specifici-ties 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 tar-gets 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 ver-tebrate 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 differ-ent strains of a pathogen species, which suggests that the interior of plant

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 mamma-lian cytokines and interferons13,14 (Box 2).

Here we outline the present ideas about NLR activation and signal-ing in plants and discuss functional analogies to the immune system in animals. We illustrate how combined population and molecular genet-ics 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 NLRsEarly 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 receptor- effector 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 inter-preted as evidence of direct binding of cognate effectors, which allows the

Box 1 NLR-mediated host-cell death and disease resistanceRapid 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 nucleus108,109, 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 responses131. 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 response132 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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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 Prf-AvrPto 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 immu-nocomplexes, 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 polymor-phisms 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 effec-tors 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

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 comple-ment. 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, desig-nated the ‘guardee’, which is the target of an effector-mediated modifica-tion. 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 effec-tor 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

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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 membrane-associated 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.

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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 oligomerizationA sequence of intramolecular conformational changes and the replace-ment 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 gain-of-function mutations are frequently found in the sequence encoding the central domain of plant and human NLRs64,65. In humans, most promi-nent 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 effec-tor, 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 (autoinhibi-tory) 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,

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 bac-terial 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 sub-stances, such as uric acid crystals (which are associated with the inflamma-tory 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 interconnec-tion 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 cathep-sins 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 pneu-mophila59, 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 poly-morphic 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.

Box 2 NLR action and phytohormone-dependent defense signalingSalicylic 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 network13. 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.

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initiation to the N-terminal CC or TIR modules. But how good is the evidence for this? Overexpression of the TIR region from different TIR-type 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 regula-tory protein with a signaling function in disease resistance mediated by several TIR-type NLRs82–84. This suggests that the TIR-dependent auto-activity 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 effector-dependent immunity in the context of full-length MLA. Thus, it is likely that only the properly folded CC homodimer with its characteristic sur-face-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 over-expression of particular TIR or CC modules leads to nonphysiological

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-apop-tosis 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 initiationBecause 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

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.

a b

Box 3 Functional diversification of allelic NLRs in plantsThirteen 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 unsuccessful140.

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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 pro-teins 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 gener-ally 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 estab-lished. Given the approximately 150 NLRs encoded by the Arabidopsis genome6, this indicates the existence of up to 150 unique NLR signal-initiation interfaces. If so, this shifts the conundrum of receptor-specific signal transduction to the next downstream components. Specific associa-tions 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 recep-tor signal initiation has also been documented for the N-terminal death domain of mammalian NLRs (Fig. 1b). Genetic evidence suggests that sig-nal-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 interac-tions 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,

heterotypic associations with full-length NLR members of the same sub-class, 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 correspond-ing 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 con-vergence 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

Pre-activation complex

Endogenousprotein

(guardee or decoy)

CC,TIR,other Signal

initiation

LRR

Post-activation complex

Effector

?

NB-ARC

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.

Box 4 The role of N-terminal interactors of plant NLRsThe CC region of Arabidopsis RPM1 associates with RIN4 at the plasma membrane as part of a recognition complex55, 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).

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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 ani-mals123,124. Arabidopsis SRFR1 specifically and negatively regulates TIR-type 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. syrin-gae 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 intrigu-ing 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 remarksMendelian 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 instru-mental 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 func-tion 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.

ACKNOWLEDGMENTSSupported 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 INTERESTSThe 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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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 mitogen-activated 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 inflam-masomes 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 indepen-dently of ASC recruitment107.

Compartment-specific receptor subfunctionsCoordinated 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 parti-tioning or its cognate viral elicitor inside plant cells has shown that recep-tor 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 criti-cal 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 nucleus-derived signal is generally needed for Arabidopsis TIR-type NLR func-tions 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 cyto-plasm, 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 recruit-ment of transcription factors and histone-modifying enzymes to the pro-moter 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 NLRsIn few cases, different NLRs act together to confer resistance in response to the identical effector116,117. Remarkably, the pair of TIR-type NLRs

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NLR functions in plant and animal immune systems: so far and yet so closeDetection of non-self and ‘modified self’ by NLRsBox 1 NLR-mediated host-cell death and disease resistanceFigure 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 membrane-associated 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.Activation of NLRs by receptor oligomerizationBox 2 NLR action and phytohormone-dependent defense signalingNLR-mediated signal initiationFigure 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.Box 3 Functional diversification of allelic NLRs in plantsabFigure 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.Box 4 The role of N-terminal interactors of plant NLRsCompartment-specific receptor subfunctionsCrosstalk between NLRsConcluding remarksACKNOWLEDGMENTSCOMPETING FINANCIAL INTERESTS