Nuclear Trafficking During Plant Innate Immunity

Molecular Plant

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Volume 1

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Number 3

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Pages 411–422

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May 2008

REVIEW ARTICLE

Nuclear Trafficking During Plant Innate Immunity Jun Liu and Gitta Coaker1 Department of Plant Pathology, The University of California, Davis, CA, USA

ABSTRACT Land plants possess innate immune systems that can control resistance against pathogen infection. Conceptually, there are two branches of the plant innate immune system. One branch recognizes conserved features of microbial pathogens, while a second branch specifically detects the presence of pathogen effector proteins by plant resistance (R) genes. Innate immunity controlled by plant R genes is called effector-triggered immunity. Although R genes can recognize all classes of plant pathogens, the majority can be grouped into one large family, encoding proteins with a nucleotide binding site and C-terminal leucine rich repeat domains. Despite the importance and number of R genes present in plants, we are just beginning to decipher the signaling events required to initiate defense responses. Recent exciting discoveries have implicated dynamic nuclear trafficking of plant R proteins to achieve effector-triggered immunity. Furthermore, there are several additional lines of evidence implicating nucleo-cyctoplasmic trafficking in plant disease resistance, as mutations in nucleoporins and importins can compromise resistance signaling. Taken together, these data illustrate the importance of nuclear trafficking in the manifestation of disease resistance mediated by R genes.

INTRODUCTION The ability to perceive and initiate a defense response against pathogenic microorganisms is paramount to the success of all life. Unlike animals, plants lack a circulating immune system recognizing microbial pathogens. Plant cells are more autonomous in their defense mechanisms and rely on the innate immune capacity of each cell and systemic signals that disseminate from infection sites (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006). Conceptually, there are two different branches of the plant innate immune system. However, the activation of either branch results in a largely overlapping set of responses, including transcriptional reprogramming of the plant cell to arrest pathogen growth. One branch, termed PAMP-triggered immunity (PTI), consists of extracellular surface receptors that recognize pathogen-associated molecular patterns (PAMPs). PAMPs are conserved microbial features such as bacterial flagellin, Ef-Tu and fungal chitin that fulfill a function crucial to the lifestyle of an organism (Gomez-Gomez and Boller, 2000; Kaku et al., 2006; Zipfel and Felix, 2005; Zipfel et al., 2006). Relatively few PAMP receptors have been identified to date. Those that have been characterized possess extracellular leucine-rich repeats and intracellular kinase domains (Gomez-Gomez and Boller, 2000; Kaku et al., 2006; Zipfel et al., 2004, 2006). The activation of PTI results in the induction of mitogen-activated protein (MAP) kinase signaling, transcriptional reprogramming mediated by WRKY transcription factors, production of reactive oxygen species and callose deposition (reviewed in Nurnberger and Kemmerling, 2006). Collectively, these responses prevent pathogen growth.

PTI results in transcriptional reprogramming mainly mediated by antagonizing members of plant-specific WRKY transcription factors (TFs). WRKYs bind to promoters of defense-related genes through specific interaction of the WRKY domain with pathogen response elements termed W boxes (reviewed in Eulgem and Somssich, 2007). Interestingly, the promoters of WRKY genes are enriched in W boxes, implicating the presence of sophisticated feedback loops between different family members (Dong et al., 2003). WRKY TFs can act as both positive and negative regulators of disease resistance; the inactivation of defense-suppressing WRKY proteins is an important aspect of plant innate immunity (reviewed in Eulgem and Somssich, 2007). This complicated WRKY web may ensure rapid and efficient signal amplification while enabling proper context-dependent gene expression. The second branch of the plant innate immune system consists of resistance (R) proteins that specifically recognize the presence of corresponding pathogen effector proteins. This branch of the immune system is termed effector-triggered immunity (ETI). From an evolutionary perspective, it is hypothesized that microbial pathogens developed effector proteins in an attempt to suppress PTI (Chisholm et al., 2006). In turn, plants developed R proteins specifically recognizing these effectors, thus restoring disease resistance resulting in ETI. 1 To whom correspondence should be addressed. E-mail glcoaker@ucdavis. edu, fax 530 752-5694, tel. 530 752-6541.

ª The Author 2008. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssn010, Advance Access publication 1 April 2008

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ETI reactivation of resistance responses is hypothesized to be mediated by cytoplasmic proteins and transcriptional reprogramming required to initiate defense responses involves signaling from the cytoplasm to the nucleus (Burch-Smith et al., 2007; Shen et al., 2007; Tameling and Baulcombe, 2007; Wirthmueller et al., 2007). Although both branches of the innate immune system result in disease resistance, R protein activation generates a faster and stronger response, culminating in programmed cell death surrounding the infection site. This programmed cell death is a hallmark of ETI and is termed the hypersensitive response (HR). Different plant R proteins can recognize effectors from all classes of pathogens (viruses, bacteria, fungi, oomycetes and nematodes). R proteins can either directly or indirectly recognize the presence of their cognate pathogen effectors. Because the majority of plant R proteins are intracellular, it is hypothesized that pathogen effectors must also be localized inside plant cells. Viral pathogens replicate intracellularly and bacterial pathogens utilize a specialized protein secretion system, termed the Type III, to deliver their effectors into host cells. Oomycete pathogens possess very large collections of pathogen effectors, many of which contain the N-terminal RxLR amino acid motif that is involved in their translocation into host cells (Kamoun, 2006). Surprisingly, this host targeting signal is conserved between oomycete plant pathogens and the human malarial parasite Plasmodium falciparum (Morgan and Kamoun, 2007; Win et al., 2007). Despite the diversity of pathogens recognized, most plant R genes can be grouped into one large family, encoding proteins with a Nucleotide Binding Site (NB) and C-terminal Leucine Rich Repeat (LRR) domains; there are 149 NB-LRR proteins in Arabidopsis (Meyers et al., 2003). These NB-LRR proteins are broadly related to the animal Apoptosis regulators CATERPILLER/NOD/ NLR proteins and STAND ATPases (Leipe et al., 2004; Ting and Davis, 2005). This homology indicates that R protein activation may require ATP binding or hydrolysis (Marquenet and Richet, 2007; Tameling et al., 2002, 2006). The NB-LRR proteins can be further subdivided into two main subclasses, based on distinct N-terminal domains, which influence the requirement for downstream signaling responses. One class contains a coiledcoil (CC) domain, while the other class possesses significant homology to the cytosolic portion of the TOLL/interleukin-1 receptors (TIR; Dangl and Jones, 2001). Multiple autoactivating mutants in plant R genes map to the linker region between the NB and LRR domains, which possesses a potential ATP-binding site (reviewed in Shen and Schulze-Lefert, 2007). This has let to a model in which R proteins usually exist in an auto-repressed state, bound to ADP. The direct or indirect detection of cognate effector proteins is hypothesized to induce a conformational change, enabling the ATP-binding site to exchange ADP for ATP. ATP binding is then thought to trigger a second conformational change that enables the R protein’s N-terminus (TIR, CC) to interact with downstream targets (Takken et al., 2006). How do R proteins initiate the disease resistance signaling cascade? Despite the number of R proteins present in plants,

little is known about the signaling events required to initiate effector-triggered resistance. Recently, multiple exciting discoveries have implicated dynamic nuclear re-localization of plant R proteins after effector recognition, suggesting that R proteins may directly regulate transcription factors to induce defense signaling (Burch-Smith et al., 2007; Shen et al., 2007; Tameling and Baulcombe, 2007; Wirthmueller et al., 2007). Several genetic loci required for ETI are also imported into the nucleus from the cytoplasm (Dong, 2004; Palma et al., 2005; Wiermer et al., 2005; Zhang and Li, 2005). Taken together, these data suggest that dynamic protein import into the nucleus in response to R protein activation is important to initiate disease resistance signaling. In this review, we will focus on recent exiting discoveries implicating the nucleus as a key player in R protein signaling. Additional components required for plant innate immunity that are nuclear localized will also be re-evaluated in light of the new data surrounding R protein signaling mechanisms.

NUCLEO-CYTOPLASMIC TRAFFICKING The nuclear envelope is present in all eukaryotic cells and consists of a double-layer membrane that separates the nucleus from the cytoplasm. While the composition of animal and yeast nuclear envelopes has been studied in detail, knowledge of the plant nuclear envelope is still at an early stage, although most components of nucleo-cytoplasmic trafficking have been identified in plants (Meier, 2007). For the purpose of this review, we will focus on the current knowledge of how trafficking occurs using data primarily obtained in yeast and mammals. Proteins transported into the nucleus do so by traveling through nuclear pores. Nuclear pores are large protein complexes that span the nuclear envelope and the proteins that make up the nuclear pore complex are called nucleoporins. Nuclear pore complexes (NPCs) serve as a molecular sieve to transport water-soluble molecules across the nuclear envelope. Molecules up to 60 kD can passively diffuse through the NPC, but molecules over 50 kD diffuse at a very slow rate. Efficient transport of larger molecules greater than 50 kD requires active transport under the assistance of importin receptors (Gasiorowski and Dean, 2003). Proteins with a nuclear localization signal (NLS) are targeted for active nuclear import. The amino acid sequences of several NLS are known and contain a conserved stretch of basic residues. The first NLS to be discovered was the bipartite NLS, which consists of two clusters of basic amino acids separated by a short spacer around 10 amino acids in length (Dingwall et al., 1988). Monopartite NLS are characterized by a short stretch of basic amino acids. Furthermore, there are several other types of NLS, such as the acidic M9 domain of hnRNP A1 (Mattaj and Englmeier, 1998). The classical scheme for active nuclear import of cargo proteins containing a NLS begins with importin a binding to the cargo’s NLS and acting as a bridge for importin b to attach. This

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trimeric importin a/b/cargo complex is directed toward the NPC and translocated into the nucleus. The small GTPase Ran (Rasrelated nuclear protein) is also required for nucleo-cytoplasmic import and export. The Ran GTP versus GDP gradient maintains the directionality of transport: Ran GTP is required for disassociating import cargos and binding export cargos inside the nucleus; Ran GTP hydrolysis in the cytoplasm is required for dissociation of export cargo complexes (Figure 1; Gasioroswski and Dean, 2003). Export receptors such as AtXPO1 in Arabidopsis (Haasen et al., 1999), CseI in Saccharomyces cerevisiea (Cook et al., 2005) and CAS in mammals (Kutay et al., 1997) work to-

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gether with Ran to transport importin a back into the cytoplasm, where importin a can bind new cargo proteins destined for the nucleus (Figure 1).

NUCLEO-CYTOPLASMIC TRAFFICKING OF PLANT RESISTANCE PROTEINS R proteins are found in a variety of subcellular locations, from the inner face of the plasma membrane to the cytosol and nucleus. An emerging picture in plant innate immunity is that R protein nuclear localization and direct interaction with WRKY

Figure 1. Model for Protein Import through Nuclear Pore Complexes. Cytoplasmic proteins possessing a nuclear localization signal are transported into the nucleus by importin a and importin b. The trimeric importin a/b/cargo complex is then targeted to the nuclear pore complex. Export receptors like AtXPO1 in Arabidopsis can work together with Ran GTP to transport importin a back into the cytoplasm. Ran GTP hydrolysis in the cytoplasm then disassociates export cargo complexes.

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transcription factors may be a common mechanism required for activating this branch of the innate immune system (reviewed in Wiermer et al., 2007). WRKYs are primarily plant-specific transcription factors associated with the transcriptional reprogramming that occurs during the innate immune response (Eulgem and Somssich, 2007). One classical example of this concept can be illustrated by RRS1 controlling broad-spectrum disease resistance in Arabidopsis to Ralstonia solanacearum expressing the effector PopP2 (Table 1). The effector PopP2 contains a bipartite nuclear localization signal and is targeted to plant nuclei. RRS1 contains a fusion of TIRTIR-NB-LRR and WRKY domains (Deslandes et al., 2003). Furthermore, RRS1 possesses a NLS and localizes to the nucleus in a PopP2-dependent manner (Deslandes et al., 2003). These data suggest that, upon activation, RRS1 is able to localize to the nucleus and act as a transcriptional regulator. Four additional studies have highlighted the importance of R protein nuclear localization within the last year for N, MLA,

RPS4, and RX, resulting in a revised model for R protein-mediated defense activation (Burch-Smith et al., 2007; Shen et al., 2007; Tameling and Baulcombe, 2007; Wirthmueller et al., 2007). In this model, R proteins recognize the presence of pathogen effectors in the cytoplasm and function to initiate transcriptional reprogramming of the plant cell in the nucleus. Together, these studies highlight a mechanism in which plants can balance the need for rapid induction of defense responses without the threatening constitutive autoimmune activation. If this concept can be expanded to R proteins in general, this could unravel the activation mechanism for ETI in plants.

MLA The barley Mla resistance locus consists of CC-NB-LRR proteins such as MLA1, MLA6, and MLA10 that recognize isolatespecific effectors from the powdery mildew fungus Blumeria graminis f. sp. hordei (Halterman et al., 2001; Halterman and Wise, 2004; Shen et al., 2003; Zhou et al., 2001). Shen

Table 1. Summary of Cloned R Genes Possessing the Predicted Nuclear Localization Signals (NLSs). R gene

NLS type

R protein class

Species

Reference

RPS2

Monopartite (1)/bipartite (1)

CC-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

RPM1

Monopartite (1)/bipartite (1)

CC-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

RPW8

Bipartite (1)

CC-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

RPP13

Bipartite (2)

CC-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

RGC1

Monopartite (1)/bipartite 1)

CC-NB-LRR

Solanum tuberosum

van der Vossen et al. (2000)

HRT

Monopartite (3)/bipartite (1)

LZ-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

Prf

Monopartite (1)

LZ-NB-LRR

Solanum pimpinellifolium

Reviewed by Martin et al. (2003)

Mi

Monopartite (1)

LZ-NB-LRR

Solanum lycopersicon

Reviewed by Martin et al. (2003)

RPP1

Monopartite (2)/bipartite (3)

TIR-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

RPS5

Monopartite (1)

TIR-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

RPS4

Monopartite (5)/bipartite (2)

TIR-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

RPP5

Monopartite (1)

TIR-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

SNC1

Monopartite (1)

TIR-NB-LRR

Arabidopsis thaliana

Reviewed by Zhang et al. (2003)

RPP4

Monopartite (2)

TIR-NB-LRR

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

N

Monopartite (1)

TIR-NB-LRR

Nicotiana tabacum

Reviewed by Martin et al. (2003)

L

Monopartite (1)

TIR-NB-LRR

Linum usitatissimum

Reviewed by Martin et al. (2003)

M

Monopartite (1)

TIR-NB-LRR

Linum usitatissimum

Reviewed by Martin et al. (2003)

P

Monopartite (5)

TIR-NB-LRR

Linum usitatissimum

Reviewed by Martin et al. (2003)

Bs2

Monopartite (1)

NB-LRR

Capsicum chacoense

Reviewed by Martin et al. (2003)

Xa1

Monopartite (1)

NB-LRR

Oryza Sativa

Reviewed by Martin et al. (2003)

Cf-2

Monopartite (1)

TM-LRR

Solanum pimpinellifolium

Reviewed by Martin et al. (2003)

Cf-4

Monopartite (3)

TM-LRR

Solanum hirsutum

Reviewed by Martin et al. (2003)

Cf-5

Monopartite (1)/bipartite (1)

TM-LRR

Solanum esculentum

Reviewed by Martin et al. (2003)

Cf-9

Monopartite (3)

TM-LRR

Solanum pimpinellifolium

Reviewed by Martin et al. (2003)

RRS1-R

Monopartite (10)/bipartite (3)

TIR-NB-LRR-WRKY

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

Mlo

Monopartite (2)

Novel

Hordeum vulgare

Reviewed by Martin et al. (2003)

RTM2

Bipartite (1)

Jacalin homology

Arabidopsis thaliana

Reviewed by Martin et al. (2003)

CC, coiled-coil domain; NB, nucleotide-binding site; LRR, leucine-rich repeat domain; TIR, TOLL/interleukin 1 receptor; LZ, leucine zipper; TM, transmembrane domain. Amino acid sequences from multiple cloned R genes were subjected to the pSORTII subcellular localization predictor, which can determine the presence of bipartitite and monopartite NLSs (Nakai and Horton, 1999).

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and colleagues (2007) show that MLA-mediated responses require nuclear localization of MLA. Biochemical fractionation of MLA proteins revealed that the majority of the protein resides in the cytoplasm, but a small amount can also be detected in the nucleus (Bieri et al., 2004; Shen et al., 2007). In support of this observation, florescence microscopy analysis of MLA10 tagged with a yellow florescent protein (YFP) co-localized to both the cytoplasm and nucleus in barley epidermal cells. MLA10-YFP-expressing cells were able to mount

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a defense response to B. graminis expressing the effector AvrA10, while MLA10-YFP fused to a nuclear export signal (NES) was unable to accumulate in the nucleus or initiate defense signaling in response to AvrA10. Purification of intact nuclei after pathogen inoculation revealed that nuclear levels of MLA10 increased only in a resistance response. These exciting sets of experiments indicate that nuclear localization of MLA during pathogen infection is required for the manifestation of ETI (Figure 2).

Figure 2. Nuclear Activity of MLA Immune Receptors. Barley MLA receptors recognize and confer resistance to corresponding Blumeria graminis f sp. horei effectors (e.g. MLA10 recognizes AvrA10). Pathogen effector recognition induces nuclear associations between MLA and WRKY transcription factors. Some WRKYs are activators of defense signaling, whereas other WRKYs repress defense signaling. HvWRKY1/2 represses defense signaling and MLA removes HvWRKY1/2 repression. CC, coiled-coil domain; KD, kinase domain; LRR, leucine-rich repeat domain; NB, nucleotide-binding site; TM, transmembrane domain. Figure adapted from Dangl (2007). Reprinted with permission from AAAS.

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Shen and colleagues (2007) were also able to directly link MLA to WRKY transcription factors. Using the conserved CC1–46 domain of MLA, the authors detected a direct interaction with the transcription factors HvWRKY1 and HvWRKY2 by yeast two-hybrid. Moreover, transient overexpression of both WRKY transcription factors compromised ETI mediated by several different MLA alleles. Optimization of the Forster resonance energy transfer measurement enabled them to elegantly detect a specific association between MLA10 and HvWRKY2 in the nucleus only the in presence of AvrA10. HvWRKY1 and HvWRKY2 are closely related transcription factors and members of a small clade within the large WRKY family. They concluded that activated MLA acts to repress HvWRKY1/2, as overexpression of these WRKY transcription factors compromised MLA-specified immune responses (Figure 2). These exciting results indicate that not only does MLA function inside the nucleus, but also directly links this R protein to the interaction with WRKY transcription factors only in the presence of its cognate effector. MLA blocks the repressor activity of HvWRKY1/2, thus enabling rapid defense gene activation in response to pathogen perception (Figure 2). If these findings can be generalized for multiple resistance proteins, the signal transduction cascade linking R protein activation and the transcriptional induction of defense-related genes may be surprisingly short. In addition, the discovery that the R protein MLA can interfere with negative regulators of PTI illustrates that defense signaling mediated by PTI and ETI can be very similar and may only differ with respect to the amplitude of the response.

N Tobacco N is a TIR-NB-LRR R gene that confers resistance to Tobacco mosaic virus (TMV; Whitham et al., 1994). During TMV infection, N recognizes the presence of p50, the 50-kD helicase domain of the TMV replicase (Abbink et al., 2001; Erickson et al., 1999). Using a combination of biochemical cell fractionation, immunoprecipitation and florescence microscopy, Burch-Smith et al. (2007) were able to detect both N and p50 in the cytoplasm and nucleus. Like MLA, N’s nuclear localization is required for its function. Fusing a nuclear export signal onto N inhibited its ability to accumulate in the nucleus and mount a defense response in the presence of p50. However, p50 can elicit N-mediated defense responses even when solely expressed in the cytoplasm. This indicates that N initially recognizes p50 in the cytoplasm, and activated N can subsequently move to the nucleus, initiating defense signaling (Figure 3). N and p50 can associate in planta and N’s TIR domain is required for this association (Burch-Smith et al., 2007). Thus, N’s TIR domain may act as an adaptor, linking two distinct R protein functions: pathogen effector perception and innate immune signaling. What is the function of nuclear and cytoplasmic pools of N? A set of plant-specific transcription factors were previously identified as interacting with N, suggesting that, like MLA, N may also directly link to transcription factors in the nucleus after pathogen recognition (Liu et al., 2004). However, there

has also been a significant amount of work showing the requirement of MAP kinase cascades for N-mediated responses (Jin et al., 2003; Liu et al., 2003). The suppression of all three known components in the MEK2-SIPK/WIPK pathway in tobacco suppresses N gene-mediated TMV resistance. These three components are: MEK2 (upstream MAPKK), WIPK (upstream MAPK), and SIPK (protein kinase; Jin et al., 2003). It is possible that N is able to signal in the cytoplasm through the activation of MAP kinase cascades as well as directly link to transcription factors in the nucleus. Alternatively, transcriptional reprogramming in the nucleus could lead to the induction of MAP kinase signaling cascades in the cytoplasm.

RPS4 RPS4 is a TIR-NB-LRR R gene that confers resistant to P. syringae pv. tomato strain DC3000 expressing the AvrRps4 effector (Gassmann et al., 1999). Using a combination of cell biology and genetic analyses, the majority of RPS4 was shown to associate with endomembranes, with the minority of RPS4 protein localized to the nucleus (Wirthmueller et al., 2007). Furthermore, RPS4’s nuclear accumulation is dependent upon its C-terminal NLS and is required to trigger the immune response (Table 1). The AvrRps4 effector is present in the cytoplasm, indicating that, like N, RPS4 may recognize the AvrRps4 effector outside the nucleus (Wirthmueller et al., 2007). Wirthmeuller and colleagues (2007) also investigated the function of EDS1—a genetic locus present in the nucleus and cytoplasm that is required for RPS4 function. The presence of EDS1 did not influence RPS4 protein levels or localization, indicating that EDS1 functions after RPS4 activation but upstream of RPS4-induced transcriptional reprogramming of the nucleus.

RX Potato RX is a CC-NB-LRR R protein which mediates resistance to Potato virus X by recognizing the viral coat protein (Bendahmane et al., 1995, 1999). RX-mediated resistance is also referred to as extreme resistance, due to the rapid induction of RX during infection and the ability of RX to suppress viral accumulation without exhibiting an HR (Bendahmane et al., 1995, 1999). RX is able to confer extreme resistance to Potato virus X in both potato (Solanum tuberosum) and Nicotiana benthamiana. Using a proteomics approach, RX was implicated in nucleocytoplasmic trafficking through its association with a Ran GTPase activating protein (RanGAP) (Sacco et al., 2007; Tameling and Baulcombe, 2007). S. tuberosum and N. benthamiana RanGAP2 proteins are highly homologous and both proteins can interact with RX’s CC domain. Moreover, silencing RanGap2 abolished RX-mediated resistance. RanGAP proteins regulate the activity of the GTPase Ran, an essential component for nucleo-cytoplasmic trafficking through the nuclear pores (Figure 1) (Meier, 2007). Thus, RanGAP2 association with RX may direct RX into the nucleus. Like MLA and N, RX was also detected in the nucleus (Tameling and Baulcombe, 2007). RX does not have a predicted NLS. Therefore, RX’s interaction with

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Figure 3. A Working Model of N’s Activation and Nuclear Localization. The R protein N recognizes the presence of the Tobacco Mosaic Virus p50 helicase replicase. N’s recognition of p50 likely occurs in the cytoplasm, where p50 associates with N’s TIR domain and the association is bridged by unknown host factor(s). P50’s association with the TIR domain of N induces a conformational change, enabling p50 to bind to both the NB and LRR domains of N, facilitating N’s oligomerization and nuclear import. TIR, Toll-interleukin 1 receptor; NB, nucleotide-binding site; LRR, leucine-rich repeat domain.

RanGAP2 may serve as a signal for protein transport into the nucleus. RanGAP2 may itself serve as a carrier to facilitate the transport of RX into the nucleus or may fold RX in such a manner that it can interact with another carrier protein to facilitate nuclear translocation. RanGAP2 was also found to associate with homologues of RX that share high homology between their CC domains, such as RX2 and Gpa2, which confer resistance to the nematode Globodera pallida and the bacterium Xanthomonas campestris,

respectively (Bendahmane et al., 2000; Sacco et al., 2007; Tameling et al., 2002; van der Vossen et al., 2000). It will be interesting to assess whether these interactions are specific and required for RX2 and Gpa2-mediated resistance. As RX originates from S. tuberosum, future experiments addressing the biological relevance of RanGAP proteins in S. tuberosum during inoculation with Potato Virus X will provide important information about the relevance of this interaction in agronomically important species.

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SNC1 Snc1 (suppressor of npr1-1, constitutive)—a protein containing classical R gene architecture—possesses TIR-NB-LRR motifs. Snc1 was originally identified in a mutant screen and is a constitutively active R gene (Zhang et al., 2003). Two suppressors of the snc1 phenotype—mos3 and mos6 (modifier of snc1)—have highlighted an essential role of nucleo-cytoplasmic trafficking for the defense responses mediated by snc1. MOS3 encodes a protein with high homology to human nucleoporin 96 and a MOS3–green florescent protein fusion localizes to the nuclear envelope, the correct subcellular localization for nucleoporins (Zhang and Li, 2005). Nucleoporins are the main components of the nuclear pore complex and mediate bidirectional nucleo-cytoplasmic transport (Meier, 2007). In addition to compromising defense responses mediated by snc1, mos3 plants were also compromised in basal and effector-triggered immune responses (Zhang and Li, 2005). Mos6 encodes an importin a homolog and partially suppresses the constitutive activation of immune responses in snc1 (Palma et al., 2005). Arabidopsis mos6 mutants suppressed SA accumulation and PR gene expression in snc1 plants. Given the importance of Mos3 and Mos6 for snc1’s constitutive defense activation phenotype, it will be important to carefully assess snc1’s subcellular localization. It is plausible that the point mutation in snc1 may result in constitutive nuclear localization, whereas wildtype SNC1 resides in the cytoplasm.

The Significance of R Protein Nucleo-Cytoplasmic Trafficking How do R proteins get inside the nucleus? MLA, N, and RX all lack the presence of a classical bipartite nuclear localization signal. NB-LRR R proteins are large in size (over 60 kD) and cannot simply diffuse through nuclear pores. Thus, R proteins must be actively transported into the nucleus from the cytoplasm. R proteins could interact with carrier protein(s) that facilitate their targeting to the nuclear pore. Alternatively, these R proteins could possess ‘loose’ NLS or nuclear localization signals that have yet to be discovered. N possesses a monopartite pat4 nuclear localization signal near its C-terminus, which could possibly be the signal for its nuclear localization (Table 1). We hypothesize that, as the field of nuclear localization advances in plant systems, novel NLS sequences will be uncovered which may explain how some R proteins gain entry into the nucleus. In yeast, 43% of known nuclear proteins do not possess a detectable NLS (Lange et al., 2007). Taking this into account, plant R proteins could gain entry into the nucleus in the absence of a discernable NLS, either through their association with ‘carrier’ proteins or by NLS-independent nuclear import pathways. How prevalent is R protein nuclear localization? Do the majority of NB-LRR proteins recognizing diverse pathogens localize to the nucleus to activate defenses? Given the recent discoveries of R protein nucleo-cytoplasmic trafficking, it is possible that this may represent a common mechanism facili-

tating rapid induction of defense responses. The fact that the four R proteins that have been shown to act in the nucleus and are constitutively present in the nucleus at low levels indicates that a key site of action may not be where the majority of the protein is localized. To date, members of both the CC and TIR classes of R proteins require nuclear localization for function. Based on the evidence emerging over the last year, it would be prudent to experimentally re-examine the subcellular localization of characterized R proteins. To determine the prevalence of NLS in existing R genes, we subjected them to the pSORTII subcellular localization predictor (Nakai and Horton, 1999). Multiple R proteins possess NLSs, including both bipartite and monopartite NLSs (Table 1). Undoubtedly, future experiments will assess the prevalence of R protein function inside the nucleus. It is important to note that low levels of N, MLA, RPS4, and RX are present in the nucleus in the absence of pathogen infection. Upon effector perception, R proteins likely change conformation, enabling novel intra-molecular interactions. There may be a threshold level of R protein abundance in the nucleus that is required to initiate effective induction of defense responses. Alternatively, only activated R proteins may be able to interact with nuclear transcription factors.

PARALLELS BETWEEN PLANT AND ANIMAL NB-LRR NUCLEO-CYTOPLASMIC TRAFFICKING In vertebrates, microbial molecules are sensed inside host cells by the innate immune receptors known as NOD-leucine-rich repeats (NOD-LRR), NOD-like receptors (NLRs), NACHT-LRR and CATERPILLER proteins. These vertebrate proteins are structurally similar to plant R proteins; all have the NB-LRR domain structure (Ting and Davis, 2005; Ting et al., 2006). Genetic studies have revealed that mutations in members of the CATERPILLER family are associated with various immunological and inflammatory disorders. CIITA is the founding member of the CATERPILLER family, has recently been implicated in nucleo-cytoplasmic trafficking, and can directly activate transcription factors (reviewed in Ting et al., 2006). CIITA is the master regulator of the major histocompatibility complex class II genes, which play central roles in adaptive immunity and antigen presentation (Zika and Ting, 2005). By either directly or indirectly interacting with DNA-binding transcription factors in the nucleus, CIITA is able to reprogram the transcriptome. The LRR domain is involved in oligomerization, nuclear import, nuclear export, and transcriptional reprogramming (reviewed in Ting et al., 2006). However, the role of CIITA in sensing microbial patterns is unclear. These seminal discoveries indicate that a mammalian CATERPILLER protein moves from the cytoplasm to the nucleus in order to rapidly activate key components of the host defense response. These breakthroughs raise a series of pressing scientific questions about how far we can translate this knowledge to other NB-LRR proteins. Do the LRR domains of plant R

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proteins generally function in oligomerization and nuclear import? Can plant R proteins also act as master regulators of transcription factor families, or is their interaction more specific? We anticipate that the answers to these questions will not only help us to understand if and how other NB-LRR proteins act as transcriptional activators, but could also help to illuminate unsuspected functions.

GENETIC LOCI AND THEIR IMPORTANCE IN NUCLEAR TRAFFICKING Systemic acquired resistance (SAR) is a type of broad-spectrum disease resistance that is induced in response to pathogen infection. Induction of SAR requires the signaling molecules methyl salicylate and salicylic acid (SA). The Arabidopsis NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) is an essential regulator of SA-induced expression of PR genes, which possess antimicrobial activity (Dong, 2004). Activated NPR1 is localized to the nucleus, with NPR1’s NLS required for nuclear import (Kinkema et al., 2000; Mou et al., 2003). In response to SA, NPR1 is translocated into the nucleus, where it acts as a transcriptional regulator, interacting with TGA and WRKY transcription factors to activate the expression of thousands of genes (Wang et al., 2006). In the absence of SA, NPR1 exists in the cytoplasm as an oligomer held together by disulphide bonds. The presence of SA induces a change in the cellular redox potential, resulting in NPR1 monomerization. Monomeric NPR1 is then transported into the nucleus (Kinkema et al., 2000; Mou et al., 2003). NPR1 appears to be constitutively poised for activation with the nuclear membrane serving as the activation barrier, enabling plants to balance the need for rapid induction of defense responses without the negative affects of inappropriate activation. Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and its interacting partner, PHYTOALEXIN DEFICIENT 4 (PAD4), act as a regulatory node controlling aspects of plant innate immunity (Wiermer et al., 2005). EDS1 is primarily required for defense responses mediated by the TIR-NB-LRR class of R proteins, but some CC-NB-LRR proteins also require EDS1. In addition to PAD4, EDS1 can associate with SENESCENCEASSOCIATED GENE101 (SAG101) (Feys et al., 2005). Together, these three proteins are required for the hypersensitive response generated by TIR-NB-LRR R proteins and restricting the growth of virulent pathogens (Feys et al., 2005). Using a combination of biochemical fractionation, immunoprecipitations and florescent resonance energy transfer experiments, the existence of an EDS1–SAG101 complex was detected inside the nucleus that is distinct from the EDS1–PAD4 complex present in both the nucleus and cytoplasm (Feys et al., 2005). These sets of experiments suggest that the changes in the composition and localization of EDS1 complexes in response to pathogen infection may be crucial for activating an appropriate defense response. Like NPR1, EDS1 can be present in either the cytoplasm or nucleus. It is plausible that the subcellular localization of EDS1 may be controlled in a similar manner as

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NPR1. Based on this hypothesis, EDS1 localization may also shift in response to changes in the cellular redox potential, enabling EDS1 nuclear localization.

EFFECTORS AND NUCLEAR LOCALIZATION Plant R proteins can recognize effectors from diverse pathogens. A limited number of viral, bacterial and oomycete effectors have been shown to be nuclear localized. For example, viruses can utilize host replication machinery to proliferate and DNA viruses replicate in the nucleus. Viral proteins required for transcription, replication, and encapsulation typically possess NLSs. In addition, some viral proteins may shuttle between the nucleus and cytoplasm and possess both NLSs and nuclear export signals. For example, the beet necrotic yellow vein virus p25 protein contains both an NLS and a nuclear export signal and exists in the cytoplasm and nucleus (Vetter et al., 2004). The p25 protein can bind importin a and its nuclear localization is required for symptom development (Vetter et al., 2004). Similarly, nuclear import of two oomycete effectors from Phytopthora infestans, Nuk6 and Nuk7, is dependent upon a importins (Kanneganti et al., 2007). Taken together, these data suggest that pathogen effectors can possess host targeting signals, usurp host targeting proteins, and localize to the nucleus. Most characterized effectors are from Gram-negative bacteria, which utilize the Type III secretion system to deliver their effectors into host cells during infection (reviewed in Mudgett, 2005). Bacterial effectors that have been demonstrated to be nuclear localized typically contain one or more NLSs. AvrBs3, a Xanthomonas campestris pv. vesicatoria effector, is the prototypic member of a large family (reviewed in Gurlebeck et al., 2006). After delivery into the plant cell, AvrBs3 dimerizes in the cytoplasm and its NLSs are critical for import into the nucleus (Gurlebeck et al., 2005; Szurek et al., 2002). AvrBs3 family members possess a putative leucine zipper and acidic activation domain, which are hallmarks of transcription factors. Recently, AvrBs3 was shown to act as a plant transcription factor and induces hypertrophy in susceptible genotypes by binding a conserved element in the upa20 promoter, a master regulator of cell size (Kay et al., 2007). In resistant pepper genotypes, the AvrBs3 binds to the promoter of its cognate R gene Bs3 and activates Bs3 transcription in the nucleus (Romer et al., 2007). Taken together, these results indicate that AvrBs3 and likely other members of this family promote bacterial virulence in susceptible genotypes by acting as eukaryotic transcription factors that initiate cellular reprogramming of the plant cell. Conversely, in resistant genotypes, the expression of the cognate Bs3 R gene is induced by the AvrBs3 transcriptional activator.

DISCUSSION Several recent scientific discoveries have forced the field to reevaluate previous paradigms. The new model of plant pathogen

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perception includes R proteins recognizing their cognate pathogen effectors in the cytoplasm. Effector perception may result in altered intra and inter-molecular R protein interactions, including oligomerization. Activated R proteins can then cycle into the nucleus and directly bind transcriptional repressors of innate immunity, resulting in transcriptional reprogramming of the plant cell. In light of R protein nucleo-cytoplasmic trafficking, re-examining the same phenomenon in proteins required for ETI and SAR (e.g. NPR1 and EDS1) indicates that the plant immune system is poised for rapid activation with relatively short signaling pathways. Future studies will determine the extent of R protein nuclear function. A significant number of both TIR-NB-LRR and CC-NBLRR protein models in Arabidopsis contain bipartite or monopartite NLSs (51 and 39 respectively; Shen and Schulze-Lefert, 2007). This indicates that R protein nuclear localization is a common occurrence. It will be important to decipher if immune receptors cycle continuously between the nucleus and cytoplasm, or if nuclear import is unidirectional. If immune receptors continuously cycle through the nucleus, differences between import and export rates could account for the low levels of R proteins found in the nucleus as opposed to the cytoplasm. A mechanism likely exists to limit the abundance of R proteins inside the nucleus in the absence of effector perception. R proteins may interact with diverse transcription factors in the nucleus in order to prevent pathogen disruption of immune signaling. The targets of R proteins in the nucleus will likely become a focus of future research.

NOTE ADDED IN PROOF While this paper was in press, a paper was published (Caplan, J.L., Mamillapalli, P., Burch-Smith, T.M., Czymmek, K., and Dinesh-Kumar, S.P. (2008) Chloroplastic protein NRIP1 mediates innate imune receptor recognition of a viral effector) describing NRIP1, a protein required for N meditated resistance that interacts with both N’s TIR domain and p50 (Figure 3). NRIP1 is normally localized to the chloroplast, but is recruited to the cytoplasm and nucleus by the p50 effector.

FUNDING J. Liu and G. Coaker are supported by start-up funds provided by the University of California, Davis.

ACKNOWLEDGMENTS We thank S. Chisholm for critically reading the manuscript. We apologize to our colleagues whose work we were unable to cite due to page limitations. No conflict of interest declared.

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