Intracellular immune sensors of microbial structures in plants and animals. Graphic representation of the tripartite modular structure of human NLRs, the apoptotic regulators human Apaf-1 and C. elegans CED-4, and R proteins. NB and ATPase activity is mediated by the central NB-ARC domain. The C-terminal LRRs are believed to sense, directly or indirectly, microbe-derived ligands. Structurally diverse N-terminal effector domains include the CARD, PYR, BIR, TIR, CC, and BEAF and BED. A WRKY DNA-binding domain is located at the C-terminus of RRS1. AD, activation domain.
View full figure (132 KB)EMBO Members Review
- The EMBO Journal (2007) 26, 4293 - 4301
- doi:10.1038/sj.emboj.7601854
Published online: 13 September 2007
Subject Categories:
Rumble in the nuclear jungle: compartmentalization, trafficking, and nuclear action of plant immune receptors
Qian-Hua Shen1 and Paul Schulze-Lefert1
- Department of Plant Microbe Interactions, Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, Köln, Germany
Correspondence to:
Paul Schulze-Lefert, Department of Plant Microbe Interactions, Max-Planck-Institut für Züchtungsforschung, Carl-von-Linne-Weg 10, Köln 50829, Germany. Tel.: +49 221 5062350; Fax: +49 221 5062313; E-mail: schlef@mpiz-koeln.mpg.de
Received 1 June 2007; Accepted 23 August 2007
Abstract
Plants and animals have evolved structurally related innate immune sensors inside cells to detect the presence of microbial molecules. An evolutionary ancient folding machinery becomes engaged for the synthesis of autorepressed receptor forms in both kingdoms. The receptors act as regulatory signal transduction switches and are activated upon direct or indirect perception of non-self structures. Recent findings indicate that nucleo-cytoplasmic partitioning and nuclear activity is critical for the function of several plant immune sensors, thereby linking receptor function to transcriptional reprogramming of host cells for pathogen defense. This implies short signalling pathways and reveals parallels with regulatory control mechanisms of animal steroid receptors.
Keywords:
- disease resistance,
- innate immunity,
- NB-LRR proteins,
- nucleo-cytoplasmic trafficking,
- pathogen recognition
Introduction
Introduction
Top of pagePlants have evolved two classes of immune receptors to detect non-self molecules. One class consists of membrane-resident pattern recognition receptors (PRRs) that detect microbe-associated molecular patterns (MAMPs). MAMPs are evolutionarily conserved structures that include components of fungal cell walls such as chitin (N-acetyl-chitooligosaccharide oligomers), most likely lipopolysaccharides (LPS) from gram-negative bacteria, as well as short peptides derived from bacterial flagellin or the elongation factor EF-Tu (Zipfel and Felix, 2005). Few MAMP receptors have been isolated to date, but include the Arabidopsis plasma membrane-resident receptor-like kinases FLS2 and EFR, recognizing flagellin and EF-TU-derived peptides flg22 and elf18, respectively (Gomez-Gomez and Boller, 2000; Zipfel et al, 2006). In rice, the plasma membrane-anchored CEBiP chitin receptor contains two extracellular LysM domains, a module implicated in peptidoglycan-binding, but lacks an intracellular kinase domain (Bateman and Bycroft, 2000; Kaku et al, 2006). MAMP-triggered signalling pathways leading to termination of microbial pathogenesis are genetically poorly defined. However, biochemical evidence points to close links between MAMP-triggered immune responses and changes of ion fluxes across and production of reactive oxygen species (ROS) on the outer surface of the plasma membrane within minutes of MAMP perception, induction of mitogen-activated protein (MAP) kinase signalling, as well as transcriptional activation of early defense-response genes. During interactions with virulent pathogens, the PRR-triggered defense system confers only weak immune responses that allow moderate pathogen growth (Chisholm et al, 2006). This immune weakening is mediated by pathogen-delivered effectors and involves the direct or indirect suppression of MAMP-triggered signalling (Fujikawa et al, 2006; He et al, 2006; Melotto et al, 2006; Nomura et al, 2006; de Torres-Zabala et al, 2007).
Plant resistance (R) proteins define a second mainly intracellular immune receptor class that have the capacity to detect directly or indirectly isolate-specific pathogen effectors, encoded by avirulence (AVR) genes. Like PRR-triggered immune responses, R protein-conditioned immunity is also linked to ROS accumulation and to defense gene activation, but differs both quantitatively and kinetically from the former, typically leading to host cell death at attempted invasion sites (Shirasu and Schulze-Lefert, 2000; Tao et al, 2003; Caldo et al, 2004). This 'hypersensitive response' is thought to limit the spread of infection. Because PRR- and R protein-triggered output responses are similar, it is possible that the signalling pathways converge.
Rather than repeating recent reviews on effector perception mechanisms by R proteins, we focus on recent insights in post-recognition R protein signalling, receptor compartmentalization and dynamics, as well as emerging links to the transcriptional machinery. In vertebrates, microbial molecules are detected inside cells by a class of sensors of the innate immune system known as NOD-leucine-rich repeats (NOD-LRR), NOD-like receptors (NLRs), NACHT-LRR, or CATERPILLER proteins that are structurally related to plant R proteins. We discuss the engagement of evolutionarily conserved proteins for immune receptor function in both kingdoms in the context of shared receptor folding/stabilization mechanisms. Finally, we compare emerging regulatory features of intracellular immune receptor function with steroid receptor regulation in vertebrates.
Intracellular immune sensors act as signal transduction switches
Top of pageA central nucleotide binding (NB) domain and C-terminal LRRs are common structural modules found in plant R and vertebrate NLRs (Figure 1). In contrast, a structurally diverse range of domains was apparently co-opted during evolution N-terminal to the NB domain, including coiled coil (CC) or TOLL/interleukin-1 receptor (TIR) domains in plants and in vertebrates a caspase recruitment domain (CARD), or pyrin domain (PYD), or baculovirus IAP repeats (BIRs). The central NB domain is part of a larger domain, called NB-ARC, due to its occurrence in plant R proteins, the apoptotic regulator human apoptotic protease-activating factor 1 (APAF-1), and its Caenorhabditis elegans homolog CED-4 (van der Biezen and Jones, 1998). NB-ARC domain-containing proteins belong to the family of STAND (signal transduction ATPases with numerous domains) NTPases that are found in archaea, bacteria, fungi, plants, and animals (Leipe et al, 2004). STAND ATPases are modular proteins and display a wide range of fusions to domains involved in protein–protein or protein–DNA interactions, small-molecule-binding domains, as well as catalytic domains involved in signal transduction (Leipe et al, 2004). These proteins are considered to act as regulatory signal transduction switches. A critical aspect of this switching is reversible, NTP hydrolysis-powered, conformational changes that are relayed to effector domains. STAND NTPases are unusual, because the regulatory switch, scaffolding, and occasionally, sensory as well as signal-generating moieties are integrated into a single multidomain protein (Leipe et al, 2004).
The crystal structures of APAF-1 and CED-4 revealed four NB-ARC subdomains, the nucleotide-binding site (NBS) plus three ARC subdomains (ARC1–ARC3) (Riedl et al, 2005; Yan et al, 2005) (note that ARC3 is absent in R proteins and substituted by a short linker of yet unknown structure). Importantly, the nucleotide in APAF-1 and CED-4 is bound at the interface of NBS, ARC1, and ARC2 subdomains and, depending on the presence of ATP or ADP, brings about markedly different conformers. A highly conserved MHD-motif (hxhHD) of plant R proteins is located in the ARC2 subdomain (Takken et al, 2006). The histidine next to the aspartate in this motif directly interacts with the 
-phosphate in APAF-1 (Riedl et al, 2005). Mutagenesis of either the histidine or aspartate in several R proteins as well as human NOD2 results in autoactivation (Bendahmane et al, 2002; Shirano et al, 2002; Tanabe et al, 2004; Howles et al, 2005; Tameling et al, 2006), indicating that these residues are important to keep the receptors in an inactive form. Biochemical analysis of two autoactivating mutations of the tomato I-2 R protein, which confers resistance to the fungal pathogen Fusarium oxysporum, showed in vitro markedly reduced ATP hydrolysis but did not affect nucleotide binding (Tameling et al, 2006). This suggests that the ATP bound form is the 'on state' whilst ATP hydrolysis switches the protein back to the 'off state' (Figure 2A).
Figure 2.
Intracellular immune sensors act as a regulatory signal transduction switch and are translocated into the nucleus. (A) In the absence of cognate microbial structures, the immune sensor is in an autorepressed, ADP-bound resting state. Direct or indirect perception of a microbial effector is thought to induce first a conformational change in the NB-ARC domain allowing exchange of ADP by ATP. This is believed to trigger a second conformational change in the N-terminal effector domain, thereby activating the receptor. The ATPase activity of the NB-ARC domain switches the conformation of the protein back to its resting state. (B) Translocation of immune receptors into the nucleus could either involve continuous cycling or is unidirectional. In the latter scenario, a presumed nuclear degradation pathway might explain disproportionately low levels of nuclear receptor pools.
View full figure (119 KB)Autoactivating mutations in plant R proteins and human NOD2 also map in the linker region between ARC2 and the LRR region (Zhang et al, 2003), as well as in the N-terminal part of the LRRs (Bendahmane et al, 2002; Shirano et al, 2002; Tanabe et al, 2004; Takken et al, 2006). This points to the existence of additional receptor regions that keep the protein in an autorepressed form in the absence of a cognate pathogen effector. Indeed, domain swap experiments between the highly sequence-related potato CC-NB-LRR-type Rx and GPA2 R proteins produced autoactive variants upon inappropriate pairings of ARC2 and LRR domains (Rairdan and Moffett, 2006), suggesting that intramolecular interactions between these two domains regulate the receptor's transition from an autorepressed to an active state. The importance of intramolecular domain–domain interactions in Rx is also illustrated by the reconstitution of effector-dependent Rx activity following in planta coexpression of nonoverlapping receptor fragments (Moffett et al, 2002). Together, this has led to a model in which the direct or indirect recognition of pathogen effectors by the polymorphic LRR region initiates a first conformational change (Figure 2A). This facilitates exchange of ADP by ATP, which in turn is thought to trigger a second conformational change that renders the respective N-terminal effector domain (CC, TIR, CARD, PYR, BIR) accessible for associations with downstream targets. Subsequent ATP hydrolysis switches the receptor back to its autorepressed form (Takken et al, 2006).
Nuclear action of R proteins
Top of pageHow plant NB-LRR proteins activate immune responses following recognition of pathogen-derived effectors has been a major question since the molecular isolation of the founding members of this protein family (Bent et al, 1994; Whitham et al, 1994). Recent findings suggest that members of the CC- and TIR-type receptor families function in the nucleus. Allelic barley MLA CC-type receptors recognize isolate-specific effectors of the grass powdery mildew fungus, Blumeria graminis f sp hordei (Ridout et al, 2006). Fractionation of cell extracts using transgenic plants that express native levels of epitope-tagged MLA as well as visualization of a fluorochrome-marked MLA in living epidermal cells localized the majority of the receptor to the soluble cytoplasmic fraction and approximately 5% to the nucleus (Bieri et al, 2004; Shen et al, 2007). Perturbation of nucleocytoplasmic MLA10 partitioning by expression of a receptor fusion protein containing a nuclear export signal (NES), which enhances nuclear export over import, abrogated MLA10-specified disease resistance (Shen et al, 2007). Similarly, adding a NES to the tobacco TIR-type N receptor, which conditions immunity against the tobacco mosaic virus (TMV) upon recognition of the p50 TMV replicase, impaired both N nuclear accumulation and TMV disease resistance (Whitham et al, 1994; Burch-Smith et al, 2007). Nuclear action of MLA and N was unexpected, because both proteins lack a canonical nuclear localization signal (NLS). Unlike this, the Arabidopsis TIR-type RPS4 protein, conditioning immunity to Pseudomonas syringae strains expressing avrRps4 (Gassmann et al, 1999), contains a bipartite NLS, and this targeting signal is required for both nuclear import and disease resistance (Wirthmueller et al, 2007). Similar to barley MLA, less than 10% of total cellular RPS4 was found in Arabidopsis nuclei preparations, whereas the bulk of the receptor associates with endosomes. Re-inspection of all 71 annotated Arabidopsis TNL and 54 CNL subfamily members (Meyers et al, 2003) reveals a widespread potential for nuclear localization of other R proteins; using the WoLF PSORT subcellular localization prediction (http://wolfpsort.org/), 51 TNL and 39 CNL protein models contain predicted monopartite or bipartite NLSs. Given the fact that in yeast 43% of known nuclear proteins enter the nucleus without discernible NLSs (Lange et al, 2007), the utilization of NLS-dependent and seemingly NLS-independent nuclear import pathways for plant R proteins is not surprising.
Transcriptional reprogramming of plant cells upon pathogen attack is extensive, affecting between 3 and 12% of the 24 000 tested Arabidopsis genes upon fungal or bacterial challenge, respectively (Nishimura et al, 2003; Thilmony et al, 2006). How the perception of non-self structures by PRRs and R proteins leads to transcriptional activation of defense-response genes has been a long-standing question. In this context, nuclear activities of barley MLA, tobacco N, and Arabidopsis RPS4 reveal novel insight. Quantitative fluorescence lifetime imaging of fluorochrome-tagged receptor was employed to visualize in vivo in nuclei an effector-dependent physical association between the MLA10 receptor and two WRKY transcription factors (HvWRKY1 and HvWRKY2 TFs; (Shen et al, 2007), suggesting that the TFs serve as immediate downstream targets of the activated receptor. This protein–protein association is mediated by the invariant N-terminal CC domain of allelic MLA receptors. Because the polymorphic C-terminal LRR region of MLA has been shown to determine recognition specificity (Shen et al, 2003), it is possible that this region senses, directly or indirectly, the presence of powdery mildew effectors, while the N-terminal CC of the activated receptor acts as a signal relay moiety to the WRKY TFs. Accordingly, different structural modules at opposite ends of the receptor might account for sensory and signal transmission subfunctions. Whilst it remains to be seen whether MLA and RPS4 proteins detect the corresponding effectors in the cytoplasm and/or nucleus, the cytoplasmic pool of tobacco N appears to detect the TMV p50 viral effector. When the p50 effector was fused to the NES, thereby depleting the nuclear p50 pool and enforcing cytoplasmic localization, plant cells retained the ability to trigger N-mediated disease resistance (Burch-Smith et al, 2007). Thus, sensory and signal transmission activities of N might take effect in different compartments.
Tobacco N interacts with two squamosa promoter-like (SPL) TFs that are required for TMV disease resistance (D Kumar, personal communication). As viral effector recognition by N occurs in the cytoplasm, the N interacting SPL TFs could serve as targets of the activated receptor. The SPL gene family represents a group of structurally diverse transcription factors found apparently only in plants (Cardon et al, 1999). Notably, loss-of-function mutations in the Arabidopsis SPL14 gene render mutant plants insensitive to the mycotoxin fumonisin B1, which elicits an apoptotic form of cell death in wild-type plants as well as tissue-cultured plant and animal cells (Gilchrist, 1997; Stone et al, 2005). This raises the question whether fumonisin B1- and N receptor-triggered cell death processes are mechanistically related and involve modifications of SPL TF activities. Likewise, the effector-dependent association between barley MLA10 and WRKY TFs appears to contribute to receptor-triggered disease resistance and host cell death at attempted fungal infection sites (Shen et al, 2007). However, the WRKY TFs interacting with MLA act as repressors of MAMP-triggered immune responses and might have a role in preventing 'chronic inflammatory responses' and/or to dampen immune responses below a threshold that is detrimental to attacked plant cells (Xu et al, 2006; Figure 3A). We hypothesized that MLA receptors may interfere with the WRKY repressor function, thereby derepressing MAMP-triggered immune responses. The derepression could amplify MAMP-triggered immune responses and, in principle, would be sufficient to drive plant cells into suicide. Thus, the effector-triggered MLA WRKY association could serve as nexus to integrate signals generated by PRRs and R proteins (Figure 3B). A similar regulatory logic might help to explain previous in planta experiments with autoactive forms of the flax TIR-NB-LRR protein L6 (Howles et al, 2005). Wild-type L6 confers typical race-specific immunity associated with localized cell death to strains of the flax rust fungus that carry the cognate avirulence gene, designated AvrL567. In recovered transgenic plants expressing autoactive L6 defense-related gene expression is chronically activated without signs of cell death. However, when the transgenic plants were challenged with flax rust isolates that are virulent on wild-type L6 plants, effective immunity was observed that was accompanied by an L6-like cell death response. Thus, while autoactive L6 alone is unable to drive plant cells into suicide, MAMPs released during fungal attack might trigger cell death-associated immune responses because of the simultaneous presence of autoactive L6.
Figure 3.
Nuclear action of MLA links effector-specific and MAMP-triggered immune responses. (A) One or several MAMP receptors initiate PAMP signalling via intracellular MAPK cascades, which in turn stimulate the induction of unknown WRKY transcriptional activators (pink color) and WRKY1/2 repressors (blue color). The WRKY repressors are thought to prevent chronic defense gene activation. Autorepressed MLA receptors are folded by RAR1, SGT1, and cytosolic HSP90 and might continuously cycle between nucleus and cytoplasm. (B) Integrated PAMP- and MLA-triggered immune response upon coactivation of one or several MAMP receptors and MLA by cognate powdery mildew effectors (designated AVRA). Activated MLA stimulates nuclear association with WRKY1/2 repressors, thereby derepressing MAMP-triggered immunity. Derepression of basal defense responses is thought to amplify expression of defense-related genes (bold arrow) and might drive attacked host cells into cellular suicide. Whether AVRA is directly or indirectly recognized by the cytoplasmic and/or nuclear MLA pool remains unknown.
View full figure (187 KB)Unlike direct links between MLA or N receptor function and the transcriptional machinery, nuclear RPS4 activity requires EDS1, a protein of unknown biochemical function(s) that lacks known chromatin- or DNA-binding domains and resides in both cytoplasmic and nuclear compartments (Feys et al, 2005; Wirthmueller et al, 2007). RPS4-triggered immunity, but not nucleo-cytoplasmic partitioning or receptor stability, is abolished in an eds1 null mutant background. Together with an almost complete breakdown of RPS4/EDS1-dependent activation/repression of approximately 130 defense-related genes in eds1 plants (Bartsch et al, 2006; Wirthmueller et al, 2007), this suggests that EDS1 acts as intermediary positive signal transducer between the receptor and defense gene expression.
Further evidence for transcription machinery-associated functions of plant immune sensors comes from a functional analysis of Arabidopsis RRS1, which conditions disease resistance to the bacterial pathogen Ralstonia solanacearum expressing the cognate effector PopP2 (Deslandes et al, 2003). RRS1 is unusual because it encodes a TIR-NB-LRR R protein with a C-terminal WRKY domain (Figure 1). The latter is shared by all WRKY transcription factor family members and is known to bind to cis-active DNA elements, termed W-boxes (Ulker and Somssich, 2004; Yamasaki et al, 2005). The type III effector PopP2 carries a bipartite nuclear localization signal and is specifically targeted to host cell nuclei. Transient gene expression experiments in Arabidopsis protoplasts using fluorochrome-tagged RRS1 and PopP2 demonstrated that nuclear visualization of RRS1 requires coexpression of PopP2, while expression of RRS1-GFP alone did not produce a fluorescence signal (Deslandes et al, 2003). As RRS1 and PopP2 were also shown to interact in yeast two-hybrid experiments, it is possible that the association with PopP2 either induces conformational changes in RRS1-GFP, thereby producing a detectable fluorescence signal, or that RRS1-GFP forms a heterocomplex, which is resistant to degradation. A 3 bp insertion mutation in RRS1 (synonym SHL1) that results in the addition of a single amino acid in the WRKY domain, thereby impairing its DNA-binding activity, leads to chronic expression of defense genes and occasional cell death in the absence of the parasite (Noutoshi et al, 2005). One interpretation is that in healthy plants, the wild-type protein must bind to DNA to repress plant defense gene expression. In this scenario, the association between RRS1 and PopP2 could serve as a trigger to sequester the R protein away from DNA, thereby allowing defense gene expression. Note that this is conceptually similar to the proposed derepression of MAMP-triggered immune responses following effector-induced associations between barley MLA10 and WRKY1 or WRKY2 repressors (Shen et al, 2007). Similarly, the plasma membrane-tethered Arabidopsis RPM1 CC-NB-LRR protein, which recognizes the P. syringae effector AvrRpm1, can interact with a DNA polymerase II accessory protein, TIP49a, that acts as an inhibitor of plant immune responses (Holt et al, 2002). Similar to the results obtained for barley MLA, full-length RPM1 does not interact with TIP49a in yeast, whereas the N-terminal CC-NB part alone does. This might be explained by both full-length R proteins adopting a conformation that masks the N-terminal interacting residues in yeast. This is consistent with the idea that the association between TIP49a and RPM1 is a post-activation event. However, it remains to be shown whether RPM1 must enter the nucleus to associate with TIP49a.
The Arabidopsis genome contains another R gene homolog (At4g12020), in which an N-terminal WRKY DNA-binding domain is fused to a TIR-NB-LRR protein. This deduced protein contains an additional C-terminal kinase domain. Although no biological function has been assigned to the WRKY-TIR-NB-LRR-kinase to date, it is of note that the Populus trichocarpa genome contains 40 NB-LRR gene models, not present in Arabidopsis, which carry an N-terminal BEAF and DREF DNA-binding finger (BED) DNA-binding zinc-finger domain (Aravind, 2000; Tuskan et al, 2006). This domain is also present at the N-terminus of the rice Xa1 NB-LRR R protein to Xanthomonas oryzae (Figure 1; Yoshimura et al, 1998). Thus, it is possible that a subgroup of plant immune receptors has acquired direct DNA-binding capacity by domain co-option involving WRKY or BED domains. To date, there is only one example of nuclear activity for a human NOD-like receptor family member: the CIITA protein contains an N-terminal CARD domain and acts through direct association with DNA-binding proteins to regulate expression of all major histocompatibility class II and other genes important in antigen presentation (Figure 1; Ting et al, 2006). CIITA function appears to involve 'promoter loading' of MHC class II genes. Unfortunately, its potential role in sensing microbial structures remains still unclear.
Nucleo-cytoplasmic shuttling or unidirectional nuclear import?
Top of pageAs barley MLA, tobacco N, as well as Arabidopsis RPS4 each localizes to the cytoplasm and nucleus in healthy plants, nucleo-cytoplasmic partitioning is an intrinsic effector-independent feature of these receptors. This partitioning is expected to engage the nuclear import and export machinery. Indirect evidence for this comes from genetic experiments in which a gain-of-function mutation in an Arabidopsis TIR-NB-LRR gene was used to study the requirements of an 'autoimmunity' phenotype (Zhang et al, 2003; Palma et al, 2005; Zhang and Li, 2005). Mutant snc1 plants express the autoactive SNC1 protein carrying a single-amino-acid substitution between the NB LRR domains, leading to chronic activation of defense responses and disease resistance to bacterial and oomycete pathogens (Zhang et al, 2003). Recessive mutations in two suppressor loci of the snc1 genotype, MOS3 and MOS6, each affects components required for protein passage through the nuclear pore. MOS3 is homologous to vertebrate nucleoporin 96 (Nup96) and resides at the nuclear rim (Zhang and Li, 2005). Vertebrate Nup96 and the yeast homolog, C-Nup145p, serve as components of the conserved Nup107–160 nuclear pore subcomplex, which is localized to both sides of the nuclear pore and regulates nuclear pore complex assembly and mRNA export (Vasu and Forbes, 2001; Walther et al, 2003). MOS6 encodes importin 
3 (Palma et al, 2005), a family of proteins known to function as adapters by binding to NLS-containing cargo proteins and to importin . The latter interacts with Nups to traverse nuclear pore complexes, thus implying mechanistically linked functions for MOS3 and MOS6 in nucleo-cytoplasmic trafficking. Functional specialization of importin family members is indicated by the fact that importin 3 represents one of eight importin homologs present in the Arabidopsis genome (Palma et al, 2005). The biological significance of snc1 suppressors comes from fully or partially restored susceptibility to virulent bacterial and oomycete pathogens in mos3 and mos6 plants, respectively (Palma et al, 2005; Zhang and Li, 2005). One intriguing possibility is that MOS3 Nup96 and MOS6 importin 3 are required for nuclear import of the autoactive SNC1 protein. Alternatively, they might serve as 'downstream' components of a presumed nuclear SNC1 activity, for example, as gatekeepers for mRNA export of SNC1 target genes.
A physical association between the CC-NB-LRR-type Rx R protein, conditioning immunity to the PVX virus, and Nicotiana benthamiana Ran GTPase-activating protein 2 (NbRanGAP2) directly links R protein function to nucleo-cytoplasmic trafficking (Tameling and Baulcombe, 2007). Affinity purification of epitope-tagged Rx coupled to protein mass spectrometry revealed an association with NbRanGAP2, but not with the closely related NbRanGAP1. Importantly, gene silencing of NbRanGAP2 partially compromises Rx-dependent viral disease resistance. RanGAP proteins are conserved in eukaryotes and are known to regulate the activity of the small GTPase Ran (Ras-related nuclear protein), which in turn is pivotal for trafficking of macromolecules through nuclear pores (Meier, 2007). The above-mentioned importin complex, loaded with NLS-containing cargo, dissociates in the nucleus upon binding of RanGTP, thereby permitting cytoplasmic reshuttling of importin and (note that RanGDP localizes to the cytoplasmic side of the nuclear envelope; (Merkle, 2001; Xu and Massague, 2004; Meier, 2007). Vertebrate RanGAP and plant RanGAP contain kingdom-specific domains, but appear to be both anchored to the outer surface of the nuclear envelope through different nuclear envelope-associated proteins (Rose and Meier, 2001). Like barley MLA and tobacco N, Rx lacks an obvious NLS and colocalizes to the cytoplasm and nucleus (J Bakker, personal communication). Thus, it is conceivable that NbRanGap2 plays a direct role in the nuclear passage of Rx: by carrying Rx into the nucleus or by mediating loading of Rx to other NLS-containing carriers. If this were true, then gene silencing of NbRanGAP2 is expected to deplete the nuclear Rx pool. Alternatively, Rx could form a preformed recognition complex with NbRanGAP2 in healthy plants. In this scenario, the PVX coat protein, which is recognized by Rx (Farnham and Baulcombe, 2006), might target NbRanGAP2 for viral dissemination. Viral manipulation of NbRanGAP2 would then be indirectly sensed by Rx.
If the above discussed immune receptors cycle continuously between nucleus and cytoplasm, then differential nuclear import and export rates or a cytoplasmic retention mechanism could account for the observed 
16-fold excess of cytoplasmic barley MLA and Arabidopsis RPS4 receptor pools (Shen et al, 2007; Wirthmueller et al, 2007). Alternatively, if nuclear import is unidirectional, then low levels of the nuclear receptor could be the consequence of a specific nuclear degradation mechanism. As the cycle of ATP/ADP binding and ATP hydrolysis in the NBS of R proteins is likely operating at a low level in the absence of cognate effectors (Figure 2A; Takken et al, 2006), thereby running the risk of autoactivation, a presumptive nuclear receptor degradation mechanism could serve as additional safeguard to prevent inappropriate signalling and/or might have a role in signal desensitization (Figure 2B and below).
Folding, stabilization, and degradation of plant and animal immune sensors
Top of pageGenetic and biochemical experiments revealed evolutionarily conserved proteins in vertebrates and plants that appear to serve critical functions in maintaining 'optimal' preactivation receptor levels, possibly by coupling receptor folding and degradation pathways. The co-chaperone-like proteins RAR1 and SGT1, as well as the cytosolic HSP90 chaperone were originally identified in plants by mutational screens as essential components of a subset of R protein-triggered immune responses to diverse plant pathogens (Shirasu and Schulze-Lefert, 2003). Loss of disease-resistance function in rar1 or sgt1 or hsp90 mutant plants typically results in a severe depletion of R protein levels in the absence of parasite (Tornero et al, 2002; Hubert et al, 2003; Lu et al, 2003; Belkhadir et al, 2004; Bieri et al, 2004; Holt et al, 2005; Azevedo et al, 2006). Specific mutations in HSP90, inactivating its intrinsic ATPase activity, and direct physical associations between the chaperone and R proteins suggest that the latter are HSP90 'clients' and become stabilized by the chaperone (Figure 3A and B; Hubert et al, 2003). As SGT1 and (metazoan) RAR1 share structural similarities with co-chaperones and bind to each other as well as to HSP90 (Azevedo et al, 2002; Shirasu and Schulze-Lefert, 2003; Takahashi et al, 2003; Bieri et al, 2004; Liu et al, 2004), the former two are thought to act as co-chaperones, possibly by positively modulating HSP90 activity on its R protein clients. A link to protein degradation comes from the finding that RAR1 and SGT1 each interact with subunits of the COP9 signalosome, a multiprotein complex of the ubiquitin-proteasome pathway (Serino and Deng, 2003), and from an association of SGT1 with SCF ubiquitin ligase components (Azevedo et al, 2002; Liu et al, 2002). The receptors themselves could serve as degradation targets. As R protein levels are typically decreased in recessive rar1 and sgt1 single mutants, the corresponding wild-type genes could antagonize a default receptor degradation pathway such that receptor folding and degradation processes are coupled. A seemingly antagonistic control of R protein stability by RAR1 and SGT1, as inferred from a reduction and partial recovery of R protein levels in Arabidopsis rar1 single and rar1 sgt1b double mutants, respectively, could alternatively be explained by a compensatory SGT1b-like activity supplied by the closely related SGT1a homolog (Holt et al, 2005; Azevedo et al, 2006). Repressors of immune responses/cell death that associate with the receptors before or after effector recognition, serve as additional candidate targets of the COP9/SCF ubiquitin degradation machinery (Azevedo et al, 2002; Liu et al, 2002). Thus, it will be interesting to examine the fate of barley WRKY1/2 repressors or Arabidopsis TIP49a after receptor activation.
Recent findings show that human homologs of SGT1 and cytosolic HSP90 form complexes with the CARD domain-containing NOD1 and NOD2 immune sensors as well as with several other NLRs including NALP3 (Hahn, 2005; da Silva Correia et al, 2007; Mayor et al, 2007). The latter contains an N-terminal PYR instead of a CARD domain. Application of geldanamycin, an HSP90 inhibitor, or depletion of SGT1 by small interfering RNA from cultured cells revealed a requirement for bacterial peptidoglycan-triggered NOD1 and NOD2 immune sensor function. Substantial decreases of NOD1 or NALP3 levels in geldanamycin-treated cells that could be antagonized by lactacystin, a proteasome inhibitor, suggests that shared biochemical mechanisms contribute to the folding/stability of signalling-competent immune sensors in animals and plants (Hahn, 2005; Mayor et al, 2007). Functional dependence on SGT1 and HSP90 of receptors carrying unrelated N-terminal domains (TIR, CC, CARD, PYR) as well as direct binding of SGT1-HSP90 complexes to their LRRs indicate that folding of the signalling-competent form occurs primarily via the polymorphic C-terminal receptor region. Despite these advances in assigning RAR1, SGT1, and HSP90 a function in folding/stability of preactivated immune sensors, it remains possible that they fulfill additional roles in post-activation signalling.
Both controlled folding and nucleo-cytoplasmic trafficking of intracellular immune receptors are regulatory features that are strikingly reminiscent of animal steroid receptor regulation (Pratt and Toft, 2003; Pemberton and Paschal, 2005). A cytoplasmic hetero-complex consisting of several heat shock proteins—including HSP90, HSP70, and HSP40—as well as co-chaperones forces an opening of the steroid-binding cleft, driven by heat-shock protein ATPase activity, such that the binding pocket can be accessed by a steroid ligand. Activation occurs in a stepwise manner, driven by ATP hydrolysis, to produce first a 'primed complex' and then a steroid-binding competent complex. Translocation of cytoplasmic steroid hormone receptor complexes to the nucleus upon binding of cognate steroids and docking to hormone response DNA elements is a hallmark of this receptor family (McKenna and O'Malley, 2002). Recent data suggest that steroid-binding may regulate a chaperone-dependent step that occurs after recognition of the NLS in these receptors (Davies et al, 2002; Freedman and Yamamoto, 2004). Nucleocytoplasmic shuttling of steroid receptors also provides a nexus for crosstalk with kinase stress pathways (Shank and Paschal, 2005). For example, epidermal growth factor signalling through a MAP kinase pathway leads to phosphorylation of the progesterone receptor, MAP kinase-dependent nuclear export, and subsequent degradation in the cytoplasm (Qiu et al, 2003).
Conclusion
Top of pageIt remains to be seen how many plant NB-LRR proteins function in the nucleus. The widespread occurrence of NLSs in Arabidopsis TIR- and CC-type receptor subfamilies is an indication that their nuclear location might not be an exception. Direct targeting of the transcriptional machinery by NB-LRR proteins as in the case of MLA receptors implies a short signalling pathway that may not depend on authentic signalling components. This could explain why mutational approaches in plants have failed so far in identifying signalling mutants that exclusively compromise NB-LRR receptor function. Given the functional and structural similarities of plant R and vertebrate NLR immune sensors, it will not be surprising if nuclear pools exist for NLR family members besides CIITA. Derepression of MAMP-triggered immune responses through MLA receptor interference with WRKY repressors is likely to be only one of several potential convergence points between MAMP- and R protein-triggered signalling pathways. Convergence points could also be generated by MAMP-triggered and MAP kinase-dependent R protein phosphorylation, in turn modulating effector-triggered receptor activity and/or nucleo-cytoplasmic receptor partitioning. In this context, nuclear translocation of a plant MAP kinase upon treatment of cell cultures with an oomycete-derived MAMP deserves special note (Ligterink et al, 1997). If nuclear action of R proteins is a widespread phenomenon, one would expect that evolution favored diverse interception points with the transcriptional machinery to avoid an Achilles' heel for immune sabotage by pathogens. Thus, whether different nuclear immune sensors target the same, different, or overlapping chromatin sites and how this translates into spatio-temporal changes of defense gene expression patterns could become a focus of future experimentation.
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