Characterization of Vitis vinifera NPR1 homologs involved in the regulation of Pathogenesis-Related gene expression
© Le Henanff et al; licensee BioMed Central Ltd. 2009
Received: 10 February 2009
Accepted: 11 May 2009
Published: 11 May 2009
Grapevine protection against diseases needs alternative strategies to the use of phytochemicals, implying a thorough knowledge of innate defense mechanisms. However, signalling pathways and regulatory elements leading to induction of defense responses have yet to be characterized in this species. In order to study defense response signalling to pathogens in Vitis vinifera, we took advantage of its recently completed genome sequence to characterize two putative orthologs of NPR1, a key player in salicylic acid (SA)-mediated resistance to biotrophic pathogens in Arabidopsis thaliana.
Two cDNAs named VvNPR1.1 and VvNPR1.2 were isolated from Vitis vinifera cv Chardonnay, encoding proteins showing 55% and 40% identity to Arabidopsis NPR1 respectively. Constitutive expression of VvNPR1.1 and VvNPR1.2 monitored in leaves of V. vinifera cv Chardonnay was found to be enhanced by treatment with benzothiadiazole, a SA analog. In contrast, VvNPR1.1 and VvNPR1.2 transcript levels were not affected during infection of resistant Vitis riparia or susceptible V. vinifera with Plasmopara viticola, the causal agent of downy mildew, suggesting regulation of VvNPR1 activity at the protein level. VvNPR1.1-GFP and VvNPR1.2-GFP fusion proteins were transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, where they localized predominantly to the nucleus. In this system, VvNPR1.1 and VvNPR1.2 expression was sufficient to trigger the accumulation of acidic SA-dependent Pathogenesis-Related proteins PR1 and PR2, but not of basic chitinases (PR3) in the absence of pathogen infection. Interestingly, when VvNPR1.1 or AtNPR1 were transiently overexpressed in Vitis vinifera leaves, the induction of grapevine PR1 was significantly enhanced in response to P. viticola.
In conclusion, our data identified grapevine homologs of NPR1, and their functional analysis showed that VvNPR1.1 and VvNPR1.2 likely control the expression of SA-dependent defense genes. Overexpression of VvNPR1 has thus the potential to enhance grapevine defensive capabilities upon fungal infection. As a consequence, manipulating VvNPR1 and other signalling elements could open ways to strengthen disease resistance mechanisms in this crop species.
Grapevine (Vitis vinifera) is a major fruit crop worldwide that is susceptible to many microbial infections, especially by fungi, thus requiring an intensive use of phytochemicals. The economic costs and negative environmental impact associated with these applications led to search for alternative strategies involving activation of the plant's innate defense system. In order to efficiently limit the losses due to diseases, it is therefore necessary to have a thorough knowledge of grapevine disease resistance mechanisms.
Plants have developed a two-layered innate immune system for defense against pathogens. Primary innate immunity, the first line of defense of plants, is achieved through a set of defined receptors, that recognize conserved microbe-associated molecular patterns . In order to defend themselves against pathogens that can suppress primary defense mechanisms, plants have developed a secondary defense response that is triggered upon recognition of race-specific effectors. Resistance proteins monitor these effectors and subsequently trigger secondary defense responses that often culminate in localized cell death response associated with additional locally induced defense responses, that block further growth of the pathogen . After recognition of the invading microorganism, induced resistance to different types of pathogens is achieved through a network of signal transduction pathways in which the small molecules salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) act as secondary messengers . These regulators then orchestrate the expression of sets of downstream defense genes encoding antimicrobial proteins or enzymes catalyzing the production of defense metabolites. Plant resistance to biotrophic pathogens is classically believed to be mediated through SA signalling . SA accumulation as well as the coordinated expression of Pathogenesis Related (PR) genes encoding small proteins with antimicrobial activity are also necessary to the onset of Systemic Acquired Resistance (SAR) in plants. SAR is a plant immune response that establishes a broad spectrum resistance in tissues distant from the site of primary infection .
In the past years, considerable progress has been made in the model plant Arabidopsis thaliana in identifying genes that affect regulation of defense gene expression. Several key plant defense regulators especially involved in the SA signalling pathway have been cloned and characterized . The npr1 mutant was isolated in a genetic screen for plants that failed to express PR2 gene after SAR induction . NPR1 (Nonexpressor of PR genes 1) has been identified as a key positive regulator of the SA-dependent signalling pathway and is required for the transduction of the SA signal to activate PR gene expression and Systemic Acquired Resistance . The NPR1 gene was cloned in 1997 and shown as encoding a novel protein containing ankyrin repeats involved in protein-protein interactions . NPR1 is constitutively expressed and levels of its transcripts increased only two-fold following SA treatment, suggesting that it is regulated at the protein level . Indeed, NPR1 activity is regulated by redox systems which have been recently identified . Inactive NPR1 is present as cytosolic disulfide-bound oligomers in the absence of pathogen attack. Following SA induction, oligomeric NPR1 is reduced to active monomers . NPR1 monomers are translocated to the nucleus where they interact with the TGA class of basic leucine zipper transcription factors, leading to the expression of SA-dependent genes [3, 9]. Recent studies have also involved WRKY transcription factors in SA defense responses downstream or in parallel with NPR1 .
In Arabidopsis, the NPR1-dependent SA pathway controls the expression of PR1, PR2 (β-1.3-glucanase) and PR5 (thaumatin-like) genes. In contrast, induction of distinct defense genes encoding the defensin PDF1.2 and the PR3 (basic chitinase) proteins is controlled by JA/ET dependent pathways .
Originally, the npr1 mutant was thought to be only deficient in SA-mediated defense. However, it was shown that NPR1 plays a role in other defense signalling pathways. In npr1, the establishment of Induced Systemic Resistance (ISR) in leaves by non-pathogenic root rhizobacteria is blocked. Interestingly, this resistance response is independent of SA but requires ET and JA signalling . Apart from NPR1, Arabidopsis genome contains five NPR1-related genes called AtNPR2 to AtNPR6 . Members of the AtNPR family encode proteins sharing two domains involved in mediating protein-protein interactions: the Broad Complex, Tramtrack and Bric a brac/Pox virus and Zinc finger (BTB/POZ) domain in the N-terminal and the Ankyrin Repeat Domain (ARD) in the middle of the protein. Whereas AtNPR1 to AtNPR4 have been implicated in signalling of defense responses, AtNPR5 and AtNPR6 (called AtBOP1 and AtBOP2) form a distinct group involved in the regulation of developmental patterning of leaves and flowers .
AtNPR1 has been over-expressed in Arabidopsis, rice, tomato and wheat, resulting in enhanced bacterial and fungal resistance [7, 14–16]. Moreover, homologs of AtNPR1 have been cloned and characterized in several crop plants including rice , apple , banana  and cotton . In rice, over-expression of OsNPR1 conferred disease resistance to bacterial blight, but also enhanced herbivore susceptibility in transgenic plants . Similarly, over-expression of the Malus NPR1 in two apple cultivars resulted in activation of PR genes and enhanced resistance to Erwinia amylovora and to two important fungal pathogens of apple .
In grapevine, many studies described the induction of PR proteins and the production of stilbenes after infection [21, 22]. However, signalling pathways and regulatory elements leading to the induction of these responses remain to be characterized in this species. Recently, two genes encoding transcription factors of the WRKY family and potentially involved in grapevine resistance to pathogens have been characterized. Overexpression of VvWRKY1 and VvWRKY2 in tobacco conferred reduced susceptibility to different types of fungi [23, 24].
Recent completion of Vitis vinifera genome sequencing in a highly homozygous genotype and in a heterozygous grapevine variety has led to the identification of putative resistance genes and defense signalling elements [25, 26]. Based on conserved domain analyses, the grape genome was found to contain a number of genes showing a nucleotide binding site (NBS) and a carboxy-terminal leucine-rich repeat (LRR) typical of resistance (R) genes . Besides putative R genes, the grape genome contains several candidate genes encoding putative signalling components for disease response, with similarity to Arabidopsis EDS1, PAD4, NDR1 and NPR1 . A possible role of the two grapevine regulatory elements sharing sequence similarity to the Arabidopsis SA signalling components NDR1 and EDS1 was recently described by our group .
Given the pivotal role of AtNPR1 in plant defense, we decided to take advantage of data from grapevine EST databases and genome sequencing to identify two genes encoding proteins with similarity to AtNPR1, that we called VvNPR1.1 and VvNPR1.2. Expression of these genes was studied after treatment with benzothiadiazole (BTH, a SA analog) and after inoculation of two resistant or susceptible Vitis species with Plasmopara viticola, the causal agent of downy mildew. Nuclear localization of VvNPR1.1 and VvNPR1.2 was demonstrated by expressing GFP fusions. To get further insight into VvNPR1 function, the two genes were transiently overexpressed in both N. benthamiana and Vitis vinifera leaves and consequences on PR gene induction were studied.
Identification and sequence analysis of two NPR1-like genes in Vitis vinifera
At the beginning of this study, the grapevine genome was not entirely sequenced. The nucleic acid sequence of AtNPR1 (At1g64280) was used to search an EST database of abiotically stressed Vitis vinifera cv Chardonnay leaves (EST Analysis Pipeline, ESTAP, ). Two ESTs with significant similarity to AtNPR1 were identified. Sequence comparison of these two EST with data from grapevine genome sequencing project  enabled us to obtain the two full-length cDNAs, named VvNPR1.1 (GSVIVT00016536001) and VvNPR1.2 (GSVIVT00031933001). Amino acid sequence comparison of VvNPR1.1 and VvNPR1.2 showed that the two proteins display 47% identity and 66% similarity. Completion of V. vinifera genome sequencing has revealed only two genes related to "defense" AtNPRs (K. Bergeault, unpublished results).
VvNPR1.1 and VvNPR1.2 encode putative proteins of 584 and 587 amino acids respectively (Figure 1B). According to PROSITE tool , VvNPR1.1 and VvNPR1.2 are predicted to have the same overall organization as members of the AtNPR family, with an amino terminal BTB/POZ domain and a central ankyrin repeat domain (Figure 1B). In addition, the carboxy terminal domains of VvNPR1.1 and VvNPR1.2 are rich in basic amino acids typical of nuclear localization signals (NLS, Figure 1C). Kinkema et al.  showed that five residues in the C-terminus of AtNPR1 are essential for its nuclear translocation and constitute the NLS1. Four of these five amino acids are conserved in VvNPR1.1 (Figure 1C), whereas some lysine residues have turned into arginine in VvNPR1.2. Basic amino acids of the second NLS in AtNPR1 have been shown to be not necessary for nuclear targeting  and are less conserved among the different homologs even in the two grapevine proteins (Figure 1C).
VvNPR1.1 and VvNPR1.2expression following BTH treatment in grapevine leaves
Expression patterns of VvNPR1.1 and VvNPR1.2during compatible and incompatible interactions with Plasmopara viticola
We have next investigated whether the expression of VvNPR1.1 and VvNPR1.2 could be modulated after pathogen infection and whether their expression was differentially affected during compatible or incompatible interactions. Grapevine and related species exhibit a wide spectrum of resistance to the biotrophic pathogen Plasmopara viticola, the downy mildew agent. Two different Vitis species, the resistant Vitis riparia cv Gloire de Montpellier and the susceptible Vitis vinifera cv Chardonnay, were challenged with Plasmopara viticola or water as control. The expression patterns of VvNPR1.1 and VvNPR1.2 were determined after inoculation using real-time quantitative PCR. The expression of each gene after inoculation was calculated as fold induction compared to H2O-inoculated leaves at the same time point as described by Pfaffl et al .
Transcript accumulation of VvNPR1.1 and VvNPR1.2 was then quantified after P. viticola infection. As shown in Figure 3B and 3C, no significant change in the expression of these two genes was detectable for either genotype. Other studies from our group have shown that constitutive expression of VvNPR1.1 and VvNPR1.2 was also not affected by infection with Botrytis cinerea or with Pseudomonas syringae pv pisi (data not shown). Taken together, expression studies suggest that VvNPR1.1 and VvNPR1.2 are not regulated at transcriptional level upon pathogen infection.
Subcellular localization of VvNPR1.1 and VvNPR1.2
Transient expression of VvNPR1.1 and VvNPR1.2 in N. benthamianatriggers the accumulation of acidic PR1 and PR2 but not of PR3
Similar results concerning PR protein expression were observed after infiltration of N. benthamiana with Agrobacterium harbouring the coding regions of AtNPR1, VvNPR1.1 and VvNPR1.2 under the control of the 35S CaMV promoter in a pBinplus vector devoid of GFP (data not shown).
Transient expression of AtNPR1 and VvNPR1.1 in grapevine leaves enhances accumulation of VvPR1transcripts
Together, these results show that transient expression of both AtNPR1 and VvNPR1.1 in Vitis vinifera is able to enhance expression of a grapevine defense gene known to be controlled by the SA signalling pathway in model plants.
In order to characterize defense response signalling components in grapevine, we identified two homologs of AtNPR1 in Vitis vinifera cv Chardonnay. Our study provides the first elements for the functional characterization of VvNPR1.
Expression studies of VvNPR1.1 and VvNPR1.2 showed that these genes are constitutively expressed and that expression can be further enhanced by treatment with BTH, a SA analog. Induction of NPR1 genes by treatment with SA or its analogs has been described in a number of plant species including Arabidopsis, mustard, apple, rice, banana and cotton [4, 17–20, 35]. Interestingly, VvNPR1.2 is the most responsive to BTH induction and forms a phylogenetically related group with MpNPR1, AtNPR3 and AtNPR4 which are also highly induced by BTH or INA (another SA analog) respectively [18, 36]. In rice, it has been shown that OsNPR1 is more rapidly induced in the incompatible interactions leading to resistance than in the compatible interactions leading to disease . Similarly, MNPR1A from banana was induced earlier and to higher levels after infection in a Fusarium oxysporum tolerant cultivar than in a sensitive one . To evaluate if VvNPR1 expression could be differentially regulated during compatible or incompatible interactions between Vitis species and Plasmopara viticola, we examined the expression of both genes after inoculation of susceptible Vitis vinifera cv Chardonnay or resistant Vitis riparia cv Gloire de Montpellier with downy mildew. The expression of a gene encoding a stilbene synthase, an enzyme involved in the synthesis of phytoalexins, which is known to be stimulated by P. viticola infection was also studied as a positive control. We detected a faster induction of STS gene expression after inoculation of the resistant genotype (Vitis riparia), consistent with an earlier induction of defense genes in incompatible versus compatible interactions . However, no significant changes in transcript levels were detected for both VvNPR1.1 and VvNPR1.2 after infection with downy mildew. Overall, the constitutive expression of VvNPR1 and the absence of transcriptional regulation after pathogen infection suggest that VvNPR1 activity is regulated at the protein level in grapevine, as previously described in Arabidopsis .
In order to address VvNPR1 function, particularly its subcellular localization and its ability to regulate defense gene expression, we first used an heterologous system for transient expression by agroinfiltration of N. benthamiana leaves. This method has been described as a rapid and efficient system for the in vivo analysis of plant transcription factors and promoters of PR genes . The predicted amino acid sequences of VvNPR1.1 and VvNPR1.2 were found to contain a putative nuclear localization signal (NLS1) in their C terminus. Consistently, transiently expressed VvNPR1-GFP and AtNPR1-GFP fusion proteins were localized predominantly to the nucleus, even in the absence of the SAR inducer SA. Constitutive nuclear localization was also revealed by transient expression of AtNPR1-GFP after bombardment of epidermal onion cells . By contrast, in stable transformants, exclusive nuclear localization of AtNPR1-GFP, which is required for activation of PR gene expression, was triggered only after treatment with a SAR inducer or infection with a pathogen . Similarly, Arabidopsis lines overexpressing AtNPR1 under the control of the constitutive 35S CaMV promoter and grown under non-inducing conditions have not revealed an increase in the basal level of PR genes, indicating that AtNPR1 is essentially inactive in the absence of pathogen infection. NPR1-overexpressing plants will thus not activate SA-dependent defense responses until they are challenged with a pathogen .
In this study, we showed by transient expression that VvNPR1.1 and VvNPR1.2 are functional in triggering the accumulation of acidic PR1 and PR2 in N. benthamiana. This effect was obtained in the absence of an exogenous inducer and correlated with the nuclear localization of VvNPR1.1 and VvNPR1.2. It is likely that agroinfiltration of N. benthamiana leaves itself induces a biotic stress that activates responses related to SAR, including targeting of NPR1 proteins to the nucleus. This hypothesis is supported by a higher basal level of PR proteins in empty vector-agroinfiltrated leaves compared to leaves infiltrated with water (Figure 5). Similarly, it has been reported that Agrobacterium-mediated transient assays of stress-inducible PR promoters have relatively high levels of GUS activity in water and mock-treatments . Finally, it appears that both grapevine NPR1 are active in N. benthamiana, in agreement with the ability of AtNPR1 to activate defense responses in other plant species such as rice and wheat [14, 16]. Induction of PR protein accumulation was rather specific of defense markers that have been demonstrated to be SA-specific in tobacco . Conversely, NPR1 expression had no significant effect on basic chitinase (PR3) accumulation. In Arabidopsis, PR3 represents an SA- independent marker whose expression is controlled by the JA/ET pathway . Moreover, class I basic chitinase expression is activated by overexpression of an ethylene-responsive transcription factor (ERF) in tobacco cells .
In order to gain further information on VvNPR1 activity in a homologous system, we used a recently described method of Agrobacterium-mediated transient gene expression in Vitis vinifera . This system circumvents the time consuming process of generating stable transgenic lines in grapevine. In this study, we provide a first example of successful use of Agrobacterium-mediated transient expression for functional analysis of signalling elements in grapevine. AtNPR1 and VvNPR1.1 were successfully expressed at relatively high level in leaves of V. vinifera cv Syrah after agroinfiltration. Transient expression of these two signalling genes resulted in increased VvPR1 gene expression in both uninoculated and in P. viticola inoculated leaves. In inoculated tissues, the expected stimulation of PR1 expression by P. viticola was observed; however, PR1 expression was further enhanced in infected leaves overexpressing AtNPR1 or VvNPR1.1. It is likely that the activity of the NPR1 proteins is enhanced by P. viticola inoculation. Moreover, it appeared that VvNPR1.1 had a stronger activity than AtNPR1 on induction of PR1 expression in grapevine.
Transient expression in N. benthamiana and V. vinifera shows that VvNPR1.1 and VvNPR1.2 have a positive activity on the expression of PR1 and PR2 genes (Figure 5). It is thus likely that as in other plant species, VvNPR1 controls the expression of a set of SA-responsive defense genes in grapevine. However, it remains to be determined if VvNPR1.1 and VvNPR1.2 perform different functions in grapevine defense. Arabidopsis genome contains 3 additional genes closely related to AtNPR1, which are likely involved in plant defense responses , and 2 other more distant genes, AtBOP1 (AtNPR5) and AtBOP2 (AtNPR6), with functions in the control of growth asymmetry in leaf and floral patterning . Among NPRs involved in plant defense, phylogenetic analysis revealed that AtNPR1 and AtNPR2 form a subgroup, whereas AtNPR3 and AtNPR4 form a distinct pair . Interestingly, grapevine genome sequencing revealed only two genes related to "defense" AtNPRs. VvNPR1.1 belongs to the subgroup comprising AtNPR1 and AtNPR2, and VvNPR1.2 forms a distinct subgroup with AtNPR3, AtNPR4 and MpNPR1-1 from apple (Figure 1). Curiously, a hallmark of this second subgroup is a high inducibility of gene expression by BTH or its analogs [[18, 36] and this study]. Different members of the AtNPR family appear to mediate different functions in plant defense. AtNPR1 has been identified as a key positive regulator of SA-dependent gene expression that is required for SAR establishment as well as for basal resistance to virulent pathogens . On the other hand, AtNPR3 and AtNPR4 have been proposed to act as negative regulators of plant defense, since the double npr3npr4 mutant shows elevated basal PR1 expression and enhanced resistance to virulent bacterial and oomycete pathogens . However, the negative regulation of defense mechanisms by AtNPR3 and AtNPR4 is in contradiction with another study where npr4 single mutants were shown to be more susceptible to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 . In this study, AtNPR4 was also implicated in the regulation of JA-inducible genes and in the cross-talk between the SA- and the JA-dependent signalling pathways . Even if VvNPR1.2 is closely related to AtNPR3 and AtNPR4, it is likely not acting as a negative regulator of defense genes since its expression in N. benthamiana resulted in enhanced PR1 and PR2 accumulation. Moreover, VvNPR1.2 is closely related to MpNPR1-1, whose overexpression led to activation of PR genes and resistance to bacterial and fungal pathogens in apple  (Figure 1). Therefore, phylogenetic analysis is not sufficient to predict a positive or negative control of defense responses for a given member of the NPR family. However, it is likely that the two NPR1 homologs identified in grapevine do not perform fully overlapping functions. Overexpression or silencing of the two genes in grapevine will help to clarify their respective role in resistance to different pathogens in the future.
We show here that genome sequence resources combined with transient expression in heterologous and homologous systems allow to obtain rapidly functional information on grapevine genes. The upregulation of acidic PR1 and PR2 expression by VvNPR1 both in N. benthamiana and Vitis vinifera strongly suggests that VvNPR1 is a component of the SA defense signalling pathway in grapevine. This implies the existence of highly conserved mechanisms for regulation of defense gene expression among plant species. As a consequence, overexpression of VvNPR1 and other signalling elements has the potential to enhance disease resistance in this crop species. Further work will concentrate on the search for transcription factors interacting with the two VvNPR1 proteins in grapevine, and on the analysis of pathogen tolerance in npr1 mutant and wild type Arabidopsis overexpressing VvNPR1.1 and VvNPR1.2.
Vitis vinifera cv Chardonnay 96 and Vitis riparia cv Gloire de Montpellier were obtained from ENTAV (Etablissement National Technique pour l'Amélioration de la Viticulture, Le Grau du Roi, France). Vitis vinifera cv Syrah was provided by INRA (Colmar, France). These clones were propagated on MS medium supplemented with 20 g.L-1 sucrose and 0.7% bacto-agar in a growth chamber at 25°C, under a 16/8 h photoperiod.
Four-week-old in vitro plantlets of Vitis vinifera cv Chardonnay and Vitis riparia were transferred to potting soil (Fertiligène, NFU 44–571) inside a closed translucide propagator under saturating humidity for 7 days. Plantlet acclimatization was realized by gradually raising the propagator's lid. Plants were grown in potting soil for 3 weeks (22°C, 16/8 h photoperiod, 70% humidity) before use for treatments or pathogen inoculation. Eight-week old in vitro-grown plants from Vitis vinifera cv Syrah were used for Agrobacterium infiltration experiments.
Nicotiana benthamiana plants were grown in potting soil under a 16/8 h photoperiod for 2 weeks prior to be used for Agrobacterium infiltration. Plasmopara viticola was kindly provided by Sabine Merdinoglu (INRA, Colmar, France).
Treatment of plants with chemicals and pathogens
Detached leaves of Vitis vinifera cv Chardonnay were sprayed with a BTH solution (80 mg.L-1, Bion, Syngenta Agro AG, Dielsdorf, Switzerland). Control leaves were sprayed with water. Leaves were maintained in sealed Petri dishes on humid Whatmann 3 MM paper, collected at different time points and immediately frozen in liquid nitrogen.
For Plasmopara viticola inoculation, Vitis vinifera cv Chardonnay and Vitis riparia plantlets were placed in a closed translucide propagator. Abaxial leaf surfaces were sprayed with freshly collected sporangia propagated on V. vinifera cv Chardonnay and resuspended in water at approximately 1.5 × 105 spores.mL-1. Inoculated plants were placed in a growth chamber at 21°C under obscurity for 24 h, then under a 16/8 h photoperiod for 6 days. Inoculation of Vitis vinifera cv Syrah was performed by spraying 104 spores.mL-1 on detached leaves of agroinfiltrated in vitro-cultured plantlets that were maintained in sealed Petri dishes on humid Whatmann paper under conditions described above. Leaves were collected at different time points and immediately frozen in liquid nitrogen.
Cloning of VvNPR1.1 and VvNPR1.2
The nucleic acid sequence of Arabidopsis NPR1 was used to search an EST database of abiotically stressed leaves of V. vinifera cv Chardonnay (EST Analysis Pipeline, ESTAP , using BLASTN. Two EST with significant similarity to AtNPR1 were identified. Full-length cDNA were reconstituted by searching the Genoscope database of grapevine genome sequencing with the two EST previously identified . Full-length cDNAs of VvNPR1.1 (GSVIVT00016536001) and VvNPR1.2 (GSVIVT00031933001) were amplified from reverse-transcribed RNA from SA-treated Chardonnay leaves using Pfx DNA polymerase (Invitrogen). AtNPR1 cDNA was amplified from reverse-transcribed cDNA from Arabidopsis thaliana Col-0 leaves.
For subcellular localization, the AtNPR1, VvNPR1.1 and VvNPR1.2 coding sequences were cloned by Gateway (Invitrogen) recombination reactions into the pK7FWG2 vector , upstream of eGFP.
For transient expression in N. benthamiana and grapevine leaves, full-length AtNPR1, VvNPR1.1 and VvNPR1.2 cDNAs were cloned between the CaMV 35S promoter and the 35S terminator sequences of the pUCAP-intron vector . This vector contains an intron between the promoter and the terminator sequence, which was excised and replaced by NPR1 cDNA sequences. A six histidine tag coding region was added to the 3' end of each cDNA in order to facilitate detection of transgene product. The cassette containing AtNPR1, VvNPR1.1 and VvNPR1.2 between the CaMV 35S promoter and the 35S terminator was excised by AscI/PacI digestion and cloned into the pBINplus vector .
Sequence alignment and phylogenetic analysis
Protein sequence alignment was realized using the ClustalW program. The phylogenetic tree was constructed with the Phylo_win program , using the Neighbor-Joining method. Boostrap values were obtained from 500 replicates.
Gene expression analysis by semi- quantitative PCR and real-time quantitative PCR
RNA extraction and DNase I treatment were performed as described in Chong et al. . Reverse transcription was performed on 0.5 μg of RNA with the iScript cDNA synthesis kit (Biorad), according to the manufacturer's instructions.
Sequence of primers used for semi-quantitative RT-PCR in grapevine
Forward Primer 5' → 3'
Reverse Primer 5' → 3'
Sequence of primers used for real-time PCR in grapevine
Forward Primer 5' → 3'
Reverse Primer 5' → 3'
Transient expression in tobacco and grapevine leaves
For transient expression in tobacco leaves, we used Agrobacterium tumefaciens GV3101 transformed with pK7FWG2 or pBINplus carrying AtNPR1, VvNPR1.1 or VvNPR1.2. An overnight culture of bacteria containing the appropriate construct was resuspended in the same volume of 10 mM MgCl2. Bacterial suspension's concentration was adjusted to OD600 = 0.5 with 10 mM MgCl2. Acetosyringone (200 μM final) was added to the bacterial suspension prior tobacco leave infiltration using a syringe without needle.
Transient expression in grapevine leave experiments was realized as described in Santos-Rosa et al. . A. tumefaciens C58CI culture transformed with pBINplus carrying AtNPR1-His or VvNPR1.1-His was prepared as described . Detached leaves from 8- to 10 week-old grown V. vinifera cv Syrah were submerged abaxial face down in cylindrical flasks (40 mL) containing 7 mL of bacterial culture. Leaves were covered by a disk of Miracloth. Flasks were then placed into a dessicator. Vacuum was applied for 2 min at 15 mm Hg with an oil-pump (GmbH, Type N035.3AN.18). Vacuum was applied twice for each leaf. Leaves were then placed in sealed Petri dishes on humid Whatmann paper for three days before harvest or inoculation with Plasmopara viticola as described in "Biological materials".
Subcellular localization of VvNPR1.1 and VvNPR1.2
AtNPR1, VvNPR1.1 and VvNPR1.2 in pK7FWG2 vector  were transiently transformed into Nicotiana benthamiana by agroinfiltration as described above. Agroinfiltrated leaf sectors were observed 3 days after infiltration. Images were acquired with a LSM510 confocal microscope (Carl Zeiss, software version AIM 4.2), using a 63×, 1.2 NA water immersion objective lens at 23°C. Fluorescence of free GFP or GFP fusion proteins was observed after excitation with a 488 nm laser line, using a 505–550 band-pass emission filter.
Immunoblot analysis of PR proteins
Foliar explants were harvested from N. benthamiana infiltrated with Agrobacterium carrying AtNPR1, VvNPR1.1 and VvNPR1.2 in pK7FWG2 vector, 3 days after infiltration.
Total soluble protein was extracted from leaves by grinding in liquid nitrogen and resuspending the powder in extraction buffer as described . The protein concentration of the extract was determined with the Bio-Rad protein assay. SDS PAGE was carried out according to standard procedures with 10 μg of total proteins. Proteins were electro-transfered on Immobilon P membranes (Millipore, Bedford, MA). Detection was realized with the immune-star chemiluminescent kit (Bio-Rad, Hercules, CA). The blots were probed by using polyclonal antisera raised against an acidic PR-1 isoform (PR 1b, ), a β-1.3 glucanase isoform (PR-2, ) and basic chitinases (PR-3, ) purified from tobacco. Polyclonal antisera were kindly provided by M. Legrand (IBMP, Strasbourg, France) and used at a 1:10 000 dilution. Protein loading was checked by Coomassie Blue staining of membranes. The experiment was performed twice with similar results.
non expressor of PR genes 1
systemic acquired resistance
broad complex, tramtrack and bric a brac/pox virus and zinc finger
ankyrin repeat domain
nuclear localization signal.
We are grateful to Sabine Merdinoglu (INRA, Colmar) for providing Plasmopara viticola isolate, and to Michel Legrand (IBMP, Strasbourg) for the anti-PR protein sera. Special thanks to Christophe Bertsch (LVBE, Colmar) for helpful discussions and to Pierrette Geoffroy (IBMP, Strasbourg) for taking care of N. benthamiana plants. This work was supported by the Université de Haute Alsace and by a doctoral fellowship from the French Ministry of Research to GLH.
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