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The Arabidopsis lectin receptor kinase LecRK-I.9 enhances resistance to Phytophthora infestans in Solanaceous plants

Authors

  • Klaas Bouwmeester,

    1. Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands
    2. Centre for BioSystems Genomics (CBSG), Wageningen, The Netherlands
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  • Miao Han,

    1. Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands
    2. Department of Plant Pathology, China Agricultural University, Beijing, China
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    • These authors contributed equally to this work.
  • Rosario Blanco-Portales,

    1. Wageningen UR Plant Breeding, Wageningen University, Wageningen, The Netherlands
    Current affiliation:
    1. Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba, Spain
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    • These authors contributed equally to this work.
  • Wei Song,

    1. Centre for BioSystems Genomics (CBSG), Wageningen, The Netherlands
    2. Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands
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  • Rob Weide,

    1. Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands
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  • Li-Yun Guo,

    1. Department of Plant Pathology, China Agricultural University, Beijing, China
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  • Edwin A.G. van der Vossen,

    1. Wageningen UR Plant Breeding, Wageningen University, Wageningen, The Netherlands
    Current affiliation:
    1. Keygene N.V., Wageningen, The Netherlands
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  • Francine Govers

    Corresponding author
    1. Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands
    2. Centre for BioSystems Genomics (CBSG), Wageningen, The Netherlands
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Summary

Late blight caused by the plant pathogenic oomycete Phytophthora infestans is known as one of the most destructive potato diseases. Plant breeders tend to employ NB-LRR-based resistance for introducing genetically controlled late blight resistance in their breeding lines. However, P. infestans is able to rapidly escape this type of resistance, and hence, NB-LRR-based resistance in potato cultivars is often not durable. Previously, we identified a novel type of Phytophthora resistance in Arabidopsis. This resistance is mediated by the cell surface receptor LecRK-I.9, which belongs to the family of L-type lectin receptor kinases. In this study, we report that expression of the Arabidopsis LecRK-I.9 gene in potato and Nicotiana benthamiana results in significantly enhanced late blight resistance. Transcriptional profiling showed strong reduction in salicylic acid (SA)-mediated defence gene expression in LecRK-I.9 transgenic potato lines (TPLs). In contrast, transcripts of two protease inhibitor genes accumulated to extreme high levels, suggesting that LecRK-I.9-mediated late blight resistance is relying on a defence response that includes activation of protease inhibitors. These results demonstrate that the functionality of LecRK-I.9 in Phytophthora resistance is maintained after interfamily transfer to potato and N. benthamiana and suggest that this novel type of LecRK-based resistance can be exploited in breeding strategies to improve durable late blight resistance in Solanaceous crops.

Introduction

Late blight caused by the oomycete pathogen Phytophthora infestans is one of the most serious diseases of potato. Combating late blight disease is a difficult enterprise, as the majority of potato cultivars are nowadays susceptible to P. infestans. Many of these cultivars are the end product of selective potato resistance breeding using race-specific late blight resistance (R) genes of the nucleotide binding site–leucine-rich repeat (NB-LRR) class, most of which originate from the Mexican wild potato species Solanum demissum and S. stoloniferum (Vleeshouwers et al., 2011). A small set of dominant S. demissum R genes has been used for decades by potato breeders to introgress late blight resistance into new potato cultivars Unfortunately, attempts to achieve durable resistance were not very successful due to the rapid adaptation of P. infestans to introduce R genes. Nowadays, late blight disease management largely relies on protective and curative fungicides, which can have an adverse effect on the environment and are costly. As a consequence, development of new resistant potato cultivars has received renewed attention in many modern potato breeding programmes.

As cytoplasmic NB-LRR resistance proteins are quickly overcome by P. infestans, it is essential to search for alternative sources of genetically determined resistance. One strategy is to exploit cell surface receptors that are able monitor extracellular danger signals to regulate pathogen resistance. Little is known about the molecular basis underlying the perception of these signals, yet it is generally accepted that cell surface proteins with extracellular ‘sensing’ domains play important roles in signal surveillance and response activation (Humphrey et al., 2007). Notable examples of membrane-associated receptors playing a role in defence are the receptor-like proteins (RLPs) Cf4 and Ve1 that have been shown to confer durable resistance to the fungal plant pathogens Cladosporium fulvum and Verticillium dahliae, respectively (Fradin et al., 2009; Kruijt et al., 2005). Pathogen recognition is also governed by receptor-like kinases (RLKs). Several RLKs containing extracellular leucine-rich repeat domains, for example FLS2 and EFR, are known to be essential for disease resistance; they function as pattern recognition receptors (PRRs) of microbe-associated molecular patterns (MAMPs) initiating MAMP-triggered immunity (MTI) (Monaghan and Zipfel, 2012). Other RLKs considered to operate in plant defence are the L-type lectin receptor kinases (LecRKs) (Bouwmeester and Govers, 2009). Although multiple Arabidopsis LecRK genes are induced in expression upon treatment with elicitors and pathogen infection, there are only few examples that experimentally demonstrate the involvement of LecRKs during adaptive responses to biotic stress (Bouwmeester and Govers, 2009). Previously, we presented the first functional characterization of a LecRK that functions as a potential PPR and governs defence against Phytophthora pathogens in Arabidopsis (Bouwmeester et al., 2011a). This receptor, named LecRK-I.9, was identified by its ability to interact via its extracellular lectin domain with the RGD tripeptide motif of the RXLR effector protein IPI-O from P. infestans (Gouget et al., 2006). Arabidopsis LecRK-I.9 mutant lines were found to have reduced levels of MAMP-elicited callose deposition and a destabilized continuum between the plant cell wall (CW) and plasma membrane (PM) (Bouwmeester et al., 2011a). In addition, LecRK-I.9 mutant lines show gain of susceptibility to the oomycete pathogen Phytophthora brassicae, whereas lines that overexpress LecRK-I.9 display increased resistance to P. brassicae (Bouw-meester et al., 2011a).

Recently, it was shown that next to LecRK-I.9, also LecRK-V.5 and LecRK-VI.2 play important roles in plant innate immunity in Arabidopsis. LecRK-V.5 negatively regulates MTI-mediated stomatal closure, whereas LecRK-VI.2 is positively regulating resistance to bacterial pathogens (Desclos-Theveniau et al., 2012; Singh et al., 2012). Another Arabidopsis LecRK involved in plant stress adaptation is AtLPK1 (LecRK-IV.3) (Huang et al., 2013). AtLPK1 expression was induced by salt stress, upon treatment with various plant hormones, and after inoculation with the necrotrophic fungal pathogen Botrytis cinerea. Arabidopsis lines overexpressing AtLPK1 displayed an increased tolerance to salt stress and B. cinerea infection (Huang et al., 2013).

The finding that LecRK-I.9 functions as a Phytophthora resistance component in Arabidopsis raised the question whether heterologous expression of LecRK-I.9 would lead to increased resistance to the potato late blight pathogen P. infestans in Solanaceous plants. In this study, we describe that late blight development is reduced on both potato and Nicotiana bentha-miana plants due to expression of LecRK-I.9. These results show that interfamily transfer of Arabidopsis LecRK-I.9 to Solanaceous plants confers increased resistance to P. infestans, and hence suggest that LecRK-I.9 can be used in breeding strategies to develop novel Solanaceous crops with a more durable late blight resistance.

Results and Discussion

Generation of LecRK-I.9 expressing potato plants

To test whether Arabidopsis LecRK-I.9 can confer enhanced resistance to P. infestans in potato, we generated transgenic potato lines (TPLs) expressing LecRK-I.9. The binary plasmid pK-35S-LecRK-I.9, which contains the full-length LecRK-I.9 coding sequence under control of a 35S-CaMV promoter (Bouw-meester et al., 2011a), was transferred to potato cultivar (cv.) Désirée via Agrobacterium-mediated transformation (Figure 1a). Of the 45 independent primary transformants were obtained, 41 contained and expressed the transgene as confirmed by PCR and RT-PCR, respectively (data not shown). LecRK-I.9 expression levels in three independent TPLs, named TPL30, TPL36 and TPL42, were quantified by Q-RT-PCR using total RNA isolated from potato leaves as template (Figure 1b). As expected, LecRK-I.9 mRNA was not detected in the recipient cv. Désirée. The three TPLs varied in transgene expression levels, with TPL30 having the lowest and TPL36 the highest LecRK-I.9 expression level.

Figure 1.

Ectopic expression of LecRK-I.9 in transgenic potato plants. (a) Construct used for transformation. P35S, CaMV 35S promoter; T35S, CaMV terminator; NPTII, kanamycin resistance gene; Pnos, nopaline synthase promoter; Tnos, nopaline synthase terminator; LB, left border; RB, right border. (b) Relative levels of LecRK-I.9 mRNA in transgenic potato lines measured by Q-RT-PCR. Error bars show standard deviations.

Ectopic expression of LecRK-I.9 in potato results in aberrant plant development

Transgenic potato plantlets were transferred from in vitro culture to soil, and plant growth and development were monitored during 6 weeks. In comparison with the recipient cv. Désirée, the mature leaves of TPLs exhibited a number of developmental defects. Like other potato cultivars, Désirée has so-called pinnately compound leaves, that is, leaves with pairs of leaflets along the midrib ending with a single larger terminal leaflet. In the leaves of the TPLs, we observed deformed leaf laminae which appeared more wrinkled than in Désirée leaves, especially in the three upper leaflets of the compound leaf. The severity of this phenotype differed, however, between the three TPLs and was found to be associated with the LecRK-I.9 mRNA levels. The observed phenotype was the weakest for TPL30, which has a low level of transgene expression, whereas in TPL36 and TPL42- both exhibiting higher LecRK-I.9 expression levels- the wrinkling was more severe, and such that the terminal leaflet was not separated from the two upper secondary leaflets (Figure 2). Accordingly, the leaf veins in TPL36 and TPL42 were radiating outward from a single point at the base, a venation pattern that is more associated with palmately compound leaves (Figure 2). The TPLs were also found to be markedly different in tuber morphology. Compared with the recipient cv. Désirée, which normally produces oval red-skinned tubers, the tubers of the TPLs were of smaller size, malformed, and of yellow skin colour (Figure S1).

Figure 2.

Aberrant leaf morphology of transgenic potato lines (TPLs) expressing LecRK-I.9. Compared with the recipient cv. Désirée the TPLs display a more palmate venation pattern (red asterisks) and aberrant leaf shapes with the upper three leaflets not separated (white arrows).

The altered leaf morphology found in the TPLs resembles more or less the disarrangement of digits on animal limbs, which is correlated with a lack of either the RGD-containing ECM protein laminin or laminin integrin receptors (De Arcangelis et al., 1999; Miner et al., 1998). Also, transgenic Arabidopsis lines that constitutively express LecRK-I.9 were found to be altered in leaf morphology. In comparison with their recipient line Col-0, LecRK-I.9 overexpressing lines had more compact rosettes with smaller and slightly wrinkled leaves and showed accumulation of lignin and anthocyanins (Bouwmeester et al., 2011a). These changes in phenotype might be due to the fact that overexpression of LecRK-I.9 results in cell adhesion strengthening and thus, to a much stronger attachment of the CW to the PM, thereby disturb normal plant growth.

LecRK-I.9 confers enhanced late blight resistance in potato

To investigate whether constitutive LecRK-I.9 expression in potato has an effect on late blight resistance, we tested six TPLs in detached leaf assays for their response to P. infestans. The six lines all showed enhanced resistance to one or more P. infestans isolates. On TPL30, TPL36 and TPL42 four P. infestans isolates of different origin were tested (Table 1). To standardize the infection pressure, inoculations were performed with equal concentrations of zoospores. As expected, all P. infestans isolates were capable to reach a 100% infection efficiency (IE) on the susceptible cv. Désirée, but on the TPLs, the IEs were significantly reduced (Figure 3a). For instance, the IE of isolate 88069 on TPL36 was found to be reduced to 40%, whereas the IEs on TPL42 and TPL30 were reduced to approximately 70% and 80%, respectively. Similarly, the TPLs were also less efficiently infected by the other three tested P. infestans isolates. To further assess differences in late blight resistance, we determined mean lesion areas 4 days postinoculation (dpi). As shown in Figure 3b, significant differences were found between cv. Désirée and the three TPLs for all four P. infestans isolates; that is, lesions on cv. Désirée were much larger compared with those on leaves of the TPLs. P. infestans isolate 88069, for example, was able to readily infect cv. Désirée resulting in large lesions at four dpi, whereas significant smaller lesions were observed on the three TPLs (Figure 3b). TPL30, which has low-level transgene expression, was found to be less resistant to P. infestans compared with the other two TPLs that have higher LecRK-I.9 expression levels (Figure 3c). These results indicate that constitutive expression of the Arabidopsis lectin receptor kinase LecRK-I.9 in potato enhances resistance towards P. infestans and that this resistance is correlated with the relative levels of transgene expression.

Table 1. Phytophthora infestans isolates used in this study
P. infestans isolateSolanum host of originYear of isolationCountryReference
  1. n.a., not applicable.

  2. a

    GFP expressing transformant of P. infestans H30P02.

IPO428-2S. tuberosum (potato)1992The NetherlandsFlier et al. (2003)
14-3-GFPaS. tuberosum (potato)n.a.n.a. 
88069S. lycopersicum (tomato)1988The NetherlandsVan West et al. (1998)
PIC97757 S. demissum 1997MexicoFlier et al. (2001)
Figure 3.

Enhanced late blight resistance of transgenic potato lines (TPLs) expressing LecRK-I.9. (a) Infection efficiencies of four Phytophthora infestans isolates on leaves of TPLs and the recipient cv. Désirée at 4 days postinoculation (dpi). (b) Mean lesion areas at four dpi. (c) Lesions on leaves at four dpi with P. infestans isolate 88069. Infection assays were repeated three times with similar results; results obtained from one representative experiment are shown. Data are based on at least 20 inoculation spots per potato line. Statistical significance was determined using Student's t-test (P < 0.05). Error bars represent standard deviations.

LecRK-I.9 induces transcriptional changes in defence marker genes in potato

As increased late blight resistance might be due to the fact that the TPLs have a constitutive expression of defence-related genes, we analysed relative expression levels of several potato defence marker genes by Q-RT-PCR. Clear differences in transcript levels were found between the recipient line cv. Désirée and the three TPLs. The salicylic acid (SA)-mediated defence marker genes PR1 and PR10a, and the SA signalling regulatory genes NPR1 and WRKY1 showed reduced expression in the TPLs (Figure 4). In contrast, in the TPLs, strong transcript accumulation was detected for the protease inhibitor genes PI-1 and PI-2, both of which are reported to be regulated by jasmonate (JA) or related compounds in potato (Turrà et al., 2009; Turrà and Lorito, 2011; Figure 4). This suggests that the increased resistance to P. infestans found in the TPLs does not rely on SA-mediated signalling, but is more dependent on a defence response that includes activation of protease inhibitors. It should be noted, however, that the PI-1-related gene PPI3A2 did not show a similar strong induction in expression but instead showed reduced expression, indicating that there is differential expression among protease inhibitor genes.

Figure 4.

LecRK-I.9 induces transcriptional changes in defence marker genes in potato. Relative expression levels of the potato defence marker genes PR1, PR10a, NPR1, WRKY1, PI-1, PPI3A2 and PI-2 in TPL30, TPL36 and TPL42 shown as fold changes compared with expression in cv. Désirée. Red and blue bars represent salicylic acid (SA)- and jasmonate (JA)-responsive genes, respectively. Values are average gene expression levels measured in three technical replicates. Error bars represent standard deviations.

LecRK-I.9 confers enhanced late blight resistance in Nicotiana benthamiana

Next, we explored whether LecRK-I.9 can provide resistance towards P. infestans in N. benthamiana, another Solanaceous species which can be infected by P. infestans (Shibata et al., 2010). For this purpose, we transiently expressed a GFP-tagged version of LecRK-I.9 (LecRK-I.9-GFP) in N. benthamiana leaves by agroinfiltration and first tested whether the Arabidopsis LecRK-I.9 gene behaved as expected. LecRK-I.9 expression was quantified by Q-RT-PCR, and this revealed significant LecRK-I.9 transcript levels 3 days after agroinfiltration (Figure S2). In addition, total protein extracts of agroinfiltrated N. benthamiana leaves were subjected to immunoblot analysis using anti-GFP antibody (αGFP) to determine recombinant protein accumulation. Two specific bands were detected, one with the expected molecular weight of around 120 and a larger but less stronger band that might be a complex that is not fully denatured (Figure 5a). Analysis of the two bands by mass spectroscopy showed that both contain LecRK-I.9-GFP (data not shown), which confirms accumulation of LecRK-I.9-GFP in planta. Breakdown products of the size of native GFP were also detected, suggesting partial degradation or instability of the LecRK-I.9-GFP fusion protein. Previously, we showed by confocal microscopy that LecRK-I.9-GFP localizes at the PM after transient expression in N. benthamiana (Bouwmeester et al., 2011a). To corroborate this finding, immunodetection of LecRK-I.9-GFP after subcellular protein fractionation was performed. Total protein extracts were separated by ultracentrifugation and two-phase partitioning into PM, intracellular membrane (IM) and microsomal membrane (MM) fractions. Subsequently, equal portions of the individual fractions were analysed for LecRK-I.9-GFP accumulation by immunoblotting using αGFP. LecRK-I.9-GFP is enriched in the PM fraction, but low or undetectable in the IM and MM fractions, respectively (Figure 5b). Based on these results, we conclude that LecRK-I.9-GFP properly localizes at the PM of agroinfiltrated N. benthamiana leaves. To ensure that the disease tests are not influenced by artefacts potentially arising from agroinfiltration, we quantified basal plant stress by monitoring electrolyte leakage in agroinfiltrated N. benthamiana leaves. No substantial yellowing of the LecRK-I.9-GFP agroinfiltrated zones was observed 3 days after agroinfiltration. In addition, no significant increase in conductivity was found between leaf discs expressing LecRK-I.9-GFP and GFP at both 2 and 6 days after agroinfiltration (Figure S3).

Figure 5.

Enhanced late blight resistance in LecRK-I.9 expressing N. benthamiana leaves. Immunoblot detection of GFP and LecRK-I.9-GFP in total protein extracts of agroinfiltrated N. benthamiana leaves. (b) Immunoblot detection of LecRK-I.9-GFP in the plasma membrane (PM), intracellular membrane (IM) and microsomal membrane (MS) fractions extracted form agroinfiltrated N. benthamiana leaves. Arrows in (a, b) indicate protein sizes in kDa. (c) Lesions on N. benthamiana leaves transiently expressing LecRK-I.9 or gus at four days postinoculation (dpi). Dotted lines indicate lesion perimeters. (d) Mean lesion areas at 3–6 dpi. Statistical significance was determined using Student's t-test. Asterisks indicate significant differences between the two groups (P < 0.05). Experiments were repeated three times with similar results; results from one representative experiment are shown. Error bars represent standard deviations.

For disease assays, leaves of N. benthamiana were collected 2 days after agroinfiltration, and subsequently inoculated with the same four P. infestans isolates as used in the potato detached leaf assays (Table 1). Lesions development was monitored by determining lesion areas from three dpi onwards for four successive days. Comparable IEs were found on agroinfiltrated leaves expressing LecRK-I.9-GFP and gus (data not shown). In contrast, lesions were profoundly restricted on agroinfiltrated N. benthamiana leaves expressing LecRK-I.9-GFP and much smaller compared with those on gus expressing leaves (Figure 5c, d). Significant differences were detected between the mean lesion areas as early as three dpi. Such differences were not observed when agroinfiltrated N. benthamiana leaves were inoculated with the grey mould fungus Botrytis cinerea. Both were readily infected, and no significant difference in mean lesion areas was detected between LecRK-I.9-GFP and gus expressing leaves (Figure S4). This is in line with our previous findings showing that Arabidopsis lines overexpressing LecRK-I.9 gained resistance to Phytophthora but were not altered in resistance to Botrytis cinerea and strengthens the fact that LecRK-I.9 functions as a Phytophthora resistance component (Bouwmeester et al., 2011a).

Conclusions

These results collectively show that LecRK-I.9 enhances resistance to P. infestans in the Solanaceous plants potato and N. bentha-miana. This could be due to the fact that LecRK-I.9 overexpression is strengthening cell adhesion that hinders P. infestans to penetrate cells and thereby restricts intracellular proliferation. A more robust CW–PM continuum might interfere with haustoria formation, specialized structures that P. infestans uses for nutrient uptake and for effector translocation towards host cells, respectively (Bouwmeester et al., 2011b). Alternatively, LecRK-I.9 might function in the perception of MAMPs or CW damage molecules, and as such overexpression could activate an enhanced defence response. The present study further demonstrates that the functionality of LecRK-I.9 in Phytophthora resistance is maintained after interfamily transfer to potato and N. benthamiana. Thus far, only few examples of interfamily transfer of resistance genes resulting in a change in disease phenotype have been reported. Fradin et al. (2011) showed that the tomato resistance gene Ve1 expressed as transgene in Arabidopsis provides resistance towards the fungal pathogens Verticillium dahliae and Verticillium albo-atrum. Another prominent example is the interfamily transfer of the Arabidopsis pattern recognition receptor EFR to the Solanaceous plants N. benthamiana and tomato, which lead to resistance to multiple phytopathogenic bacteria (Lacombe et al., 2010). As LecRKs are widely spread in higher plants, with 45 genes in Arabidopsis and at least 30 in potato (Bouwmeester and Govers, 2009; K. Bouwmeester, unpubl. data), it is worth to investigate whether they can be exploited for Phytophthora resistance in potato and other Solanaceous crop plants.

Experimental procedures

Potato transformation

Binary plasmid pK-35S-LecRK-I.9 (Bouwmeester et al., 2011a) was transferred to A. tumefaciens strain COR308 through electroporation. Transformed clones were selected on LB medium containing 5 μg/mL tetracycline and 100 μg/mL spectinomycin and used to transform potato tuber discs of cv. Désirée according to Hoekema et al. (1989). TPLs were selected on kanamycin, and successful gene transfer was confirmed by PCR and Southern hybridization. TPLs were maintained in vitro in climate chambers at 20 °C and a 16-h photoperiod on MS30 medium and were propagated by transplanting vegetative tissue to fresh medium with regular intervals. Potato tubers and in vitro material were used for long-term storage. To obtain fresh plant material for infection assays, in vitro plantlets or tubers were transferred to soil.

Agroinfiltration assays

The binary plasmids pS-35S-LecRK-I.9-GFP (Bouwmeester et al., 2011a), pGWB20-GUS-10Myc and pBIN+-GFP-HA (Rairdan and Moffett, 2006) were introduced by electroporation into Agrobacterium tumefaciens strain AGL1. Transformants were selected on LB mannitol medium containing the appropriate antibiotics, and subsequently grown as described previously (Champouret et al., 2009). Agrobacterium cultures were suspended in MMA induction buffer [10 mm MES (pH 5.6), 10 mm MgCl2, 100 μm acetosyringone] to an OD600 of 1.0 and co-infiltrated at a 1 : 1 ratio with an Agrobacterium strain containing the silencing suppressor P19 into four to five week old N. benthamiana leaves.

Expression analysis by Q-RT-PCR

Total RNA was isolated from potato and N. benthamiana leaves using an RNeasy® Plant Mini Kit (Qiagen, Germantown, MD). Synthesis of cDNA was conducted using an oligo(dT) primer and a M-MLV reverse transcriptase kit (Promega, Madison, WI) according to the manufacturer's instructions. Q-RT-PCR was performed on an ABI7300 PCR machine (Applied Biosystems, Foster City, CA) using SensiMix SYBR Green I (Bioline, London, UK). Gene expression levels were determined using appropriate primers and normalized with respect to actin gene expression (Table S1). Relative expression levels reflect fold-increase compared with the recipient line cv. Désirée, or to untransformed N. benthamiana.

Protein extraction and immunoblot analysis

N. benthamiana leaves were harvested 2–3 days after agroinfiltration and ground in liquid nitrogen to a fine powder. One gram of powered leaves was resuspended in 2 mL of extraction buffer [0.6% (w/v) CHAPS, 50 mm Tris-HCl (pH 8.0) and 150 mm NaCl + protease inhibitor (Roche, Almere, the Netherlands, cat no. 11836170 00)] and homogenized using a potter tube. Extracts were incubated for 30 min on ice and centrifuged for 10 min at 16 g at 4 °C to remove debris. Protein fractions were isolated using the protocol of Larsson and Widell (2000) with minor modifications. Immunopurification was performed using GFP-Trap beads (ChromoTek, Planegg-Martinsried, Germany) according to the manufacturer's protocol. GFP beads were subsequently collected by 720 g centrifugation, washed for 7 times in 1 mL extraction buffer, boiled in Laemmli loading buffer for 5 min and centrifuged for 10 min at 16 g, after which the supernatant was collected for further analysis. Protein samples were run on a 10% SDS–PAGE gel, and proteins were blotted onto PVDF membrane. Membranes were probed with a 1 : 2000 (v/v) dilution anti-GFP antibody (Roche, cat. no.11814460001), and immunodetection was performed using SuperSignal West Femto Maximum Sensitivity Substrate chemiluminescence substrate (Thermo Scientific, Rockford, IL).

Pathogen growth and inoculum preparation

P. infestans isolates (Table 1) were routinely grown on rye agar medium supplemented with 20 g/L sucrose at 18 °C. P. infestans zoospores were isolated as described in Champouret et al. (2009). Botrytis cinerea B05.10 was cultured on malt extract agar. Isolation of conidia and inoculum preparation was performed as previously described by Benito et al. (1998). P. infestans and B. cinerea inoculum concentrations were adjusted before leaf inoculation to 2 × 105 zoospores per mL and 1 × 106 conidiospores per mL, respectively.

Plant growth and pathogen inoculations

Potato and N. benthamiana plants were grown in potting soil under standard greenhouse conditions. Potato cultivar Désirée (R0) was used as nontransgenic control. Foliar resistance was assessed in detached leaf assays using potato leaves of 7-week-old plants and N. benthamiana leaves harvested two days after agroinfiltration (Vleeshouwers et al., 1999). Inoculation with P. infestans was performed by placing 10 μL of a zoospore suspension on the abaxial side of the leaf. Inoculated leaves were incubated at 18 °C and for the first 24 h kept in the dark. At four dpi, IEs and lesion areas were determined as previously described (Vleeshouwers et al., 1999). Inoculation with B. cinerea was performed by applying 3 μL drops of a conidia suspension on the adaxial leaf sides. Lesion areas were measured at two dpi.

Electrolyte leakage assay

Leaf discs (Ø 8 mm) were obtained from agroinfiltrated N. bentha-miana leaves using a cork borer. Six leaf discs were floated on 4 mL sterile distilled water at room temperature under continuous shaking at 250 rpm on a LaboTech shaker. After 2 h, conductivity was measured using a Mettler Toledo InLab®741 ISM conductivity probe (Mettler Toledo, Tiel, the Netherlands). Subsequently, samples containing the leaf discs were autoclaved and measured for conductivity. Electrolyte leakage ratios were calculated by dividing conductivity values of samples before autoclaving with the conductivity values of samples after autoclaving.

Acknowledgements

We would like to acknowledge Tsuyoshi Nakagawa, Patrick Smit and Wladimir Tameling for providing plasmids, Annelies Loonen for technical assistance, and Henk Smit and Bert Essenstam at Unifarm for excellent plant care. Miao Han and Rosario Blanco-Portales were supported by the China Scholarship Council (CSC) and a postdoctoral fellowship from the Agricultural Ministry of Andalucia, respectively. This research was financially supported by the Dutch Ministry of Economic Affairs, Agriculture and Innovation grant LNV427 (Parapluplan Phytophthora) and by the EU-BioExploit grant FOOD-CT-2005-513959, and performed within the Centre for BioSystems Genomics (CBSG) which is part of The Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research.

Conflict of interest

The authors have no conflict of interest to declare.

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