Characterization of Vitis vinifera NPR1 homologs involved in the regulation of Pathogenesis-Related gene expression
BMC Plant Biology
Characterization of Vitis vinifera NPR1 homologs involved in the regulation of Pathogenesis-Related gene expression
Galle Le Henanff 2
Thierry Heitz 1
Pere Mestre 0
Jerme Mutterer 1
Bernard Walter 2
Julie Chong 2
0 Laboratoire de Genetique et Amelioration de la Vigne, INRA et Universite de Strasbourg (UMR1131) , 28 rue de Herrlisheim, 68000 Colmar , France
1 Departement Reseaux Metaboliques chez les Vegetaux, IBMP du CNRS (UPR2357) , 12 rue du general Zimmer, 67000 Strasbourg , France
2 Laboratoire Vigne, Biotechnologies et Environnement (LVBE , EA3991) , Universite de Haute Alsace , 33 rue de Herrlisheim, 68000 Colmar , France
Background: 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. Results: 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. Conclusion: 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
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 (-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
leucinerich 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).
Amino acid sequence comparisons showed that
VvNPR1.1 has a higher identity with AtNPR1 (55%
identity and 75% similarity) than VvNPR1.2 (40% identity
and 61% similarity with AtNPR1). VvNPR1.1 and
VvNPR1.2 were also compared to NPR1 homologs in
different plant species. Phylogenetic analysis (Figure 1A)
reveals that VvNPR1.1 groups closely with tobacco and
tomato NPR1 proteins (86% and 85% similarity
respectively), with NPR1 from monocots and with AtNPR1 and
AtNPR2. VvNPR1.2 forms a discrete group with NPR1
from apple (87% similarity), AtNPR3 and AtNPR4.
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 VvNPR1.2 expression following BTH
treatment in grapevine leaves
In Arabidopsis, AtNPR1 is constitutively expressed and
can be further stimulated by SA or
2.6-dichloroisonicotinic acid (INA) treatment and by infection with
Hyaloperonospora parasitica . In order to study the expression
profile of the two grapevine NPR1 genes, detached leaves
of Vitis vinifera cv Chardonnay were treated with a
solution of BTH (a SA analog). We also monitored the
expression of a grapevine PR1 gene, a SAR marker, whose
sequence is the most closely related to Arabidopsis
SAdependent PR1 (GSVIVT 00038575001,). As shown
in Figure 2, VvPR1 expression was strongly stimulated by
BTH as soon as 12 h posttreatment compared to
watertreated leaves where VvPR1 expression was almost
undetectable. VvNPR1.1 was constitutively expressed in
watertreated leaves, but expression was only slightly
upregulated by BTH treatment (Figure 2). Interestingly,
VvNPR1.2, whose expression was also detectable in
VvNPR1.1 (584 aa)
VvNPR1.2 (587 aa)
SYPEKGTVKERRQKRMRYNELKNDVKKAYSKDK---VARSCLSSSS--PASSLREALENPT--------------------FCiogmurpear1ison of VvNPR1.1 and VvNPR1.2 with other NPR1 homologs and members of Arabidopsis thaliana NPR family
Comparison of VvNPR1.1 and VvNPR1.2 with other NPR1 homologs and members of Arabidopsis thaliana
NPR family. (A) Phylogenetic tree generated with the Phylo_win program using the neighbour-joining method . Sequence
alignment was previously realized using the ClustalW tool. Accession numbers are: AtNPR1 (At1g64280), AtNPR2
(At4g26120), AtNPR3 (At5g45110), AtNPR4 (At4g19660), AtBOP1 (At3g57130), AtBOP2 (At2g41370), Nicotiana tabacum
(NtNPR1, AAM62410.1), Oryza sativa cv. japonica (OsNPR1, AAX18700.1), Lycopersicon esculentum (LeNPR1, AAT57637.1),
Musa acuminata (MNPR1A, ABI93182.1; MNPR1B, ABL63913.1), Malus domestica (MpNPR1-1, ACC77697.1) and Vitis vinifera
(Genoscope accession numbers: VvNPR1.1, GSVIVP00016536001; VvNPR1.2, GSVIVP00031933001). Bootstrap values based
on 500 replicates are indicated beside the branches. (B) Schematic representation comparing the structure of AtNPR1,
VvNPR1.1 and VvNPR1.2, including the positions of the BTB/POZ domain, the ankyrin repeat domain (ARD) and the nuclear
localization signals (NLS). (C) Multiple alignment of putative nuclear localization signals (NLS) at C-terminus of NPRs from
different plant species. Basic amino acids are highlighted in grey and residues essential for AtNPR1 nuclear localization  are
highlighted in black.
expression was enhanced much earlier in resistant V.
riparia, where transcripts began to accumulate 12 h after
inoculation and were stimulated about 20-fold at 2 days.
In contrast, maximal induction of VvSTS expression was
measured 5 days after inoculation in V. vinifera cv
Chardonnay (Figure 3A). Thus, VvSTS transcript accumulation
was delayed in susceptible V. vinifera cv Chardonnay
compared to resistant V. riparia.
hpt 0 12 24 48 72 96 12 24 48 72 96
tEFrxiegpaurtemrseesin2otn patterns of VvNPR1.1 and VvNPR1.2 upon BTH
Expression patterns of VvNPR1.1 and VvNPR1.2 upon
BTH treatment. Detached leaves of Vitis vinifera cv
Chardonnay were sprayed with a solution of BTH (80 mg.L-1) or
water as control. Samples were collected at different time
points. Hpt: hours post treatment; 0: untreated leaves at the
beginning of the experiment. Actin (VvACT) was used as an
internal control. Primer sequences are listed in table 1.
trol leaves, was further induced by BTH and peaked
between 12 to 48 h after treatment (Figure 2). These
results show that, as observed in Arabidopsis, VvNPR1.1
and VvNPR1.2 are constitutively expressed in grapevine
and that VvNPR1.2 expression can be further enhanced by
a SAR inducer.
Expression patterns of VvNPR1.1 and VvNPR1.2 during
compatible and incompatible interactions with
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
realtime 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 .
Five days after inoculation with P. viticola, a number of
necrotic spots were observed on leaves of the resistant
species V. riparia, whereas sporangia covered almost the
entire leaf surface of the susceptible V. vinifera (data not
shown). Expression of a stilbene synthase gene (VvSTS)
was determined as a positive control of defense gene
induction by P. viticola infection. As expected,P. viticola
inoculation triggered VvSTS expression in both
susceptible and tolerant Vitis species (Figure 3A). However, VvSTS
wPRe1e.n2gdruarpienvginaecaonmdExpression patterns of VvNPR1.1 and VvNPR1.2
during a compatible or an incompatible interaction
between grapevine and Plasmopara viticola. Leaves of
plantlets of Vitis vinifera cv Chardonnay (grey bars) and Vitis
riparia cv Gloire de Montpellier (dark bars) were inoculated
with Plasmopara viticola (1.5 105 spores mL-1). Control
leaves were sprayed with water. Leaves were collected at
different time points as indicated. Hpi: Hours post inoculation.
Transcript levels of each gene (Stilbene synthase VvSTS (A);
VvNPR1.1 (B); VvNPR1.2 (C)) were normalized to actin
transcript levels. The fold induction indicates normalized
expression levels in inoculated leaves compared to normalized
expression levels observed in water-treated leaves at the
same time point. Expression ratio at the beginning of the
experiment (0) is set to 1. Mean values and standard
deviations were obtained from 2 duplicate experiments.
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
Subcellular localization of VvNPR1.1 and VvNPR1.2
The amino acid sequences of both VvNPR1.1 and
VvNPR1.2 were found to contain a putative nuclear
localization signal (NLS1) in the C terminus of the protein
(Figure 1C). To determine the subcellular localization of
VvNPR1.1 and VvNPR1.2, the coding regions of
VvNPR1.1, VvNPR1.2, and AtNPR1 were fused to
5'-terminus of eGFP under the control of the CaMV 35S promoter.
The resulting constructs were introduced into Nicotiana
benthamiana following transient transformation by
agroinfiltration. Leaf sectors of agroinfiltrated N.
benthamiana were observed 3 days after infiltration for GFP
fluorescence by confocal microscopy (Figure 4). GFP
fluorescence levels were comparable with the 3
constructions studied. Control leaves expressing free GFP yielded
a weak fluorescence predominantly visible in the
cytoplasm (Figure 4A and 4B). As described previously ,
the AtNPR1-GFP fusion protein fluorescence strongly
labelled the nucleus (Figure 4C and 4D). Consistent with
the presence of the NLS1, VvNPR1.1-GFP and
VvNPR1.2GFP fusion proteins were localized to the nucleus and to
a lesser extent to the cytoplasm both in mesophyll and
epidermal cells (Figure 4E and 4F). Localization of GFP
fluorescence to nucleus was further observed in cells from
peeled epidermis transiently transformed with VvNPR1.1
(Figure 4G and 4H). Treatment of N. benthamiana leaves
with SA 48 h before observation did not influence the
localization of the fusion proteins (data not shown).
Transient expression of VvNPR1.1 and VvNPR1.2 in N.
benthamiana triggers the accumulation of acidic PR1 and
PR2 but not of PR3
To investigate if VvNPR1.1 and VvNPR1.2 could control
the expression of PR genes (especially the PR1 gene), PR
protein accumulation was analyzed after transient
expression of AtNPR1-GFP, VvNPR1.1-GFP and VvNPR1.2-GFP.
Leaves of N. benthamiana were analyzed 3 days after
agroinfiltration for PR protein production by Western blot
with anti sera raised against tobacco PR proteins. PR
proteins were undetectable in untreated leaves (Figure 5).
Transient expression of AtNPR1-GFP, VvNPR1.1-GFP and
VvNPR1.2-GFP was sufficient to trigger accumulation of
acidic PR1, in contrast to expression of empty vector
(encoding free GFP) which produced no signal (Figure 5).
SFuigbucerlelu4lar localization of VvNPR1.1 and VvNPR1.2
Subcellular localization of VvNPR1.1 and VvNPR1.2.
N. benthamiana leaves were infiltrated with A. tumefaciens
GV3101 containing empty vector (pK7FWG2) encoding free
GFP (A, B), or AtNPR1 (C, D), VvNPR1.1 (E, G, H), and
VvNPR1.2 (F) in pK7FWG2. Confocal images were captured
3 days after infiltration. GFP images (A, C, E, F, G) and
differential contrast images (B, D, H) of N. benthamiana epidermal
cells were compared to show the subcellular localization of
GFP, AtNPR1-GFP, VvNPR1.1-GFP and VvNPR1.2-GFP. Bar
= 10 M.
In order to determine if another marker of the SA pathway
could be enhanced by VvNPR1 expression, the same
analysis was performed to detect acidic -1.3 glucanase (PR2).
Agroinfiltration of vector alone triggered the expression of
PR2 compared to infiltration with H2O (Figure 5).
However, transient expression of AtNPR1-GFP, VvNPR1.1-GFP
and VvNPR1.2-GFP induced a stronger accumulation of
PR2 compared to infiltration with empty vector (Figure
5). In order to determine if PR protein induction by
AtNPR1 and VvNPR1 is specific of SA signalling, we
analyzed the accumulation of basic chitinase (PR3), a
SAindependent marker whose expression is controlled by
the JA/ET pathway in Arabidopsis . Anti-PR3 serum
recognized two proteins of 32 and 34 kDa corresponding to
the two basic chitinase isoforms described in tobacco
[, Fig 5]. Similarly to PR2, agroinfiltration with empty
vector triggered the expression of PR3 compared to
infiltration with H2O (Figure 5). However, in contrast to PR1
and PR2, expression of AtNPR1-GFP, VvNPR1.1-GFP and
VvNPR1.2-GFP did not modify significantly PR3
accumulation compared to empty vector (Figure 5).
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
ItFnriadgnuuscriteeinot5neoxfpPrRes1siaonndoPfRV2v NacPcRu1m.1ualantdioVnviNnPNR.1b.2enthamiana by
Induction of PR1 and PR2 accumulation in N.
benthamiana by transient expression of VvNPR1.1 and
VvNPR1.2. N. benthamiana leaves were infiltrated with water
(H2O) or A. tumefaciens GV3101 containing VvNPR1.1,
VvNPR1.2, or AtNPR1 in pK7FWG2 or empty vector. Leaves
were harvested 3 days after agroinfiltration. Soluble proteins
were extracted, submitted to SDS-PAGE and probed with
sera against tobacco PR1, PR2 or basic chitinases (PR3).
Transient expression of AtNPR1 and VvNPR1.1 in
grapevine leaves enhances accumulation of VvPR1
Heterologous expression in N. benthamiana showed that
VvNPR1.1 and VvNPR1.2 were able to trigger the
accumulation of acidic PR1 and PR2 in the absence of pathogen
inoculation. To evaluate the effect of VvNPR1 expression
in a homologous system (Vitis vinifera), we used a recently
described protocol of transient gene expression by
vacuum agroinfiltration in grapevine . AtNPR1 and
VvNPR1.1, which is the most closely related to AtNPR1,
were transiently expressed in leaves of V. vinifera cv Syrah,
a genotype showing high efficiency of transient expression
. Gene expression was first analyzed 3 days after
agroinfiltration. Grapevine leaves were also later
inoculated with P. viticola 3 days after agroinfiltration and
analyzed 2 days after oomycete inoculation. To confirm that
AtNPR1 and VvNPR1.1 were expressed in agroinfiltrated
grapevine leaves, we monitored the accumulation of full
length transgene-derived mRNAs of AtNPR1 and
VvNPR1.1 by RT-PCR as shown in Figure 6. No PCR
amplification was revealed when omitting the reverse
transcription step (data not shown).
Real time quantitative PCR was used to study the
expression of VvPR1 and VvSTS in grapevine leaves expressing
AtNPR1 and VvNPR1.1, 3 days after agroinfiltration. As
shown in Figure 7A, infiltration with empty vector
stimulated the expression of VvPR1, probably because of the
agroinfiltration stress. Interestingly, in leaves expressing
AtNPR1 and VvNPR1.1, a stronger increase in VvPR1
transcript accumulation was measured (Figure 7A). In
contrast, no significant increase in VvSTS transcript
accumulation was measured in leaves expressing AtNPR1
and VvNPR1.1 compared to H2O-infiltrated leaves (Figure
7B). In another experiment, we inoculated grapevine
leaves with P. viticola 3 days after agroinfiltration and
analyzed gene expression 2 days after inoculation. VvPR1
expression was induced by fungal infection as expected.
Consistent with the results obtained in uninoculated
leaves, VvPR1 stimulation in infected leaves was clearly
higher in leaves expressing AtNPR1 and VvNPR1.1 than in
leaves preinfiltrated with control Agrobacterium (Figure
7C). Although VvSTS expression was stimulated 3 fold by
infection, no significant effect on its expression was
observed when leaves were preinfiltrated with the
different constructs (Figure 7D).
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
gFDrieagtpueercvetiino6enloeafvAetsNPR1 and VvNPR1.1 transgene expression in
Detection of AtNPR1 and VvNPR1.1 transgene
expression in grapevine leaves. Leaves from in vitro grown V.
vinifera cv Syrah were infiltrated with A. tumefaciens transformed
with pBIN+ carrying AtNPR1 or VvNPR1.1. Control plants
were infiltrated with water. Infiltrated leaves were challenged
with P. viticola 3 days after agroinfiltration. Total RNAs were
extracted 3 days after agro-infiltration (uninoculated) and 2
days after P. viticola inoculation. Full-lenght mRNA from each
transgene was specifically amplified after reverse
transcription with primers listed in table 1. VvACT was used as internal
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
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
EFxigpurersesi7on of VvPR1 and VvSTS after transient overexpression of AtNPR1 and VvNPR1.1 in grapevine leaves
Expression of VvPR1 and VvSTS after transient overexpression of AtNPR1 and VvNPR1.1 in grapevine leaves. (A,
B) Expression levels of VvPR1 (A) and VvSTS (B), in uninoculated leaves, 3 days after agro-infiltration. (C, D) Expression levels of
VvPR1 (C) and VvSTS (D) in uninoculated and inoculated leaves. Leaves were infiltrated with Agrobacterium carrying the different
constructs and expression of VvPR1 and VvSTS was analyzed 3 days later (grey bars). Three days after agroinfiltration, leaves
were inoculated with P. viticola and expression of genes of interest was analyzed 2 days after inoculation (black bars). Fold
induction indicates expression levels in agroinfiltrated leaves compared to the expression in non-inoculated water-infiltrated
leaves, which is set to 1. Mean values and standard deviations were obtained from 2 duplicate experiments.
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
ethyleneresponsive 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
Forward Primer 5' 3'
Reverse Primer 5' 3'
a Genbank accession number
b Genoscope Grape Genome Browser number
ling 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
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
crosstalk between the SA- and the JA-dependent signalling
pathways . Even if VvNPR1.2 is closely related to
Forward Primer 5' 3'
Reverse Primer 5' 3'
a Genbank accession number
b Genoscope Grape Genome Browser number
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
MpNPR11, 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'Amlioration 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
25C, 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
(Fertiligne, NFU 44571) 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
(22C, 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,
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
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 21C 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
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 g of RNA with the iScript cDNA
synthesis kit (Biorad), according to the manufacturer's
Semi-quantitative RT-PCR was performed by using
recombinant Taq DNA polymerase (Invitrogen, Cergy Pontoise,
France). Control reactions to normalize RT-PCR were
done with primers derived from grapevine actin
sequences. PCR on serial dilutions of cDNA were
performed at 55C and 29 cycles to define semi-quantitative
conditions that resulted in amplification linear to RNA
amounts. The experiments were performed twice with
similar results. Primers used for PCR are listed in table 1.
For real time PCR, reactions were carried out on the
iCycler system (Biorad, Marnes-la-Coquette, France). PCR
reactions were carried out in triplicates in a reaction buffer
containing 1 iQ SYBR Green Supermix, 0.2 mM of
forward and reverse primers and 10 ng of reverse transcribed
RNA in a final volume of 25 l. Thermal cycling
conditions were: 30s at 95C followed by 40 cycles of 15s at
94C, 30s at 60C and 30s at 72C. Acquisition
temperatures were 83C for VvPR1 and 77C for VvACT, VvSTS,
VvNPR1.1 and VvNPR1.2. The calibration curve for each
gene was obtained by performing real-time PCR with
serial dilutions of the cloned cDNA fragment (from 102 to
108 cDNA copy number). The specificity of the individual
PCR amplification was checked using a heat dissociation
curve from 55 to 95C following the final cycle of the
PCR. The results obtained for each gene of interest at each
time point were normalized to the expression of a
reference gene (VvACT1) and fold induction compared to H2O
treatment was calculated as described by Pfaffl et al .
Mean values and standard deviations were obtained from
2 duplicate experiments and are representative of 2
independent experiments. Primers used for real-time
quantitative PCR are listed in table 2.
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 M final) was added to the bacterial
suspension prior tobacco leave infiltration using a syringe
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
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 23C. Fluorescence of
free GFP or GFP fusion proteins was observed after
excitation with a 488 nm laser line, using a 505550 band-pass
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 g 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 -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.
NPR1: non expressor of PR genes 1; SA: salicylic acid;
BTH: benzothiadiazole; INA: 2.6-dichloroisonicotinic
acid; JA: jasmonic acid; ET: ethylene; PR: pathogenesis
related; SAR: systemic acquired resistance; BTB/POZ:
broad complex, tramtrack and bric a brac/pox virus and
zinc finger; ARD: ankyrin repeat domain; NLS: nuclear
GLH carried out most of the experiments, ie, gene cloning
and phylogenetic analyses, expression studies, transient
expression in grapevine, and participated in transient
expression in N. benthamiana.
TH participated in the design of the study, gave advices for
GFP localization experiments and helped to draft the
PM participated in the design of the study and helped for
transient expression in V. vinifera.
JM did the confocal microscopy observations.
JC carried out transient expression in N. benthamiana and
western blot analyses, performed conceptual and
experimental design and drafted the manuscript.
All authors read and approved the final manuscript.
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
Universit de Haute Alsace and by a doctoral fellowship from the French Ministry
of Research to GLH.
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