du2015

Immune activation mediated by the late blight resistance proteinR1 requires nuclear localization of R1 and the effector AVR1

Yu Du, Jeroen Berg, Francine Govers* and Klaas Bouwmeester*Laboratory of Phytopathology, Wageningen University, Wageningen, the Netherlands

Author for correspondence:Francine GoversTel: +31 317 483138

Email: [email protected]

Received: 18 December 2014Accepted: 3 February 2015

New Phytologist (2015)doi: 10.1111/nph.13355

Key words: effector-triggered immunity(ETI), nucleocytoplasmic partitioning,nucleotide-binding leucine-rich repeat (NLR),Phytophthora infestans, RXLR effector.

Summary

Resistance against oomycete pathogens is mainly governed by intracellular nucleotide-bind-ing leucine-rich repeat (NLR) receptors that recognize matching avirulence (AVR) proteins

from the pathogen, RXLR effectors that are delivered inside host cells. Detailed molecular

understanding of how and where NLR proteins and RXLR effectors interact is essential to

inform the deployment of durable resistance (R) genes. Fluorescent tags, nuclear localization signals (NLSs) and nuclear export signals (NESs) wereexploited to determine the subcellular localization of the potato late blight protein R1 and the

Phytophthora infestans RXLR effector AVR1, and to target these proteins to the nucleus or

cytoplasm. Microscopic imaging revealed that both R1 and AVR1 occurred in the nucleus and cyto-plasm, and were in close proximity. Transient expression of NLS- or NES-tagged R1 and AVR1

in Nicotiana benthamiana showed that activation of the R1-mediated hypersensitive response

and resistance required localization of the R1/AVR1 pair in the nucleus. However, AVR1-

mediated suppression of cell death in the absence of R1 was dependent on localization of

AVR1 in the cytoplasm. Balanced nucleocytoplasmic partitioning of AVR1 seems to be a

prerequisite. Our results show that R1-mediated immunity is activated inside the nucleus with AVR1 inclose proximity and suggest that nucleocytoplasmic transport of R1 and AVR1 is tightly regu-

lated.

Introduction

During evolution, plants have developed two classes of immunereceptors for defence against a wide range of pathogens. The firstclass consists of pattern recognition receptors (PRRs), which arelocalized at the plant plasma membrane and can detect so-calledpathogen-associated molecular patterns (PAMPs). This detectionleads to PAMP-triggered immunity (PTI) which inhibits patho-gen colonization (Chisholm et al., 2006). The second class ofimmune receptors comprises the intracellular resistance (R) pro-teins, which detect pathogen-derived effectors and mediate effec-tor-triggered immunity (ETI) (Hardham & Cahill, 2010). ETI isoften associated with a strong hypersensitive response (HR) at thehost infection site to hamper pathogen colonization. Most intra-cellular R proteins share a central nucleotide-binding (NB)domain and a C-terminal leucine-rich repeat (LRR) domain andare known as nucleotide-binding leucine-rich repeat (NLR) pro-teins. NLR proteins can be divided in two subclasses according totheir N-terminal domain composition (Belkhadir et al., 2004;Tameling & Takken, 2008; Chang et al., 2013). One subclass hasa Toll/interleukin-1 receptor (TIR) domain preceding the NB

domain (T-NLR), while the other has a coiled-coil (CC) domain(C-NLR). These two subclasses probably use different proteincomplexes for downstream immune signalling (Century et al.,1997; Aarts et al., 1998; Feys et al., 2005; Wiermer et al., 2005).Plant NLRs can be in an OFF or ON state (Takken &

Tameling, 2009). When NLRs are in the OFF state, they areautorepressed and folded by a conserved chaperone complexthat contains HSP90 and its cochaperones RAR1 and SGT1(Shen & Schulze-Lefert, 2007; Shirasu, 2009). Upon effectorperception, NLR proteins switch to the ON state and thistriggers nuclear-associated immune responses such as shuttlingof proteins across the nuclear membrane, export of mRNAfrom the nucleus, and activation or repression of transcription(Shen & Schulze-Lefert, 2007; Shen et al., 2007; Elmore et al.,2011; Chang et al., 2013). It thus seems that NLRs are mainlyactive in the nucleus and likely to be nuclear localized. Consis-tent with this, Meyers et al. (2003) found in an in silico analy-sis that 72% of the Arabidopsis NLRs contain a nuclearlocalization signal (NLS) and are predicted to localize to thenucleus. Even several NLRs lacking a predicted NLS sequencewere found to be nuclear localized, for example MLA10 ofbarley (Hordeum vulgare) and Rx of potato (Solanumtuberosum) (Shen et al., 2007; Tameling et al., 2010).*These authors contributed equally to this work.

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Not all NLRs, however, are restricted to the nucleus; some ofthem also localize to the cytoplasm (Elmore et al., 2011). Todetermine if a specific subcellular localization of NLR proteins isrequired for activation of immune signalling, one can perturb thenucleocytoplasmic partitioning of NLRs by artificial modifica-tion with either an NLS that targets the protein to the nucleus ora nuclear export signal (NES) that excludes the protein from thenucleus (Wen et al., 1995; Matsushita et al., 2003). In this way itwas shown that immune responses mediated by the barley pow-dery mildew R protein MLA10 and the tobacco mosaic virus Rprotein N are only activated when these NLRs are localized tothe nucleus, the site where they interact with transcription factorsto regulate defence gene expression (Burch-Smith et al., 2007;Shen et al., 2007; Caplan et al., 2008). Also, the ArabidopsisNLR proteins RPS4 and SNC1 have to be in the nucleus to trig-ger immune responses (Wirthmueller et al., 2007; Cheng et al.,2009). However, some NLRs are activated outside the nucleus.For potato Rx, which confers resistance against potato virus X(PVX), tightly regulated nucleocytoplasmic partitioning is essen-tial for immunity. This partitioning is mediated by RanGAP2which acts as a cytoplasmic retention factor probably via physicalinteraction with Rx (Tameling & Baulcombe, 2007; Slootweget al., 2010; Tameling et al., 2010). Arabidopsis RPM1, an NLRprotein conferring resistance to the bacterium Pseudomonassyringae, activates immunity at the plasma membrane (Gao et al.,2011).

Phytophthora infestans is a filamentous oomycete plant patho-gen that causes the devastating late blight disease of potato andtomato (Solanum lycopersicum). Late blight resistance is governedby NLR proteins. To obtain late blight resistant cultivars, potatobreeders exploit NLR genes from wild Solanum species and theyincreasingly make use of NLR-linked DNA markers to facilitateselection (Carrasco et al., 2009; Vleeshouwers et al., 2011). Morerecently, transgenic and cisgenic approaches based on clonedNLR genes have also been applied (Jo et al., 2014; Jones et al.,2014). The first late blight R gene that was cloned was R1 origi-nating from Solanum demissum. It encodes a C-NLR that specifi-cally recognizes P. infestans isolates carrying the avirulence geneAVR1 (Ballvora et al., 2002; van der Lee et al., 2004). All clonedavirulence (AVR) genes from oomycetes that match with NLR-type R genes in a gene-for-gene manner encode effectors sharingthe four-amino acid host cell targeting motif RXLR at the N-ter-minus. Analyses of the subcellular localization of several of theseso-called RXLR effectors showed that they can be found in vari-ous subcellular compartments (Caillaud et al., 2012a). Eacheffector seems to have its own specific destination, presumably inthe close vicinity of its host target that needs to be modified or(in)activated to suppress host immunity (Caillaud et al., 2012b).Plant NLR proteins must also localize to particular subcellular

compartments in order to perceive their matching pathogeneffectors. A recent study on P. infestans resistance protein R3ashowed that both R3a and its corresponding RXLR effectorAVR3a localize to the host cytoplasm when either one of the twois present. However, when R3a and AVR3a are both presentinside the same cell, they relocalize to endosomal compartments.This relocalization is required for full immunity and only takes

place in the presence of the KI variant, and not the EM variant ofAVR3a. The latter has a virulence function but is not recognizedby R3a as an avirulence effector (Engelhardt et al., 2012). Inanother study it was found that the P. infestans RXLR effectorAVR2 colocalizes with its host target BSL1, a putative phospha-tase, and accumulates around haustoria (Saunders et al., 2012).AVR2 promotes the association of BSL1 with R2, thereby trig-gering HR. However, these authors did not address the subcellu-lar localization of R2. Does R2 also colocalize with AVR2around the haustorium and at what site in the host cell is the ter-nary complex formed? These questions remain to be answered,not only for this R-AVR pair but in general: where does an NLRprotein perceive its matching effector and is colocalization of theNLR protein and effector in the same subcellular compartmentrequired to mediate immunity?In this study, we determined the subcellular localization of the

P. infestans RXLR effector AVR1 and its matching NLR receptorR1. Both appeared to be present in the nucleus as well as thecytoplasm. To perturb their nucleocytoplasmic partitioning, R1and AVR1 were fused to NLS and NES signals and transiently(co-)expressed in Nicotiana benthamiana. The effects of this arti-ficial targeting on R1AVR1 recognition highlighted the impor-tance of proper subcellular localization of both AVR1 and R1 fortriggering R1-mediated resistance. By expressing the modifiedAVR1 genes in the absence of R1, we could also determine inwhich subcellular compartment AVR1 is most active as a viru-lence factor.

Materials and Methods

Plasmid construction

The primer pairs that were used to amplify the full-lengthAVR1 or R1 gene are shown in Supporting Information TableS1. For fusing NLS, mutated NLS (i.e. nls), NES or mutatedNES (i.e. nes) peptides to AVR1 and R1, the forward or reverseprimers were extended with sequences encoding the NLS/nls(Haasen et al., 1999) or NES/nes (Wen et al., 1995) peptides(Table S1). The PCR amplicons were cloned into the pENTR/D-TOPO entry vector (Invitrogen, Life Technologies EuropeBV, Bleiswijk, the Netherlands) and subsequently recombinedinto the binary vector pSOL2094 using Gateway LR recombi-nation. The inserts in the resulting plasmids are shown as car-toons in Fig. S1. Plasmids were transformed into Agrobacteriumtumefaciens strain AGL1. The plasmids used for bimolecular flu-orescence complementation (BiFC) assays were generated byrecombining pENTR clones with vector pCL113 (Yc) orpCL112 (Yn) (Bos et al., 2010), resulting in plasmids Yc-AVR1,Yc-AVR1-like and Yn-R1 (Fig. S1). Plasmid Yc-AVR2 was pro-vided by Petra C. Boevink (James Hutton Institute, Dundee,UK). These plasmids were transformed into A. tumefaciens strainGV3101. Plasmids for yeast two-hybrid assays were generatedby recombining pENTR clones with a bait (pDEST32) or prey(pDEST22) vector using Gateway LR recombination, resultingin plasmids pDEST32:AVR1, pDEST32:AVR1-like andpDEST22:R1.

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Agroinfiltration in Nicotiana benthamiana

Nicotiana benthamiana (Domin) plants were grown in a glass-house under standardized conditions for 5 wk. For agroinfiltra-tion we followed the procedures described by van der Hoornet al. (2000) and Champouret et al. (2009). Agrobacteriumtumefaciens strains harbouring the binary vectors were cultured inyeast extract broth with appropriate antibiotics for 18 h at 28C.The cells were collected by centrifugation, resuspended in agroin-filtration medium and adjusted to the desired concentration. Forcoexpression, A. tumefaciens strains were mixed in a 1 : 1 ratio.Agroinfiltrated plants were kept in a climate chamber at 25Cwith a 12-h photoperiod at 70% relative humidity (RH).

Infection assays

Detached leaf assays were performed as previously described(Vleeshouwers et al., 1999). Zoospores were obtained from 10-d-old cultures of P. infestans isolates 14-3-GFP and 88069 grownon rye sucrose agar medium at 18C. The zoospore suspensionwas adjusted to a concentration of 29 104 ml1. Leaves detachedfrom 5-wk-old N. benthamiana plants were inoculated on theabaxial side with 10-ll droplets of the suspension and incubatedat high humidity at 18C, with the first 24 h in the dark. Lesionsizes were determined at 6 d after inoculation (dai).

Confocal microscopy

At 2 d post infiltration (dpi), patches were cut fromN. benthamiana leaves and used for confocal imaging on a ZeissLSM 510-META 18 confocal laser scanning microscope. Excita-tion of green fluorescent protein (GFP) and mCherry fluores-cence was performed using an argon laser (488 nm) and a HeNe1 laser (543 nm), respectively. Fluorescence was captured by505530 nm (GFP), 514 nm (yellow fluorescent protein (YFP))or 600650 nm (mCherry) filters. LSM software was used fordata processing.

Protein immunoprecipitation and immunoblot analysis

Agroinfiltrated N. benthamiana leaves were ground in liquidnitrogen. To 1 g of ground leaf material, 2 ml of extraction buffer(50 mM Tris-HCl, pH 8, 150 mM NaCl and 1% NP-40, withone complete proteinase inhibitor tablet (Roche DiagnosticsNederland BV, Almere, the Netherlands) per 50 ml) was added.To immunoprecipitate proteins, 1.5 ml of total protein extractwas mixed with 15 ll of GFP-trap_A beads (ChromoTekGmbH, Planegg-Martinsried, Germany) and incubated at 4Cfor 1 h. The beads were collected and washed three times, andwere subsequently boiled for 5 min with loading buffer (300 mMTris-HCl, pH 6.8, 8.7% SDS, 5% b-mercaptoethanol, 30%glycerol and 0.12 mg ml1 bromophenol blue). Protein sampleswere separated on a sodium dodecyl sulphatepolyacrylamidegel electrophoresis (SDS-PAGE) gel and transferred to anImmune-Blot PVDF membrane (Bio-Rad Laboratories BV,Veenendaal, the Netherlands), which was incubated with a-GFP

(anti-GFP-HRP, 130-091-833, MACS antibodies; 1 : 2000 dilu-tion) and subsequently with SuperSignal West Femto MaximumSensitivity substrate (Thermo Scientific, Fisher Scientific, Lands-meer, the Netherlands).

Yeast two-hybrid assays

The prey vector pDEST22:R1 was cotransformed with either theempty bait vector (pDEST32), pDEST32:AVR1, or pDEST32:AVR1-like into yeast strain MaV203. Cotransformants were firstselected on synthetic defined (SD) agar medium lacking theamino acids Trp and Leu (SD-WL) and subsequently grown onHis-deficient SD plates (SD-WLH) supplemented with 25 mM3-amino-1,2,4-triazole (3-AT).

Ion leakage measurements and staining

Ion leakage measurement and trypan blue staining were per-formed as previously described (Bouwmeester et al., 2011, 2014).

Results

R1 localizes to the nucleus and the cytoplasm

In order to determine the subcellular localization of R1, we agro-infiltrated N. benthamiana leaves with the A. tumefaciens strainR1, containing a binary vector encoding the full-length R1 pro-tein with a GFP-tag fused to its N-terminus (construct R1 in Fig.S1). The A. tumefaciens strain mCherry, which contains a binaryvector encoding the free monomeric RFP derivative mCherry,was coagroinfiltrated with R1 and used as a marker to delineatethe nucleus and cytoplasm (Lee et al., 2008). Confocal micros-copy showed that R1 colocalized with mCherry, indicating thatR1 was present in both the nucleus and the cytoplasm (Fig. 1a).To check whether GFP-tagged R1 (Fig. S1) was still functional

as an R protein, strain R1 was coinfiltrated with strain AVR1which contained a binary vector with the AVR1 gene (constructAVR1 in Fig. S1). At 3 dpi, an HR was visible at all infiltratedsites, while coinfiltration with a control strain GFP never resultedin an HR. This showed that the GFP tag did not prevent R1mediating an AVR1-triggered HR (Fig. 1b). To further checkwhether or not there was an effect of the GFP tag on the level ofR1 activity, we compared the HRs mediated by untagged R1 andGFP-R1 at various optical densities (ODs). This showed a slightreduction in activity of the GFP-tagged R1, but in the OD rangethat we used in our assays (i.e. between 0.1 and 0.5) the HR wasclearly distinguishable (Fig. S2a). We also analysed whether theposition of the GFP tag (N-terminal or C-terminal) or the natureof the tag (GFP versus myc or HA) had an effect on R1 activity,but found no obvious differences (Fig. S2b).As the HR is not in all cases correlated to resistance (Heidrich

et al., 2011), we performed infection assays to investigate whetherthe GFP-tagged R1 was still able to confer resistance. Nicotianabenthamiana leaves expressing R1 or GUS were inoculated withP. infestans isolate 14-3-GFP which contains the AVR1 gene.Control leaves expressing GUS were successfully infected while


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leaves expressing R1 showed no lesions (Fig. 1c). Apparently, thepresence of R1 arrested the growth of P. infestans isolate 14-3-GFP, demonstrating that transiently expressed R1 is able tomediate resistance and that the GFP tag does not interfere withthe function of R1 as an R protein.

R1-mediated HR requires nuclear localization

To investigate which subcellular localization of R1 is needed forR1-mediated HR, we artificially targeted GFP-tagged R1 to thenucleus or the cytoplasm by fusing either an NLS (Haasen et al.,1999) or an NES (Wen et al., 1995) to R1 (R1NLS and R1NES,respectively). Constructs with mutated NLS (R1nls) and NES(R1nes) fused to R1 were included as controls. Visualization byconfocal microscopy revealed that the NLS was indeed capable ofefficiently targeting R1 to the nucleus. With NLS-tagged R1, thefluorescence was almost entirely concentrated in the nucleus, incontrast to the controls and NES-tagged R1, which also showedfluorescence in the cytoplasm (Fig. 2a). Moreover, the NES-tagged R1 showed a strong reduction of fluorescence in thenucleus, indicating that the NES fused to R1 was to a certainextent capable of retaining R1 in the cytoplasm. To investigatewhich subcellular localization of R1 is required for the

R1-mediated HR, we coinfiltrated the various R1 strains withAVR1 in N. benthamiana leaves, and monitored cell death 3 dlater. Of the four R1AVR1 combinations, only the one withR1NES showed no HR (Fig. 2b). As a consequence of initial diffi-culties with cloning, R1NES differed from R1NLS, R1nls and R1nes

in having its targeting signal at the N-terminus instead of theC-terminus. Later, when the cloning succeeded, we were ableto show that, irrespective of an NES N-terminal (R1NES) orC-terminal fusion (R1NES*), the results were exactly the same(Fig. S3a). As shown in Fig. 2(a), infiltration with R1NES resultedin strongly reduced levels of R1 in the nucleus, whereas in R1NLS

infiltrated leaves, the majority of the R1 protein ended up in thenucleus. Based on these observations, we conclude that R1 has tobe localized in the nucleus to induce an AVR1-triggered HR.

(a)

(b) (c)

Fig. 1 (a) Confocal microscopy imaging of Nicotiana benthamiana leaveswith transient expression of green fluorescent protein (GFP)-tagged R1shows that R1 localizes to the nucleus and the cytoplasm. R1 wascoinfiltrated withmCherry in a 1 : 1 ratio and at a final optical density (OD)of 0.1. Pictures taken at 2 d post infiltration (dpi) show cells cotransformedwith R1 (green channel; left panel) and mCherry (red channel; middlepanel) and the overlay (right panel). Bar, 10 lm. c, cytoplasm; n, nucleus.(b) GFP-tagged R1 mediates the hypersensitive response (HR) uponcoexpression with AVR1. R1 was coinfiltrated with AVR1 or GFP in a 1 : 1ratio and a final OD of 0.5. Pictures were taken at 3 dpi. (c) GFP-taggedR1 confers resistance to Phytophthora infestans isolate 14-3-GFP. GUSand R1 with an OD of 0.3 were agroinfiltrated in the left and right halvesof the leaf, respectively. At 1 dpi, each half of the leaf was inoculated withP. infestans isolate 14-3-GFP. At 6 d after inoculation, lesion developmentwas monitored and pictures were taken without (left panel) and with(right panel) UV light. The ratios show how many of the inoculated leafhalves developed lesions.

(a)

(b)

Fig. 2 Transient coexpression of AVR1 with nuclear localization signal(NLS)- or nuclear export signal (NES)-tagged R1 in Nicotiana benthamianaleaves shows that nuclear localization of R1 is required for the AVR1-triggered hypersensitive response (HR). (a) Confocal microscopy imagesshowing the subcellular localization of R1 modified with the targetingsignals NES and NLS and the mutant forms nes and nls. The fusionconstructs were agroinfiltrated with an optical density (OD) of 0.1.Pictures were taken at 2 d post infiltration (dpi). Bars, 10 lm. c, cytoplasm;n, nucleus. (b) Cytoplasmic targeted R1 loses the ability to mediate the HRwhen coexpressed with AVR1. Strains with NLS- or NES-tagged R1constructs were coinfiltrated with AVR1 in a 1 : 1 ratio and at a final OD of0.5. Pictures were taken at 3 dpi. The ratios show how many of the totalnumber of infiltrated sites developed an HR at 3 dpi in two independentexperiments.


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To investigate if nuclear localization of R1 is also requiredfor R1-mediated resistance, we performed infection assays onR1 agroinfiltrated leaves. One day after agroinfiltration ofN. benthamiana leaves with a control GFP strain, strain R1 andthe four strains carrying the modified R1 constructs, the leaveswere inoculated with zoospores from P. infestans isolate 14-3-GFP. The control leaves expressing GFP showed expandinglesions at 6 dai. As expected, leaves expressing R1 showed resis-tance to this isolate (Fig. 3a). Lesions were hardly visible, andsimilar results were obtained for leaves expressing the R1 mod-ified versions R1NLS, R1nls and R1nes. By contrast, leavesexpressing R1NES developed expanding lesions similar to thecontrol leaves infiltrated with the GFP construct (Figs 3a, S3b).Quantification showed that c. 70% of the GFP-expressingleaves and 50% of the R1NES-expressing leaves developedlesions, compared with < 11% on leaves in which R1 was tar-geted to the nucleus (R1NLS) (Fig. 3b). These results suggestthat R1 has to be in the nucleus to confer resistance. To deter-mine if the resistance mediated by nuclear localized R1 is spe-cific for AVR1-containing isolates, we also performed infectionassays with a P. infestans isolate that lacks AVR1. Upon inocu-lation with isolate 88069, all the infiltrated leaves developedexpanding lesions irrespective of the site at which R1 waslocalized (Fig. S4).

Taken together, our results show that nuclear localization ofR1 is required not only for initiating an HR triggered by AVR1,but also for arresting growth of P. infestans isolates that containAVR1. This points to a functional connection between R1-medi-ated HR and R1-mediated resistance.

AVR1 localizes to the nucleus and the cytoplasm

To investigate the subcellular localization of AVR1, we con-structed a binary vector encoding AVR1 (without a signal pep-tide) with a GFP tag fused to the N-terminus (Fig. S1) andcoexpressed strain AVR1 with the free mCherry strain inN. benthamiana leaves. Confocal microscopy showed that AVR1colocalized with mCherry, indicating that AVR1 was present inboth the nucleus and the cytoplasm (Fig. 4a). In a similar way weanalysed the subcellular localization of AVR1-like, a close homo-logue of AVR1 that is not recognized by R1, and found that thisRXLR effector also localized to both the nucleus and the cyto-plasm, albeit that the signal in the nucleus was less intense thanfor AVR1 (Fig. 4a). To test if the fluorescence originates fromfull-length GFP-tagged AVR1 and AVR1-like proteins, we iso-lated proteins from the infiltrated leaves and analysed these on animmunoblot. Probing the blot with GFP-antibodies revealedproteins of the expected molecular weights of 45 and 41 kDa,

(a)

(b)

Fig. 3 Transient expression of nuclearlocalization signal (NLS)- and nuclear exportsignal (NES)-tagged R1 in Nicotianabenthamiana leaves shows that nuclearlocalization of R1 is required for R1-mediatedresistance. Strains with NLS- or NES-taggedR1 constructs and GFP and R1 as negativeand positive controls, respectively, wereagroinfiltrated with P19 in a 1 : 1 ratio and ata final optical density (OD) of 0.3. At 1 d postinfiltration (dpi), the leaves were inoculatedwith Phytophthora infestans isolate 14-3-GFP and at 6 d after inoculation (dai) lesionswere scored. The pictures in (a) were takenat 6 dai before (upper panel) and after (lowerpanel) trypan blue staining. The graph in (b)shows the percentage of inoculated sites thatdeveloped expanding lesions. The result isthe average of three biological repeats(n 27). Error bars indicate + SD. Asignificant difference compared with thecontrol is indicated (one-sided Studentst-test: *, P < 0.05).




respectively (Fig. 4b). This showed that both effector proteins arestable in planta. To determine if the GFP-tagged AVR1 was stillfunctional as an AVR protein, we monitored the HR uponcoinfiltration with R1 in N. benthamiana leaves. In contrast toGFP-tagged AVR1-like, GFP-tagged AVR1 was able to triggeran R1-mediated HR (Fig. 4c), demonstrating that the GFP tagdoes not alter AVR1 activity or disturb specificity for R1. To testif the R1-mediated HR is hampered by the GFP tag, we com-pared the activity of untagged AVR1 and GFP-tagged AVR1 inparallel and at various ODs, but observed no differences in speedand strength (Fig. S5a). We also tested the activity of AVR1 pro-teins with either a myc, HA or mCherry tag, or a GFP-tag fusedto the C-terminus and they all triggered an R1-mediated HR(Fig. S5b).

Nuclear localized AVR1 triggers the R1-mediated HR

To test whether a specific subcellular localization of AVR1 isimportant for triggering the R1-mediated HR, AVR1 was artifi-cially targeted to the host cell nucleus or the host cell cytoplasmusing the same targeting signals as described above for R1 (Fig.S1). Confocal microscopy of leaves infiltrated with the variousAVR1 strains showed that AVR1NLS was efficiently targeted tothe nucleus, while AVR1NES was almost completely excludedfrom the nucleus (Fig. 5a). The control constructs AVR1nls andAVR1nes showed the same localization as AVR1-GFP (Fig. 5a).To confirm that the observed fluorescence was indeed derivedfrom the AVR1 fusion proteins, total protein was extracted andsubjected to immunoprecipitation and western blot analysis.Probing the blot with GFP-antibodies showed the presence ofAVR1 fusion proteins of the expected size in all four samples(Fig. 5b).To determine which subcellular localization of AVR1 is

required to trigger the R1-mediated HR, we coinfiltrated the var-ious AVR1 strains with R1 and monitored the HR at 3 dpi. Thisrevealed that the HR was less severe in leaves expressingAVR1NES, with AVR1 largely in the cytoplasm, than in leavesexpressing AVR1NLS, which targets AVR1 solely to the nucleus(Fig. 5c). With AVR1NLS, 70% of the infiltrated sites showed astrong HR. By contrast, with AVR1NES only 35% of the infil-trated sites showed an HR that was overall weaker (Fig. 5c). Thelatter could be attributable to the fact that there was lessAVR1NES produced (Fig. 5b) and therefore we performed coinfil-trations with A. tumefaciens mixtures in which the OD of R1 waskept at 0.5 but the OD of AVR1NES was increased from 0.5 to 1.Even with such a high OD which resulted in higher AVR1NES

protein levels, there was no increase of the HR (Fig. S6). More-over, decreasing the OD of the other AVR1 strains from 0.5 to0.25 did not abolish the strength of the HR, suggesting that smallamounts of AVR1 would also have been sufficient to trigger theHR (Fig. S6a). Unlike AVR1NLS, AVR1NES has the targeting sig-nal at the N-terminus and theoretically this could be anotherreason for the differences observed between the two. To excludethis, we included an extra control, namely construct AVR1nes*,which has the mutated NES peptide fused to the N-terminus ofAVR1 (Fig. S1). Similar to AVR1nes, AVR1nes* triggered an HRwhen coinfiltrated with R1 (Fig. S6a). Apparently the presence ofan extra peptide at the N-terminus of AVR1 does not abolish itsfunction and is probably not the reason that AVR1NES showed aless severe HR than AVR1NLS. Taken together, the severelyreduced HR observed in leaves expressing AVR1NES is mostlikely due to lack of AVR1 in the nucleus, and suggests that R1-mediated HR is only triggered in an efficient manner whenAVR1 is localized in the nucleus.It thus seems that both AVR1 and R1 need to localize to the

nucleus to activate immunity. To further confirm this, all possi-ble combinations of strains containing the (modified) R1 andAVR1 constructs were coinfiltrated (Fig. 6a) and the percentageof infiltrated sites showing HR was determined (Fig. 6b). Thisclearly showed that the HR was only activated when both AVR1and R1 were targeted to the nucleus.

(a)

(b) (c)

Fig. 4 Transient expression of green fluorescent protein (GFP)-taggedAVR1 and AVR1-like in Nicotiana benthamiana leaves shows that AVR1and AVR1-like localize to both the nucleus and the cytoplasm. (a)Confocal microscopy images showing the subcellular localization of AVR1(upper panel) and AVR1-like (lower panel). AVR1 or AVR1-like wascoinfiltrated withmCherry in a 1 : 1 ratio and at a final OD of 0.1. Picturestaken at 2 d post infiltration (dpi) show cells cotransformed with AVR1 orAVR1-like (green channel; left panels) andmCherry (red channel; middlepanels) and the overlay (right panels). Bars, 10 lm. c, cytoplasm; n,nucleus. (b) GFP-tagged AVR1 and AVR1-like proteins accumulate inplanta. AVR1 and AVR1-like were agroinfiltrated at an optical density(OD) of 0.3. At 3 dpi, proteins were extracted and subjected to sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) andimmunoblotting. Incubation with a-GFP shows the accumulation of GFP-tagged proteins. Molecular weight markers are indicated on the right. (c)GFP-tagged AVR1, but not AVR1-like, elicits an HR when coexpressedwith R1. AVR1 and AVR1-like were co-infiltrated with R1 in a 1 : 1 ratioand at a final OD of 0.5. Pictures were taken 3 dpi. Dashed lines indicateagroinfiltrated zones.


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Cytoplasmic localization of AVR1 is required forsuppression of CRN2-induced cell death

RXLR effectors are known to modulate host defence, for exampleby disturbing the function of specific host proteins or by sup-pressing the activity of certain compounds produced by the path-ogen itself (Bos et al., 2010; Wang et al., 2011; Anderson et al.,

2012). The latter include other RXLR effectors or elicitors of celldeath, such as the crinklers CRN1 and CRN2 and the elicitinINF1 which are known to induce cell death in N. benthamiana(Kamoun et al., 1998; Torto et al., 2003). We observed thatAVR1 was able to suppress CRN2-induced cell death inN. benthamiana (Fig. 7). This raised the question of whether ornot this potential virulence function of AVR1 is associated with aspecific subcellular localization of AVR1. To investigate this, wecoexpressed nuclear and cytoplasmic targeted AVR1 with CRN2in N. benthamiana leaves. AVR1-GFP (strain AVR1) and GFPwere used as positive and negative controls, respectively. Asshown in Fig. 7, AVR1NES was able to suppress CRN2-inducedcell death in 40% of cases, similar to results for AVR1-GFP. Bycontrast, AVR1NLS showed no suppression activity, indicatingthat the presence of AVR1 in the cytoplasm is required to sup-press CRN2-induced cell death. With a mutated version of NES(AVR1nes) the behaviour was as expected, namely similar to thatfor AVR1-GFP. However, AVR1nls, which has the mutated ver-sion of NLS, showed no suppression of cell death. It is possiblethat the mutation does not fully abolish NLS function and thatfor CRN2-induced cell death the balance between nuclear andcytoplasmic localized AVR1 is much more critical than for theR1-mediated HR. The latter is a very strong and rapid HR andthe threshold to reach the effect is probably lower.

AVR1 needs nucleocytoplasmic partitioning

When we coexpressed the various modified AVR1 constructswith the silencing suppressor P19 in N. benthamiana leaves, weobserved that high expression of AVR1NES but not AVR1NLS

always resulted in strong necrosis. Similar to AVR1NLS, themutated versions AVR1nls and AVR1nes never showed necrosis

(a) (b)

(c)

Fig. 5 Transient coexpression of R1 with nuclear localization signal (NLS)- or nuclear export signal (NES)-tagged AVR1 in Nicotiana benthamiana leavesshows that nuclear-localized AVR1 can trigger the R1-mediated hypersensitive response (HR). (a) Confocal microscopy images showing the subcellularlocalization of AVR1 modified with targeting signals NES and NLS and their mutant forms nes and nls. Strains carrying the fusion constructs wereagroinfiltrated at an optical density (OD) of 0.1. Pictures were taken at 2 d post infiltration (dpi). Bars, 10 lm. c, cytoplasm; n, nucleus. (b) The AVR1 fusionproteins are stable in planta. The various AVR1 strains were agroinfiltrated at an OD of 0.3. At 3 dpi, proteins were extracted and subjected toimmunoprecipitation and western blot analysis. Incubation of the blot with a-GFP shows the accumulation of the various AVR1 fusion proteins (blackarrow). Molecular weight markers are indicated on the right. (c) Both nuclear and cytoplasmic targeted AVR1 proteins partially lose the ability to elicit theR1-mediated HR. R1 was coinfiltrated with AVR1, AVR1NLS, AVR1nls, AVR1NES or AVR1nes, in a ratio of 1 : 1 and at a final OD of 0.5. Pictures were takenat 3 dpi. The ratios show how many of the total number of infiltrated sites in two independent experiments showed an HR at 3 dpi.

Fig. 6 Transient coexpression of nuclear localization signal (NLS)- ornuclear export signal (NES)-tagged R1 and NLS- or NES-tagged AVR1 inNicotiana benthamiana leaves shows that nuclear localization of both R1and AVR1 is required for the hypersensitive response (HR). The variouscombinations of AVR1 and R1 strains were coinfiltrated in a 1 : 1 ratio andat a final optical density (OD) of 0.5. Pictures were taken at 5 d postinfiltration (dpi). This experiment was repeated three times with similarresults. The ratios show how many of the total number of infiltrated sitesin three independent experiments showed an HR at 3 dpi.




when transiently coexpressed with P19 (Fig. 8a). To confirm thatthe necrosis induced by cytoplasmic localized AVR1 onlyoccurred when coexpressed with P19, we infiltrated GFP orAVR1NES with or without P19 and measured ion leakage at 5 dpias a way to quantify cell death (Fig. 8b). We also confirmed bywestern blot analysis that coexpression with P19 indeed resultedin higher AVR1NES levels (Fig. S7). Taken together, these datashow that the cell death was attributable to a higher accumulationof AVR1NES upon coexpression with P19 and not induced byP19 itself. Because of the fact that cytoplasmic localization ofAVR1 seems to be sufficient for its virulence functions, as shownby the suppression of CRN2-induced cell death, one wouldanticipate that concentrating all AVR1 molecules in the cyto-plasm would be ideal for P. infestans to promote infection. How-ever, the strong necrosis in the AVR1NES infiltrated leavessuggests that during infection AVR1 may also have a role in thenucleus, namely to avoid host cell death. As AVR1-GFP (strainAVR1) without any modification did not trigger necrosis whencoexpressed with P19, it seems that AVR1 requires very balancednucleocytoplasmic partitioning. To further investigate this, wetested if nuclear localized AVR1 was able to suppress the necrosistriggered by solely cytoplasmic localized AVR1. We coinfiltratedP19 and AVR1NES in combination with GFP, AVR1, AVR1NLS orAVR1nls. GFP had no effect and strong necrosis was observed inall cases. However, when AVR1 was targeted to the nucleus itcould entirely suppress the necrosis-inducing activity ofAVR1NES (Fig. 8c), suggesting that nuclear-localized AVR1 has adominant negative effect on cell death triggered by cytoplasmic-localized AVR1.

R1 and AVR1 are in close proximity in planta

As R1 and AVR1 both localize to the nucleus and cytoplasm andare both required in the nucleus for mounting an R1-mediated

HR and resistance, we hypothesized that the two proteins are inclose proximity. To investigate this, we performed BiFC assays(Bos et al., 2010). We used BiFC constructs in which the N-ter-minal portion of YFP is fused to the N-terminus of R1 (Yn-R1)and the C-terminal portion of YFP to the N-terminus of AVR1,AVR1-like or AVR2 (Yc-AVR1, Yc-AVR1-like and Yc-AVR2,respectively) (Fig. S1). Coexpression of Yn-R1 and Yc-AVR1 inN. benthamiana leaves resulted in an HR, demonstrating that theconstructs were functional (Fig. S8). These HR patches, however,are not suitable for microscopy so to avoid HR we used SGT1-silenced plants for our BiFC assays. SGT1 is a cochaperone thatfunctions in NLR-mediated immunity (Azevedo et al., 2002) andwe observed that, indeed, the R1-AVR1-mediated HR was

Fig. 7 Cytoplasmic localization enables AVR1 to suppress CRN2-inducedcell death. Strains with nuclear localization signal (NLS)- or nuclear exportsignal (NES)-tagged AVR1 constructs, and GFP and AVR1 as negative andpositive controls, respectively, were coagroinfiltrated with CRN2 inNicotiana benthamiana leaves in a 2 : 1 ratio and at a final OD of 0.6 forAVR1 and 0.3 for CRN2. Pictures were taken at 6 d post infiltration (dpi).The y-axis shows the average percentage of infiltrated sites showingCRN2-induced cell death at 6 dpi and based on three independentexperiments (n 29). Error bars indicate + SD.

(a)

(b)

(c)

Fig. 8 AVR1 needs balanced nucleocytoplasmic partitioning for properfunctionality. (a, b) AVR1NES triggers cell death when coexpressed withP19. Strains with nuclear localization signal (NLS)- or nuclear export signal(NES)-tagged AVR1 constructs were coagroinfiltrated with P19 inNicotiana benthamiana leaves in a 1 : 1 ratio and at a final optical density(OD) of 0.3. Pictures in (a) were taken at 5 d post infiltration (dpi). Theratios show how many of the total number of infiltrated sites developedthe hypersensitive response (HR). (b) Quantification of cell death triggeredby AVR1NES in the presence (1 : 1 ratio; final OD 0.3) or absence (OD 0.3)of P19. GFP was included as a control. The graph shows relative ionleakage values measured at 5 dpi. This experiment was repeated at leasttwo times with similar results. Error bars indicate + SD. (c) Nuclear localizedAVR1 suppresses AVR1NES-triggered cell death. AVR1NES and P19 wereinfiltrated in N. benthamiana leaves together with GFP, AVR1NLS, AVR1nls

or AVR1 in a 1 : 1 : 1 ratio and at a final OD of 0.3. Pictures were taken at5 dpi. The ratios show how many of the total number of infiltrated sites inthree independent experiments developed an HR.


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NewPhytologist8

compromised in SGT1-silenced plants (Fig. S8). We thenchecked whether the subcellular localization of R1 changed whenSGT1 was absent. Confocal microscopy of leaves of SGT1-silenced N. benthamiana plants that transiently expressed GFP-tagged R1 showed that also in these leaves R1 was distributedover the nucleus and cytoplasm (Fig. S9), indicating that dimin-ishing SGT1 does not change the subcellular localization of R1.Upon infiltration of strains carrying BiFC constructs, no fluores-cence was observed when Yn-R1 was coexpressed with Yc-AVR1-like or Yc-AVR2 (Fig. 9). By contrast, when coexpressed withYc-AVR1, strong fluorescence was detected (Fig. 9), indicatingthat R1 and AVR1 were in close proximity. Additional BiFCassays included NES-tagged versions of either R1 or AVR1 andhere we also observed fluorescence when coexpressing Yn-R1with Yc-AVR1NES or Yn-R1NES with Yc-AVR1 (Fig. S10).Moreover, we performed yeast two-hybrid assays to investigatethe potential of a physical interaction between R1 and AVR1 butfound no evidence for a direct interaction (Fig. S11).

Discussion

In the 2002 paper that described the cloning of the first lateblight resistance gene, R1 was suggested to be anchored to theplasma membrane based on in silico prediction of four myristoy-lation sites (Ballvora et al., 2002). In the present study, we visual-ized the subcellular localization of R1 and its matching effector

AVR1 by confocal microscopy and exploited subcellular targetingmotifs to manipulate their localization, but found no evidencefor R1 being localized to the plasma membrane. Instead, wefound that both R1 and AVR1 localized to the nucleus as well asthe cytoplasm and were in close proximity. We also found thatimmune activation (or ETI) required nuclear localization of thetwo matching partners, while immune suppression was mosteffective when AVR1 was in the cytoplasm. The latter was mea-sured by suppression of CRN2-induced cell death and in theabsence of R1. ETI was monitored by determining the HR uponcoexpression of R1 and AVR1 as well as by measuring lesiongrowth on leaves transiently expressing R1 and inoculated with aP. infestans isolate that has the AVR1 gene (R1-mediated resis-tance).The nucleocytoplasmic partitioning pattern that we observed

for R1 was similar to that reported for several other plant NLRproteins (Shen et al., 2007; Tameling et al., 2010; Hoser et al.,2013; Inoue et al., 2013). However, the nuclear localization ofR1 was not foreseen. According to a prediction by NLStradamus(Nguyen Ba et al., 2009; http://www.moseslab.csb.utoronto.ca/NLStradamus/; cut-off value of 0.3), R1 lacks a significant NLSand it is a large protein (over 150 kDa) that cannot move by pas-sive diffusion through the nuclear pores. Some nuclear-localizedNLRs, for example Arabidopsis RPS4 and SNC1 and tobacco N,contain an NLS in their sequence, but others, like barley MLA10and potato Rx, do not (Burch-Smith et al., 2007; Shen et al.,

Fig. 9 Bimolecular fluorescencecomplementation (BiFC) shows that R1 andAVR1 are in close proximity. Confocalmicroscopy images of leaves from SGT1-silenced Nicotiana benthamiana plants inwhich Yn-R1 was coexpressed with Yc-AVR1, Yc-AVR1-like or Yc-AVR2, asindicated, and combined with P19 to boostexpression are shown. Pictures were taken at2 d post infiltration. Agrobacteriumtumefaciens strains harbouring the BiFCconstructs and P19 were infiltrated in a1 : 1 : 1 ratio and at a final optical density(OD) of 0.2. YFP fluorescence is shown onthe left, bright field in the middle and theoverlay on the right. Bars, 10 lm. c,cytoplasm; n, nucleus.




2007; Wirthmueller et al., 2007; Cheng et al., 2009; Tamelinget al., 2010). How R1 enters the nucleus is currently unknown.For MLA10 and Rx, it has been found that they do not needan NLS to be imported into the nucleus. Rx uses other meansto translocate between the cytoplasm and nucleus (Tamelinget al., 2010). In fact, Rx has to move in and out of the nucleusbecause it requires balanced nucleocytoplasmic partitioning foractivation of immunity (Tameling et al., 2010). MLA10 alsoshuttles between the nucleus and cytoplasm, where it seems tofulfil distinct functions (Bai et al., 2012). Similar to R1, alsoRPS4, SNC1, N and MLA10 were found to activate immunityinside the nucleus. Here, these NLRs may bind to transcrip-tion factors and in this way regulate defence gene expression,as was shown for tobacco N and barley MLA10 (Burch-Smithet al., 2007; Shen et al., 2007; Padmanabhan et al., 2013; Pad-manabhan & Dinesh-Kumar, 2014) and also, among others,for the Arabidopsis resistance proteins RCY1 and RRS1-R.The CC domain of RCY1, an NLR protein conferring resis-tance to cucumber mosaic virus (CMV), interacts with aWRKY70 transcription factor that plays an important rolein suppression of CMV multiplication (Ando et al., 2014).RRS1-R, a NLR protein active against bacterial wilt, containsa C-terminal WRKY DNA-binding domain and functionsinside the nucleus (Bernoux et al., 2008).To show that the fluorescence patterns that we observed by

confocal microscopy are indicative of localization of R1 or AVR1and not attributable to free GFP cleaved from the fusion pro-teins, we isolated proteins from agroinfiltrated leaves for westernblot analysis. In this way we could detect the AVR1 fusion pro-teins (Figs 5b, S6) but were unable to visualize the R1 fusion pro-teins. Apparently, the R1 fusion proteins, which are relativelylarge, are unstable under the protein extraction conditions thatwe used. Others working with NLR proteins have had similarexperiences. Lukasik-Shreepaathy et al. (2012), for example,showed that truncated versions of the NLR protein Mi accumu-late at high levels, as is the case with truncated R1 versions in ourhands, but full-length Mi protein was hardly detectable. In con-trast, Engelhardt et al. (2012) were able to visualize the NLR pro-tein R3a on western blots, but in that case the tagged R3a hadlost the ability to mediate an Avr3a-triggered HR. The same wasfound for the tomato NLR protein I-2, the tagged version ofwhich had lost its functionality (Tameling et al., 2002; Ma et al.,2013). It could be that accumulation of tagged NLR proteins ishampered when they retain their function but not in cases wherean artificial tag abolishes the functionality. Despite the fact thatwe cannot visualize the R1 fusion proteins by western blot analy-sis, we have ample evidence that the R1 fusion proteins are pro-duced as full-length proteins and as such present in planta. Firstof all, all R1 fusion proteins, with the exception of R1NES, triggerthe HR in the presence of AVR1, demonstrating that the fusionproteins are produced and functional. There is no reason toassume that R1NES would behave differently when it comes totranscription and translation. Secondly, we did observe differ-ences in GFP fluorescence patterns between the various R1 fusionconstructs, whereas with free GFP all the patterns would be thesame. The fusion genes used in our assays are constructed in such

a way that GFP is fused to the N-terminal end of R1 and the tar-geting signal to the C-terminal end. As free GFP localizes to boththe nucleus and the cytoplasm, we are quite sure that thenucleus-specific signals that we observed, for example in the caseof R1NLS, represent the full-length protein. Moreover, confocalimaging showed that the localization of R1 with NES fused toeither the N-terminus or the C-terminus of R1 was the same andclearly distinct from the pattern obtained with R1NLS. As a finalargument for R1NES being produced in planta, we refer to thefact that in BiFC assays complementation was observed when co-expressing Yn-R1NES with Yc-AVR1.The complementation of YFP fluorescence in the BiFC assay

showed that R1 and AVR1 were in close proximity but evidencefor physical interaction between the two is lacking. For only afew RAVR pairs there is solid evidence for direct proteinpro-tein interaction, none of which involves interaction of an oomyc-ete RXLR avirulence effector with its matching NLR protein.RXLR effectors are in general small proteins that might be ableto passively diffuse into the nucleus through nuclear pores.Mature AVR1 is 21 kDa in size and thus below the threshold of40 kDa for passive diffusion in and out of the nucleus (Marforiet al., 2011). However, with the GFP tag fused to AVR1, thefusion protein of 45 kDa just exceeds the threshold for passivediffusion and as AVR1 has no significant NLS (according to aprediction by NLStradamus using a cut-off value of 0.3) theremight be some kind of carrier protein that mediates transportacross the membrane. To date there are only a few studies inwhich the localization of an RXLR effector was linked to its bio-logical function. The plasma membrane localization ofPhytophthora sojae Avh241 and P. infestans AVR2 was found tobe critical for inducing plant cell death or triggering the HR(Saunders et al., 2012; Yu et al., 2012). Phytophthora infestansRXLR effector PITG_03192 promotes infection when localizedto the host ER membrane where it binds two NAC transcriptionfactors to prevent relocalization into the nucleus (McLellan et al.,2013). Avr3a localizes to both the nucleus and the cytoplasmwhen expressed alone, but upon coexpression with R3a the twotogether relocalize to vesicles. Inhibition of this relocalization byWortmannin and Brefeldin A abolishes the R3a/AVR3a-medi-ated HR (Engelhardt et al., 2012), indicating that relocalizationof this R/AVR protein complex is essential for mediating immu-nity. In this study, we have found that cytoplasmic localization ofAVR1 is required for suppression of CRN2-induced cell death.However, when there is an overload of cytoplasmic AVR1 thisleads to spontaneous cell death. It thus seems that balanced nu-cleocytoplasmic partitioning of AVR1 is needed for its biologicalfunctions, as this keeps AVR1 accumulation in the cytoplasm at arelative low level to avoid spontaneous cell death. It is also possi-ble that AVR1, in addition to suppressing cell death in the cyto-plasm, has a function as a virulence effector in the nucleus. Inthis regard it is interesting to note that there are two transcriptionfactors among the putative host targets identified for AVR1 in ayeast two-hybrid screen (data not shown). In either case it wouldimply that transport of AVR1 between cell compartments is inone way or another regulated and not dependent on passive diffu-sion.


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Similar to AVR1, the fungal avirulence factor AVR2 fromFusarium oxysporum and the bacterial avirulence factors PopP2from Ralstonia solanacearum and AvrRPS4 from Pseudomonassyringae have to be present in the nucleus to activate NLR-medi-ated resistance (Bernoux et al., 2008; Heidrich et al., 2011; Maet al., 2013). In the case of the tobacco mosaic virus protein p50,however, the N-mediated HR in N. benthamiana is triggeredwhen p50 is solely localized either to the cytoplasm or to thenucleus (Burch-Smith et al., 2007). This shows that perceptionof pathogen effectors by matching NLR proteins can happen inboth the host nucleus and cytoplasm. Our study demonstratesthat, despite the nucleocytoplasmic partitioning of AVR1 andR1, nuclear localization of the pair is required for the R1-medi-ated HR. However, we cannot exclude the possibility that alreadyin the cytoplasm AVR1 is recognized by R1 as its matching aviru-lence factor, while immunity is only activated in the nucleus.Thus far it is not known how recognition in the cytoplasm iscommunicated to the nucleus. Hence, one of the next logicalsteps would be to determine what happens to R1 when it encoun-ters AVR1 in the cytoplasm. Does it translocate to the nucleusand, if so, is there a role for AVR1 in the actual translocation ordo they translocate together?

Acknowledgements

We acknowledge Patrick Smit, Petra C. Boevink and TsuyoshiNakagawa for providing plasmids, Harold Meijer for discussion,and Bert Essenstam and Henk Smit at Unifarm for excellentplant care. This research was supported by a fellowship of theChina Scholarship Council (CSC) to Y.D., an STW-VENI grantfrom the Netherlands Organization for Scientific Research toK.B. and the Food-for-Thought campaign from the WageningenUniversity Fund.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.


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Fig. S1 Cartoons showing the constructs used in this study.

Fig. S2 Coexpression of AVR1 with tagged versions of R1 inNicotiana benthamiana leaves shows that the tags do not interferewith the ability of R1 to trigger the AVR1-mediated hypersensi-tive response (HR).

Fig. S3 Cytoplasmic targeted R1 loses its function as an immunereceptor.

Fig. S4 Nicotiana benthamiana leaves expressing R1 do not con-fer resistance to an AVR1-deficient Phytophthora infestans isolate.

Fig. S5 Coexpression of R1 with tagged versions of AVR1 inNicotiana benthamiana leaves shows that the tags do not interferewith the ability of AVR1 to trigger the R1-mediated hypersensi-tive response (HR).

Fig. S6 Cytoplasmic targeted AVR1 loses the ability to triggerthe R1-mediated hypersensitive response (HR).

Fig. S7 Coexpression in Nicotiana benthamiana of cytoplasmictargeted AVR1 with the silencing suppressor P19 results in

increased levels of the AVR1NES protein and causes spontaneouscell death.

Fig. S8 Coexpression of Yn-R1 and Yc-AVR1 in Nicotianabenthamiana results in a hypersensitive response (HR) that isdependent on SGT1.

Fig. S9 Localization of R1 is independent of SGT1.

Fig. S10 Bimolecular fluorescence complementation using anuclear export signal (NES)-tagged version of AVR1 with R1and an NES-tagged version of R1 with AVR1 confirms that R1and AVR1 are in close proximity.

Fig. S11 No evidence for physical interaction between R1 andAVR1 in yeast two-hybrid assays.

Table S1 Primers used in this study

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