Nematode effectors suppress plant innate immunity

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by NB-LRR proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of Globodera rostochiensis physically associates in planta with the leucine-rich repeat (LRR) domain of a member of the SW5 resistance gene cluster in tomato ( Solanum lycopersicum ). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis . By contrast, agroinfiltration assays showed that the co-expression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several CC-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato Virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato. The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants.


INTRODUCTION
The survival and reproduction of the potato cyst nematode Globodera rostochiensis relies on the successful establishment and maintenance of a feeding site inside the root of a host plant. Secretions produced by sedentary plant-parasitic nematodes such as G. rostochiensis are thought to be instrumental in the formation of the feeding site (Haegeman et al., 2012).
The nematodes use an oral stylet to deliver these secretions into the apoplast and the cytoplasm of host cells (Hussey, 1989;Davis et al., 2008). In a susceptible host plant, a recipient host cell may respond by increasing its metabolic activity and by progressing through several cycles of endoreduplication. The concomitant local cell wall degradation and subsequent fusion with neighboring protoplasts transform the infected host cells into a multinucleate syncytium (Sobczak et al., 2009). Freshly-hatched infective juveniles of G.
rostochiensis are mobile, but as soon as feeding on the syncytium commences they lose their body wall muscles and adopt a sedentary lifestyle (De Boer et al., 1992). The syncytium functions as a metabolic sink that transfers plant assimilates from the conductive tissues in the vascular cylinder to the sedentary nematode (Jones and Northcote, 1972). A failure in syncytium formation caused, for example, by host defense responses prevents development of the feeding nematode into its reproductive stage (Sobczak et al., 2009).
The majority of plant resistance proteins are members of the NB-LRR receptor family, which consist of a central nucleotide-binding (NB) domain and a leucine-rich repeat (LRR) domain at the carboxyl terminus (Eitas and Dangl, 2010). At their amino-termini, the NB-LRR plant immune receptors either carry a coiled-coiled (CC) domain, or a Toll/Interleukin-1 receptor like (TIR) domain. The NB domain, which is also referred to as the NB-ARC (nucleotidebinding adaptor shared by APAF-1, certain Resistance proteins, and CED-4) domain, most likely changes from a closed ADP bound state to an open ATP bound state when the resistance protein detects a pathogen (Lukasik and Takken, 2009). The LRR domain is thought to act as the sensor in NB-LRR receptors, which in the absence of the cognate effector keeps the resistance protein in an autoinhibited "off" state. In this model, the recognition of a pathogen effector induces a conformational change in the LRR domain that lifts the inhibition of the NB domain in the core of the resistance protein. Artificially induced mutations in NB-LRR immune receptors suggest that the two functions of the LRR domain, pathogen recognition and negative regulation of the NB domain, reside in different parts of the domain. Several sequence exchanges and deletions at the N-terminus of the LRR domain switch NB-LRR immune receptors into a permanent effector-independent autoactive state (Rairdan and Moffett, 2006). By contrast, mutations in repeats at the C-terminus of the LRR domain do not lift the autoinhibition, but instead change the recognition specificity of NB-LRR immune receptors (Farnham and Baulcombe, 2006). The molecular mechanisms underlying effector recognition by plant immune receptors are not well understood. NB-LRR immune receptors may activate signaling pathways that lead to effector-triggered immunity when they physically associate with their cognate effectors (Krasileva et al., 2010). However, the fact that such direct interactions seem to be exceptional inspired the formulation of the 'guard' model in which immune receptors activate host defenses by detecting effector-induced perturbations in other plant proteins (Van Der Biezen and Jones, 1998). Plant immune receptors may thus efficiently expand the spectrum of disease resistances of a plant by guarding common virulence targets of multiple effectors (Chung et al., 2011). In the recently proposed intermediate 'bait-and-switch' model a pathogen effector may still directly interact with NB-LRR immune receptors but only after binding to an accessory protein that functions as co-factor for the receptor (Collier and Moffett, 2009).
There are only few examples of plant immune receptors that directly interact with their cognate pathogen effector (Jia et al., 2000;Deslandes et al., 2003;Ellis et al., 2008;Krasileva et al., 2010;Tasset et al., 2010;Chen et al., 2012). For only three of these resistance proteins a physical association with the effector was demonstrated in planta. The TIR-NB-LRR resistance protein RPP1 of Arabidopsis thaliana associates via its LRR domain with the effector ATR1 of Peronospora parasitica (Krasileva et al., 2010). This interaction results in a defense-related programmed cell death in leaves of Nicotiana tabacum. Also in Arabidopsis, the association of the TIR-NB-LRR resistance protein RRS1-R with the PopP2 effector of Ralstonia solanacearum results in immunity (Tasset et al., 2010). Similarly, the physical association of the CC domain of the resistance protein RB from potato with the IPI-O1 effector of P. infestans triggers a programmed cell death in N. benthamiana (Chen et al., 2012). Recently, we found that the effector SPRYSEC-19 of G. rostochiensis interacts in yeast with the seven C-terminal repeats of the LRR domain of the CC-NB-LRR protein SW5F of tomato (Rehman et al., 2009). The SW5 resistance gene cluster in tomato confers resistance to a broad range of tospoviruses (Boiteux and de Giordano, 1993). Five other SW5 resistance gene homologs have been identified in tomato. The homolog SW5B confers resistance to tomato spotted wilt virus (TSWV), whereas the functions of SW5A and SW5C-F are currently unknown (Spassova et al., 2001).
SPRYSEC effectors are produced as secretory proteins in the dorsal esophageal gland of G. rostochiensis that is connected via the lumen of the esophagus to the oral stylet (Rehman et al., 2009). They only consist of a SPRY/B30.2 domain, which in many different eukaryotic proteins is involved in intermolecular interactions (Rhodes et al., 2005;Tae et al., 2009). The expression of the SPRYSEC effectors in G. rostochiensis is highly upregulated in infective juveniles and during the first few days post invasion. The function of the SPRYSEC effectors in plant parasitism is not well understood. It has been shown that the coexpression of the SPRYSEC GpRBP1 from G. pallida and the CC-NB-LRR resistance protein Gpa2 from potato induces a programmed cell death in leaves of N. benthamiana (Sacco et al., 2009). This finding suggests that GpRBP1 triggers Gpa2-mediated nematode resistance. However, since both virulent and avirulent G. pallida populations harbor GpRBP1, its role in nematode resistance remains to be shown. Furthermore, it is also not clear if the Gpa2-mediated programmed cell death requires a physical association between Gpa2 and GpRBP1.
In this paper we report the functional characterization of the effector SPRYSEC-19 of G. rostochiensis, and its interaction with SW5F, in plants. We first tested the hypothesis that SPRYSEC-19 activates SW5F-dependent programmed cell death and nematode resistance.
However, co-expression of SPRYSEC-19 and SW5F by agroinfiltration in leaves of N.
benthamiana and in tomato did not trigger a defense-related programmed cell death.
Moreover, nematode infection assays on tomato plants harboring SW5F showed no resistance to G. rostochiensis. Next, we tested the alternative hypothesis that SPRYSEC-19 modulates host defense responses in plants. Our data demonstrated that SPRYSEC-19 selectively suppresses CC-NB-LRR-mediated programmed cell death and disease resistance.

SPRYSEC-19 does not trigger an SW5F-mediated programmed cell death
Previously, we showed that the effector SPRYSEC-19 of G. rostochiensis interacts with a Cterminal fragment of the LRR domain of SW5F (SW5F-LRR7-13) in a yeast-two-hybrid screen on tomato root cDNA (Rehman et al., 2009). An in vitro pull-down assay confirmed that SPRYSEC-19 and SW5F-LRR can interact without cofactors (Rehman et al., 2009). This specific association of SPRYSEC-19 and SW5F was confirmed in planta by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitations (CoIP) (Supplemental Fig. S1). The only other known physical association of a pathogen effector and the LRR domain of a resistance protein in planta triggers a defense-related programmed cell death in N. tabacum leaves (Krasileva et al., 2010). We expected that co-expression of SPRYSEC-19 and SW5F would also trigger a cell death response in agroinfiltrated leaves of N.
benthamiana. However, no local cell death was observed within 10 days after transient overexpression of SW5F with either 4MYC-tagged SPRYSEC-19 or untagged SPRYSEC-19 (Supplemental Fig. S2). The fragment of SW5F (SW5F-LRR7-13) that interacted with SPRYSEC-19 in the yeast-two-hybrid screen derived from the near-isogenic line CGR161 of S. lycopersicum cultivar MoneyMaker. We reasoned that other close homologs of SW5F either in CGR161 or in the parent cultivar MoneyMaker might be able to mediate a SPRYSEC-19-triggered cell death in N. benthamiana. A PCR using SW5F specific primers resulted in the identification of three SW5F homologues (Supplemental Fig. S3). Transient co-expression of none of the SW5F homologues with either SPRYSEC-19 (Fig. 1A) 2). Not all functional disease resistance proteins trigger a local cell death at the infection site of avirulent pathogens (Bendahmane et al., 1999;Bulgarelli et al., 2010), and SW5F might therefore still confer resistance to G. rostochiensis in tomato. To test whether SW5F mediates resistance to the population of G. rostochiensis from which SPRYSEC-19 was isolated (Ro1 line 19), we inoculated 7 days old seedlings of the tomato cultivar from which SW5F was cloned (i.e. MoneyMaker) with infective second juveniles. Three weeks postinoculation on average 29 (S.E.M ±1.1) juveniles per tomato plant developed into the adult female stage, which is consistent with a normal susceptibility to G. rostochiensis in tomato (Sobczak et al., 2005).

SPRYSEC-19 suppresses programmed cell death mediated by an SW5 homolog in N. benthamiana leaves
Next, we reasoned that SPRYSEC-19 interacts with SW5F to suppress effector-triggered activation of SW5F-mediated immune signaling. The SW5F gene has not been linked to a particular disease resistance trait, and by consequence the elicitor of the pathogen that might activate SW5F-mediated signaling is also not known. The TSWV resistance mediated by SW5B is currently the only phenotype linked to the SW5 cluster in tomato. However, the elicitor of the virus that activates SW5B has not been identified either. To be able to test if SPRYSEC-19 suppresses SW5-mediated programmed cell death, we introduced a D-to-V mutation at position 879 in SW5F and at position 857 in SW5B to make the proteins autoactive (Bendahmane et al., 2002;De La Fuente Van Bentem et al., 2005;Tameling et al., 2006;Van Ooijen et al., 2008). Only the expression of SW5B-D857V resulted in an effector-independent cell death response following agroinfiltration of N. benthamiana leaves (Fig. 3A). Co-expression of 4MYC-SPRYSEC-19 suppressed the effector-independent cell death response mediated by the SW5B-D857V mutant protein in agroinfiltrated leaves of N. benthamiana (Fig. 3B). This outcome suggested that SPRYSEC-19 suppresses SW5Bmediated activation of effector-triggered immunity.

N. benthamiana leaves
Next, we investigated whether SPRYSEC-19 also suppresses the programmed cell death mediated by other CC-NB-LRR resistance proteins. The SPRYSEC effector GpRBP-1 of the white potato cyst nematode G. pallida triggers a Gpa2-mediated cell death in N.
benthamiana (Sacco et al., 2009). To investigate a possible SPRYSEC-19 controlled suppression of Gpa2-mediated programmed cell death, we co-expressed 4MYC-SPRYSEC-19 together with GpRBP-1 and Gpa2 by agroinfiltration in leaves of N. benthamiana. GpRBP-1 transiently expressed with Gpa2 and 4MYC-GFP triggered a strong cell death response in the infiltrated leaf areas within 4-7 days post infiltration. By contrast, no local cell death was observed following the co-expression of GpRBP-1, Gpa2, and 4MYC-SPRYSEC-19 in N.
benthamiana. We therefore concluded that SPRYSEC-19 suppressed elicitor dependent programmed cell death mediated by Gpa2. Gpa2 is highly similar to the virus resistance protein Rx1 that recognizes the coat protein of the avirulent PVX strain UK106 (Cp106) (Bendahmane et al., 1995). Cp106 shares no sequence similarity with GpRBP-1 or with other SPRYSEC effectors. We used the Rx1-mediated cell death response in N. benthamiana to investigate whether SPRYSEC-19 suppresses the action of a homologous CC-NB-LRR protein that is not triggered by a SPRYSEC. As expected, co-expression of Rx1, Cp106, and 4MYC-GFP resulted in a local cell death response in agroinfiltrated leaf areas of N. benthamiana (Fig. 4). By contrast, replacing 4MYC-GFP with 4MYC-SPRYSEC-19 completely abrogated the Rx1/Cp106-triggered cell death response in N. benthamiana leaves. SPRYSEC-19 of G. rostochiensis thus also suppresses programmed cell death mediated by the CC-NB-LRR resistance proteins Gpa2 and Rx1.
We also co-expressed SPRYSEC-19 with R3a (Huang et al., 2005)  benthamiana leaves, which was not suppressed by SPRYSEC-19 (Fig. 4). Altogether, our data demonstrated that SPRYSEC-19 suppresses the programmed cell death mediated by a group of closely related CC-NB-LRR resistance proteins.

SPRYSEC-19 suppresses disease resistance mediated by Rx1
The local cell death mediated by resistance proteins may be a consequence rather than a prerequisite of disease resistance in plants (Coll et al., 2011). To determine if SPRYSEC-19 also suppresses disease resistance mediated by a CC-NB-LRR protein, we assessed the replication of the avirulent PVX strain UK106 in the presence of both the resistance protein Rx1 and SPRYSEC-19, and in the presence of Rx1 alone. To this purpose, PVX was introduced into N. benthamiana leaves by agroinfiltrating the complete viral amplicon including GFP (PVX::GFP). Virus replication was first deduced from the accumulation of GFP in mesophyll cells in infiltrated leaf areas (Fig. 5A). As expected, the co-expression of Rx1, PVX::GFP, and GUS resulted in poor accumulation of GFP in agroinfiltrated areas. However, replacing GUS with 4MYC-SPRYSEC-19 in the agroinfiltration mix led to a strong GFP signal. We also co-expressed PVX::GFP and 4MYC-SPRYSEC-19 alone in N. benthamiana mesophyll cells to demonstrate that 4MYC-SPRYSEC-19 targeted the action of Rx1 and not the replication of PVX directly (Fig. 5A). To confirm that the accumulation of GFP reflects PVX replication in mesophyll cells, we also quantified the accumulation of PVX coat protein by using a specific antibody in an ELISA on total protein extracts isolated from agroinfiltrated leaf areas (Fig. 5B). We concluded that the suppression of Rx1-mediated immune signaling by SPRYSEC-19 also results in loss of disease resistance.  6B). As expected, no amplification product of the ITS region in V. dahliae was observed in the empty vector plants three weeks post inoculation with fungal spores. These data suggest that SPRYSEC-19 suppresses a yet unidentified fungal resistance in potato, rendering these plants susceptible to an otherwise avirulent strain of V. dahliae.

Suppression of disease resistance responses by SPRYSEC-19 does not require a direct interaction with R-proteins
To investigate whether the suppression of Gpa2, Rx1, and autoactive SW5B requires a physical interaction with SPRYSEC-19, we co-expressed 4MYC-SPRYSEC-19 and the LRR domains of these proteins fused to a 4HA tag in leaves of N. benthamiana for coimmunoprecipitation. Capturing 4MYC-SPRYSEC-19 in total protein extracts of agroinfiltrated leaf areas with anti-MYC beads did not result in the co-immunoprecipitation of the LRR domains of Sw5B, Rx1, and Gpa2 ( Fig. 7). We therefore concluded that SPRYSEC-

19-mediated suppression of CC-NB-LRR-mediated programmed cell death and resistance
does not require a physical interaction of SPRYSEC-19 with the LRR domains of these resistance proteins.

DISCUSSSION
We have shown that the resistance protein SW5F of tomato interacts specifically with the effector SPRYSEC-19 of G. rostochiensis in planta. Surprisingly, this interaction did not lead to the effector-triggered activation of SW5F-mediated programmed cell death and nematode resistance. Instead, SPRYSEC-19 is the first nematode effector to demonstrate suppression of defense-related programmed cell death by some, but not all, CC-NB-LRR resistance proteins (i.e. SW5B, Rx1, Gpa2, and RGH10). The suppression of CC-NB-LRR-mediated signaling does not require a physical association between SPRYSEC-19 and these resistance proteins. Furthermore, the suppression of programmed cell death mediated by autoactive mutant CC-NB-LRR proteins suggested that SPRYSEC-19 most likely disturbs receptor-mediated immune signaling rather than effector recognition. In addition to abrogating the programmed cell death mediated by Rx1, the nematode effector SPRYSEC-19 also repressed virus resistance mediated by this CC-NB-LRR protein. Altogether, our data demonstrates that SPRYSEC-19 of G. rostochiensis functions as a suppressor of CC-NB-LRR-mediated programmed cell death and disease resistance.
SPRYSEC-19 physically associates with SW5F in planta through its interaction with seven Cterminal leucine-rich repeats of the LRR domain of SW5F. There are only a few other plant resistance proteins for which a physical interaction with a pathogen effector in planta has been demonstrated. These interactions agree with the model of effector-triggered immunity following direct recognition of effectors by plant immune receptors. Like ATR1/PPR1 and IPI-O1/RB, we expected that the physical association of SPRYSEC-19 and SW5F would also activate effector-triggered immunity to G. rostochiensis. However, the absence of SPRYSEC-19-dependent SW5F-mediated programmed cell death N. benthamiana and SW5F-mediated resistance to G. rostochiensis in tomato and potato led us to reject this hypothesis.
We have demonstrated with four different experimental designs that the physical association between SPRYSEC19 and the LRR domain of SW5F is robust. That this association does not activate effector-triggered programmed cell death and resistance may indicate that SW5F is an inactive gene duplicate of a paralogous functional CC-NB-LRR resistance protein to G. rostochiensis. In this scenario the lack of functional constraints on the SW5F gene may have rendered its activation domains (i.e. CC-NB) dysfunctional, while binding to the sensor (i.e. LRR) domain is still intact (Takken and Goverse, 2012). We tried to make SW5F, along with SW5B, constitutively active by introducing mutations at positions that switch several other CC-NB-LRR resistance proteins into a permanent "on"-state. However, these mutations only induced autoactivity in SW5B, which is thus far the only member of the SW5 cluster linked to a known resistance (Spassova et al., 2001). The lack of autoactivity in SW5F mutants therefore favors the hypothesis that SW5F is a dysfunctional paralogue of a functional nematode resistance gene.
As SPRYSEC-19 lacked any evident avirulence activity on the three SW5F homologs isolated in this study, we also reasoned that SPRYSEC-19 might interact with the LRR domain of SW5F to suppress the activation of the CC-NB-LRR-mediated immune signaling.
Using agroinfiltration assays, we have demonstrated that SPRYSEC-19 suppresses programmed cell death mediated by some, but not all, CC-NB-LRR resistance protein in N.
benthamiana. Moreover, SPRYSEC-19 suppressed none of the members of the TIR-NB-LRR and extracellular LRR classes of resistance proteins tested in this study. We found no evidence in our co-immunoprecipitations that suppression of CC-NB-LRR-mediated programmed cell death requires the binding of SPRYSEC-19 to these receptor proteins.
However, it should be noted that mostly high affinity interactions between proteins can be demonstrated with co-immunoprecipitations. We therefore cannot exclude the possibility that SPRYSEC-19 more transiently interacts with the LRR domains of the resistance proteins it suppresses.
As the suppression of autoactive mutant CC-NB-LRR proteins demonstrated, SPRYSEC19 most likely does not disturb the recognition of specific cognate pathogen effectors that activates these resistance proteins. It is nonetheless conceivable that SPRYSEC-19 is able to outcompete other SPRYSEC effectors of G. rostochiensis that trigger the activation of a functional homolog of SW5F. Such a mechanism seems to determine the virulence of P.
infestans strains on potato plants harboring the RB resistance protein (Chen et al., 2012).
Alternatively, as discussed earlier SPRYSEC-19 may also suppress CC-NB-LRR resistance proteins by targeting the immune receptors to the proteasome for degradation (Rehman et al., 2009). However, western blots of total protein extracts of agroinfiltrated leaf areas revealed no enhanced breakdown of CC-NB-LRR proteins or parts thereof in the presence of SPRYSEC-19. We therefore conclude that our current data does not support a model in which SPRYSEC-19 interacts with CC-NB-LRR resistance proteins to alter their turnover rate.
Programmed cell death in the site of pathogen infections is often associated with effectortriggered immunity in plants, but may not be required for disease resistance (Coll et al., 2011). It could therefore be argued that the suppression of programmed cell death by SPRYSEC-19 in agroinfiltration assays bears little biological significance with regard to disease resistance. Using an avirulent PVX strain that was modified to express GFP but that was still recognized and restrained by the resistance protein Rx1, we have demonstrated that SPRYSEC-19 also suppresses CC-NB-LRR-mediated disease resistance. Furthermore, our observation that the overexpression of SPRYSEC-19 in potato plants abrogated the resistance of this potato genotype to V. dahliae further supports that this effector functions as a suppressor of disease resistance.
Next to the ability to induce and maintain feeding cells, the survival and reproduction of sedentary plant-parasitic nematodes is most likely determined by their ability to suppress host defenses. The molecular mechanisms underlying the suppression of host defense responses by plant-parasitic nematodes are not known. All known plant immune receptors conferring resistance to G. rostochiensis belong to the CC-NB-LRR class of resistance proteins (Molinari, 2011). Here we showed that G. rostochiensis has evolved several SPRYSEC effectors that selectively suppress CC-NB-LRR mediated programmed cell death and disease resistance. The SPRYSECs in the potato cyst nematodes G. rostochiensis and G. pallida constitute the largest effector family found in a plant parasitic nematode to date. If the SPRYSEC effector family functions as suppressors of effector-triggered immunity, the expansion of this effector family may reflect adaptations to functional diversifications in plant immune receptors. As the SPRYSEC effector GpRBP1 of G. pallida suggests, on their turn plants may have evolved novel NB-LRR plant immune receptors (e.g. Gpa2) that recognize and neutralize SPRYSEC effectors again. It will be highly interesting to investigate if mediated resistance also involves a physical association between the LRR domain of Gpa2 and GpRBP1.
All other experiments were performed year-round on 3-week-old tomato (L. esculentum cv. MoneyMaker) or N. benthamiana plants that were grown in a greenhouse in 15cm diameter pots with potting soil.

Cloning and plasmid construction
SPRYSEC-19 was subcloned from pGBKT7-A18-2 (Rehman et al., 2009) as a BspMI-BamHI fragment an inserted jointly with the complementary oligo pair A18For + A18Rev (Supplemental Table S1) into pRAP digested with NheI-BglII. The coding regions of the mature peptides of other SPRYSECs without their native signal peptides for secretion were PCR-amplified from G. rostochiensis cDNA. The full-length SW5F genes of tomato cv.  Table S2. Expression cassettes of pRAP, including promoter, affinity tags and the gene of interest, were subcloned into binary vector pBINPLUS (Van Engelen et al., 1995) using AscI and PacI restriction sites. All SW5F genes were cloned with the 3' UTR (polyadenylation signal and terminator) of the SW5F gene from isolated from MoneyMaker (Rehman et al., 2009). Autoactive SW5 mutants were made by inserting the annealed oligo pair D879V-1 and D879V-2 (Supplemental Table S3) between the BspHI and XbaI restriction sites of the SW5 genes in pRAP. All the above described constructs were mobilized to A. tumefaciens strain MOG101 (Hood et al., 1993), which was selectively grown on 50 mg/L kanamycin and 20 mg/L rifampicin. For the expression of SPRYSEC-19 in tomato, the coding region for the mature peptide of SPRYSEC-19 without its signal peptide was PCR-amplified from G. rostochiensis cDNA using primers listed in Supplemental Table   S1 and cloned into pENTR/D-TOPO (Invitrogen, Carlsbad, CA, USA). After confirmation of the sequence by DNA sequencing, SPRYSEC-19 was subcloned to the expression vector SOL2085 (kindly provided by Patrick Smit) using LR clonase (Invitrogen, Carlsbad, CA, USA), resulting in vector SOL2085:SS19. For agroinfiltrations in tomato leaves the constructs were mobilized to A. tumefaciens strain 1D1249 (Wroblewski et al., 2005) which was selectively grown on 100 mg/L kanamycin, 100 mg/L spectinomycin, and 1 mg/L tetracyclin.

Agroinfiltrations
Agrobacterium tumefaciens harboring the individual binary vectors was grown at 28°C in YEP medium (per liter: 10 g peptone, 10 g yeast extract, 5 g NaCl) with appropriate antibiotics. The bacteria were spun down and resuspended in MMA infiltration medium (per liter: 5 g Murashige and Skoog salts, 1.95 g MES, 20 g sucrose). The bacterial solution was diluted to an OD600 of 0.5 (for infiltration in N. benthamiana) or 0.1 (for infiltration in tomato) in MMA and infiltrated in the abaxial side of the leaves using a syringe. Coinfiltration of different constructs was performed by mixing equal volumes of the bacterial suspensions to a final OD600 as described above.

Suppression of programmed cell death
The suppression of programmed cell death in leaves of N. benthamiana was assessed using the pBINPLUS with MYC tagged SPRYSEC-19 construct described above. The 4MYC:GFP construct was used as a negative control for suppression. The following pairs of resistance genes and cognate elicitors were used to induce programmed cell death in leaves: Gpa2 /

Bimolecular fluorescence complementation
The coding regions of SPRYSEC-18 and -19 without signal peptide and the coding regions of LRR7-13 of SW5B and SW5F were PCR-amplified from the pRAP vectors described above using the primers listed in Supplemental Table S4

Plant transformation
Potato S. tuberosum line V (genotype 6487-9) was transformed as described by (Van Engelen et al., 1994) using A. tumefaciens strain MOG101 with vector pBINPLUS containing SPRYSEC-19, 4MYC:SPRYSEC-19, SW5F, or 4HA:SW5F under the control of a 35S promoter (described above). Genomic DNA was extracted from plant leaves by grinding tissues in liquid nitrogen and purifying DNA with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For every construct at least four independent transformation lines were tested and for each line ten biological replicates were used.

Nematode resistance assay
Dried cysts of G. rostochiensis pathotype Ro1 Mierenbos were soaked on a 100-μm sieve in potato root diffusate to collect hatched ppJ2s (De Boer et al., 1992). Freshly hatched preparasitic second stage juveniles in suspension were mixed with an equal volume of 70% (w/v) sucrose in a centrifuge tube and covered with a layer of sterile tap water. Following centrifugation for 5 min at 1,000 × g, juveniles were collected from the sucrose-water interface using a Pasteur pipette and washed three times with sterile tap water. The . Plates were coated with a 1:1000 dilution of a polyclonal antibody against PVX to bind the antigen, and a second polyclonal antibody against PVX conjugated with alkaline phosphatase was used for detection via the phosphatase substrate p-nitrophenyl phosphate.

Verticillium dahliae resistance assay
Verticillium dahliae isolate 5361 (kindly provided by Richard Cooper) was grown on 4% potato dextrose media (Duchefa, Haarlem, The Netherlands) at 28°C for 2 weeks. Fungal spores were transferred to sterile de-ionized water to a concentration of 1 x 10 6 spores/ml.

Supplemental data
The following materials are available in the online version of this article.  Table S3. Oligonucleotides used to construct autoactive SW5 mutants.  G  a  b  r  i  e  l  s  S  H  E  J  ,  V  o  s  s  e  n  J  H  ,  E  k  e  n  g  r  e  n  S  K  ,  O  o  i  j  e  n  G  V  ,  A  b  d  -E  l  -H  a  l  i  e  m  A  M  ,  B  e  r  g  G  C  M  V  D  ,  R  a  i  n  e  y  D  Y  ,  M  a  r  t  i  n  G  B  ,  T  a  k  k  e  n  F  L  W  ,  W  i  t  P  J  G  M  D  ,  J  o  o  s  t  e  n  M  H  A  J   (  2  0  0  7  )  A  n  N  B  -L  R  R  p  r  o  t  e  i  n  r  e  q  u  i  r  e  d  f  o  r  H  R  s  i  g  n  a  l  l  i  n  g  m  e  d  i  a  t  e  d  b  y  b  o  t  h  e  x  t  r  a  -a  n  d  i  n  t  r  a  c  e  l  l  u  l  a  r  r  e  s  i  s  t  a  n  c  e  p  r  o  t  e  i  n  s . P l a n t J .  G  a  r  d  e  s  M  ,  B  r  u  n  s  T  D   (  1  9  9  3  )  I  T  S  p  r  i  m  e  r  s  w  i  t  h  e  n  h  a  n  c  e  d  s  p  e  c  i  f  i  c  i  t  y  f  o  r  b  a  s  i  d  i  o  m  y  c  e  t  e  s  -a  p  p  l  i  c  a  t  i  o  n  t  o  t  h  e  i  d  e  n  t  i  f S  c  h  o  u  t  e  n  A  ,  R  o  o  s  i  e  n  J  ,  D  e  B  o  e  r  J  M  ,  W  i  l  m  i  n  k  A  ,  R  o  s  s  o  M  N  ,  B  o  s  c  h  D  ,  S  t  i  e  k  e  m  a  W  J  ,  G  o  m  m  e  r  s  F  J  ,  B  a  k  k  e  r  J  ,  S  c  h  o  t  s  A   (  1  9  9  7  )  I  m  p  r  o  v  i  n  g  s  c  F  v  a  n  t  i  b  o  d  y  e  x  p  r  e  s  s  i  o  n  l  e  v  e  l  s  i  n  t  h  e  p  l  a  n  t  c  y  t  o  s  o  l  .  F  E  B  S  L  e  t  t  .   4  1  5  :   2  3  5  -2  4  1   S  l  o  o  t  w  e  g  E  ,  R  o  o  s  i  e  n  J  ,  S  p  i  r  i  d  o  n  L  N  ,  P  e  t  r  e  s  c  u  A  -J  ,  T  a  m  e  l  i  n  g  W  ,  J  o  o  s  t  e  n  M  ,  P  o  m  p  R  ,  v  a  n  S  c  h  a  i  k  C  ,  D  e  e  s  R  ,  B  o  r  s  t  J  W  ,  S  m  a  n  t  G  ,  S  c  h  o  t  s  A  ,  B  a  k  k  e  r  J  ,  G  o  v  e  r  s  e  A   (  2  0  1  0  )  N  u  c  l  e  o  c  y  t  o  p  l  a  s  m  i  c  d  i  s  t  r  i  b  u  t  i  o  n  i  s  r  e  q  u  i  r  e  d  f  o  r  a  c  t  i  v  a  t  i  o  n  o  f  r  e  s  i  s  t  a  n  c  e  b  y  t  h  e  p  o  t  a  t  o  N  B  -L  R  R  r  e  c  e  p  t  o  r  R  x  1  a  n  d  i  s  b  a  l  a  n  c  e  d  b  y  i  t  s  f  u  n  c  t  i  o  n  a  l  d  o  m  a  i  n  s . P l a n t C e l l 2 2 :