A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence
Journal of Experimental Botany
A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence
Yuntong Jiao 0 1 2
Weirong Xu 0 1 2
Dong Duan 3
Yuejin Wang 0 1 2
Peter Nick 3
Robert Hancock, The James Hutton Institute
0 State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A & F University , Yangling, Shaanxi 712100 , People's Republic of China
1 Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture , Yangling 712100, Shaanxi , People's Republic of China
2 College of Horticulture, Northwest A & F University , Yangling 712100, Shaanxi , People's Republic of China
3 Molecular Cell Biology, Botanical Institute 1, Karlsruhe Institute of Technology , Kaiserstr. 2, D-78133 Karlsruhe , Germany
Stilbenes are central phytoalexins in Vitis, and induction of the key enzyme stilbene synthase (STS) is pivotal for disease resistance. Here, we address the potential for breeding resistance using an STS allele isolated from Chinese wild grapevine Vitis pseudoreticulata (VpSTS) by comparison with its homologue from Vitis vinifera cv. 'Carigane' (VvSTS). Although the coding regions of both alleles are very similar (>99% identity on the amino acid level), the promoter regions are significantly different. By expression in Arabidopsis as a heterologous system, we show that the allele from the wild Chinese grapevine can confer accumulation of stilbenes and resistance against the powdery mildew Golovinomyces cichoracearum, whereas the allele from the vinifera cultivar cannot. To dissect the upstream signalling driving the activation of this promoter, we used a dual-luciferase reporter system in a grapevine cell culture. We show elevated responsiveness of the promoter from the wild grape to salicylic acid (SA) and to the pathogen-associated molecular pattern (PAMP) flg22, equal induction of both alleles by jasmonic acid (JA), and a lack of response to the cell death-inducing elicitor Harpin. This elevated SA response of the VpSTS promoter depends on calcium influx, oxidative burst by RboH, mitogen-activated protein kinase (MAPK) signalling, and JA synthesis. We integrate the data in the context of a model where the resistance of V. pseudoreticulata is linked to a more efficient recruitment of SA signalling for phytoalexin synthesis.
Basal immunity; defence; grapevine (Vitis pseudoreticulata); powdery mildew; promoter activity; SA; signalling; stilbene synthase
As one of the most ancient crops, grapevine (Vitis vinifera L.)
is of economic and cultural significance. However, this crop
is challenged by several diseases such as downy and powdery
mildew, leading to a requirement for intensive plant
protection. For instance, ~70% of European fungicide production
is used for viticulture (Eurostat, 2007). This expensive
application produces a negative ecological footprint, and is far
from sustainable due to the rapid spread of fungicide
resistance through the pathogen population. So far, resistance
breeding has been the most successful strategy for sustainable
© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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viticulture and cost reduction (Eibach et al., 2007). Resistance
factors from non-vinifera species, such as the locus Rpv3 (for
Resistance to Plasmopara viticola) or the locus Ren3 (for
Resistance to Erysiphe necator) from North American wild
grapes have been used successfully for introgression into
V. vinifera cultivars that are used commercially (Welter et al.,
2007; Di Gaspero et al., 2012). However, the success of these
resistant varieties is progressively challenged by the occurrence
of new pathogen strains that have already acquired strategies
to circumvent the resistance mediated by these resistance loci
(Peressotti et al., 2010; Gómez-Zeledón et al., 2013),
stimulating the search for alternative mechanisms of resistance.
Plant innate immunity consists of two layers (Jones and
Dangl, 2006; Boller and He, 2009) that have to be
understood from the co-evolutionary interaction between host and
pathogen. The more ancient pathogen-associated molecular
pattern (PAMP)-triggered immunity (PTI) is activated by
molecules termed PAMPs or microbe-associated
molecular patterns (MAMPs), which are ubiquitous in pathogenic
microorganisms and can be recognized by receptors at the
plasma membrane. During co-evolution with their hosts,
many pathogens have developed chemical signals, so-called
effectors, to quell PTI. This will allow them to reinstate
pathogenicity, but it will also initiate a second cycle of warfare,
whereby the host cell can recognize the effectors by
resistance proteins, predominantly nucleotide-binding-leucine-rich
repeat (NB-LRR) receptors, and initiate a second layer of
defence, termed effector-triggered immunity, or ETI (Takken
and Tameling, 2009). This ETI often (but not necessarily)
culminates in a hypersensitive response (HR) of the infected cell
as the most efficient strategy to contain the intruder.
The canonical form of ETI is linked with specific strains of
the pathogen that infect specific host genotypes based on a
coevolutionary history. In fact, both downy and powdery mildew
have co-evolved with wild grapevine species in North America,
and these grapevines have evolved resistance accompanied by
HR. When these highly advanced biotrophic pathogens were
unintentionally introduced into Europe in the second half of
the 19th century, they encountered the completely naive host
V. vinifera with devastating economic consequences. For downy
mildew, the existence of ETI in sensu stricto is still under debate,
but is supported by the recent discovery of host-specific
pathogen strains (Gómez-Zeledón et al., 2013; Rouxel et al., 2013).
In contrast, strain specificity for germplasm derived from wild
North American grapes has been shown repeatedly for
powdery mildew (Ramming et al., 2012; Feechan et al., 2015),
supporting the existence of an ETI in senso strictu.
There is a current debate about the existence of ETI against
powdery mildew in non-American grapes (Qiu et al., 2015).
This debate is mainly stimulated by claims of cell
deathrelated defence resembling a HR. It should be kept in mind,
however, that ETI has to meet three criteria: (i) it originates
from a co-evolutionary history between host and pathogen;
(ii) it is strain specific; and (iii) the cell death is not necrotic,
but programmed. The first criterion is certainly not met,
because powdery mildew arrived in China only in the 1950s
(Wang, 1993; Wang et al., 1995). The second criterion would
require a study where different strains of powdery mildew are
compared, and has, to the best of our knowledge, only been
addressed and confirmed for the North American resistance
loci (Ramming et al., 2012; Feechan et al., 2015). Even the
third criterion, cell death of the programmed type, is
controversial. For instance, the factor Ren4 identified in the Chinese
species V. romanetii confers a strain-independent
resistance based on penetration barriers (Ramming et al., 2010).
However, in a recent review (Qiu et al., 2015), unpublished
data on Ren4 are mentioned that are claimed to show a
programmed cell death-based mechanism.
Also, VvPEN1 was proposed to cause penetration resistance
against incompatible strains of powdery mildew (Feechan
et al., 2013b). Although only wild North American
grapevines have co-evolutionary history with downy and powdery
mildew, the use of non-American Vitis germplasm has great
potential. For instance, the resistance loci, Ren6 and Ren7,
from the wild Chinese grapevine species V. piasezkii confer
good immunity to powdery mildew. Whether this immunity
is caused by programmed cell death as claimed by the authors
of that study (Pap et al., 2016) does not clearly verify the fact
that such factors are highly valuable. Even if this, still
unpublished, claim is substantiated, it would remain to be elucidated
whether there is strain specificity as a central pre-condition for
ETI. The discovery of a resistance locus, Ren1, in two local
grapevine varieties ‘Kishmish Vatkana’ and ‘Kara Djandal’
from Usbekistan (Hoffmann et al., 2008), that later turned out
to be linked to a region rich in NB-LRR genes, has fostered
speculation about the presence of ETI in this Non-American
germplasm, leading to the suggestion of a potential and
hitherto overlooked co-evolution with powdery mildew (Riaz
et al., 2013). The history of these varieties that derive from the
Sultanina/Thompson Seedless lineage has been described in
detail in Coleman et al. (2009). Most NB-LRR genes encode
R-proteins, and have been proposed to derive from genes that
had been recruited into a defence context (Ellis and Jones,
1998). In fact, functions of NB-LRR proteins in cytokinin
signalling (Igari et al., 2008) or phytochrome-dependent shade
avoidance (Faigón-Soverna et al., 2006) support a scenario
where this evolutionarily ancient class of protein had
undergone a functional shift from developmental signalling
(possibly competitor sensing) to immunity (reviewed in Johnson
et al., 2003). Thus, while the presence of an NB-LRR gene in
the Ren1 locus triggering a cell death-related immunity
provides an attractive possibility, alternative mechanisms should
also be considered. For instance, the Ren1 locus also harbours
a cinnamyl alcohol dehydrogenase (Coleman et al., 2009),
a member of the phenylpropanoid pathway that has been
shown to be an important resistance factor against powdery
mildews, because it is crucial for papilla formation (Bhuiyan
et al., 2009). Thus, whether ETI for powdery mildew exists
outside of the Non-American grapes remains to be elucidated.
However, it should be kept in mind that ETI is not the only
path to success, and a co-evolutionary history is not a conditio
sine qua non for resistance.
Although the receptors triggering PTI and ETI are thought
to differ, the molecular events underlying signalling are
partially shared: these include calcium influx, activation of the
apoplastic oxidative burst, MAPK (mitogen-activated protein
kinase) cascades, and transcriptional activation (Schwessinger
and Zipfel, 2008; Nürnberger and Kemmerling, 2009; Tsuda
and Katagiri, 2010). However, PTI usually does not lead to
programmed cell death, whereas ETI in most (but not in
all) cases does (Thomma et al., 2011). Comparative studies
in grapevine cells have revealed that the differential output
of defence is associated with differences in the relative
timing of the early signalling events (Chang and Nick, 2012;
Chang et al., 2016). The phytohormones jasmonic acid (JA)
and salicylic acid (SA) have been documented to play central
roles in this context. The JA pathway is mainly induced by
and involved in regulating resistance against herbivores and
necrotrophic pathogens (i.e. a type of defence where
programmed cell death is not observed). Also in grapevine cell
cultures, accumulation of jasmonates was only observed in
PTI triggered by the PAMP flg22, but not in cell death-related
immunity triggered by the elicitor Harpin (Chang et al., 2016).
In contrast, the SA pathway is primarily activated by and
involved in mediating resistance against biotrophic pathogens
(i.e. under conditions where hypersensitive cell death occurs;
Glazebrook, 2005). SA is also required for systemic acquired
resistance (SAR) (Durrant and Dong, 2004).
Stilbenes, as the central grapevine phytoalexins, confer
resistance to a broad spectrum of pathogens (Adrian et al.,
1997; Schnee et al., 2008). In particular, the non-glycosylated
resveratrol has attracted increasing attention for its medical
benefits (Roupe et al., 2006). The first product of the
committed stilbene branch of the phenylpropanoid pathway is
synthesized from coumaroyl-CoA and malonyl-CoA by the
enzyme stilbene synthase (STS). This key enzyme belongs
to the type-III group of the polyketide synthase enzyme
superfamily, which has strongly expanded in grapevine with
48 members, at least 32 of which are potentially functional
(Parage et al., 2012). The large number of STS genes with
potential functions already indicates the importance of
stilbenes for defence in grapevine. In fact, an extensive screen
in V. sylvestris, the wild ancestor of the cultivated grapevine
V. vinifera, demonstrated a correlation between the
inducibility of resveratrol accumulation and performance against
infection with downy mildew (Duan et al., 2015). In addition,
the relationships between susceptible and resistant grapevine
varieties according to stilbene concentrations in response to
powdery mildew had been described by Schnee et al. (2008).
However, the induction of STS is not confined to basal
immunity, but is also observed in the context of cell
deathrelated defence in grapevine (Chang and Nick, 2012). STS
alleles with elevated responsiveness of their promoters might
therefore be an interesting target for future resistance
breeding. Non-American wild species of Vitis have not co-evolved
with downy or powdery mildew and thus are unlikely to launch
ETI directed against these pathogens. Nevertheless, the
abovementioned factors Ren6 and Ren7 (Pap et al., 2016), Ren4
(Ramming et al., 2010; Qiu et al., 2015), and Ren1 (Hoffmann
et al., 2008; Coleman et al., 2009) provide examples for such
factors conferring resistance to powdery mildew.
Already more than two decades ago, 18 Vitis species native
to China were probed for resistance to powdery mildew (Wang
et al., 1995). This approach had uncovered a genotype in the
Chinese wild grape V. pseudoreticulata which was resistant to
powdery mildew (Wang et al., 1995). From this genotype, a
specific STS allele was isolated that differed upon
heterologous expression in tobacco as monitored by a β-glucuronidase
(GUS) reporter. The STS promoter from V. pseudoreticulata
was not induced by methyl jasmonate (MeJA), and was not
responsive to the necrotrophic pathogen Alternaria alternata
(Xu et al., 2011). However, the same promoter was activated
after inoculation with the biotrophic pathogen Erysiphe
necator (the causative agent of grapevine powdery mildew) or by
treatment with SA (Xu et al., 2010). In contrast, the STS
promoters from the vinifera cultivars ‘Carigane’ and ‘Thompson
Seedless’ showed the inverse pattern of regulation.
When expression of different STS alleles can be
differentially activated either by necrotrophic pathogens/JA or by
biotrophic pathogens/SA, this leads to the question of whether
STS promoters can differentially recruit JA and SA signalling,
whether activation of STS is sufficient to ward off biotrophic
pathogens, at what point the two signal chains converge, and
which signalling events differ between them. We addressed
these questions using two strategies. First, we introduced
the STS promoters of V. pseudoreticulata and the reference
promoter from the V. vinifera cultivar ‘Carigane’ driving an
STS coding sequence into Arabidopsis as a heterologous
system otherwise not capable of stilbene synthesis. In the second
approach, we analysed the two promoters in a homologous
promoter–reporter system (a V. vinifera cell culture) to dissect
the upstream signals conferring the response to SA. We
provide evidence for a model where STS can be activated by two
pathways that both employ the signalling chain driving basal
immunity. One pathway passes through activation of MYB14
and depends on jasmonate, and the second pathway acts
independently of MYB14 and integrates SA into basal immunity.
This second pathway is more efficiently recruited by the STS
allele from the wild Chinese grapevine V. pseudoreticulata.
Materials and methods
The powdery mildew-resistant Chinese wild V. pseudoreticulata
accession Baihe-35-1 (Wang et al., 1995) and the powdery
mildewsusceptible V. vinifera cv. ‘Carigane’ were cultivated in the Grape
Repository of Northwest A&F University, Yangling, Shaanxi,
China. Arabidopsis ecotype Columbia were grown on Murashige
and Skoog (MS) medium agar plates at 22 °C for 10–14 d, then
transferred into a mix of peat moss, perlite, and vermiculite (3:1:1,
by vol.), and cultivated under a 16 h light/8 h dark regime at 22 °C
with 75% humidity.
Isolation of STS promoter fragments and plasmid constructs
Genomic DNA from the leaves of V. pseudoreticulata and V. vinifera
cv. ‘Carigane’ was extracted using the CTAB
(cetyltrimethylammonium bromide) protocol (Lodhi et al., 1994) and then used as template.
Two fragments encoding the STS coding region and the respective
native upstream promoter were amplified by PCR using the primers
(Supplementary Table S1 at JXB online) designed according to Xu
et al. (2011). Their chromosomal location was confirmed by a BLAST
search in the Genoscope Genome Browser (http://www.genoscope.
cns.fr/blat-server/cgi-bin/vitis/webBlat). Both genomic fragments
were inserted into the pART-CAM-S vector (Xu et al., 2014) after
restriction by XhoI and SacI. From the amplified genomic sequences,
one sense primer and two antisense primers (Supplementary Table S1)
were designed to isolate the STS promoters. The verified promoter
sequences were analysed for the presence of putative cis-elements using
the PLACE (plant cis-acting regulatory DNA elements) algorithms.
Subsequently, both amplified promoter fragments were cloned into
the SacI/NcoI site of pCAMBIA1301 (http://www.cambia.org/daisy/
cambia/585.html). Each of these constructs was individually
introduced into Agrobacterium tumefaciens strain GV3101 by
electroporation (Mersereau et al., 1990). To conduct the transient luciferase assay,
the STS promoter regions were amplified with Q5 High-Fidelity
DNA Polymerase (NEB) using the specific primers (Supplementary
Table S1) to generate attB-PCR products. After Gateway® BP and
LR recombination, the promoter regions of VpSTS and VvSTS were
transferred into the pLuc luciferase vector (Horstmann et al., 2004)
and confirmed by DNA sequencing.
Pathogen inoculation and microscopic analysis of colonization
Golovinomyces cichoracearum strain UCSC1 (Wen et al., 2011) was
maintained on living Arabidopsis mutant pad4 plants to generate
fresh inocula. Inoculation was carried out as previously described
(Xiao et al., 1997). Fungal structures in inoculated leaves were
stained blue by trypan blue and observed by bright-field microscopy
(Xiao et al., 2003).
Arabidopsis transformation and GUS assays
Arabidopsis transformation was carried out using the floral dipping
method (Clough and Bent, 1998). The T3 generation of transgenic
Arabidopsis lines were inoculated either with aseptic water or with
the pathogen and then used for histochemical and quantitative
assays of GUS activity as described by Jefferson et al. (1987);
experiments were carried out in triplicate using three independent lines.
Quantification of gene expression by qRT-PCR
Total RNA was extracted from leaf samples of transgenic Arabidopsis
using the EZNA® Total RNA kit II (Omega Bio-tech), and then
transcribed into cDNA with Prime Script Reverse Transcriptase
(TaKaRa) according to the manufacturer’sְ instructions. Real-time
quantitative PCR (qRT-PCR) was conducted by a two-step protocol
as described in Xu et al. (2011), using AtGAPDH (AT1G13440) as
the internal standard. Primers used in qPCR experiments are listed
in Supplementary Table S1; experiments were repeated three times.
Biolistic transformation and treatment
The grapevine cell suspension culture V. vinifera cv. ‘Pinot Noir’
(Seibicke, 2002) cultivated in MS medium was collected at day 4 after
subcultivation (just at the end of the cycling phase) and transiently
transformed by particle bombardment as described by Maisch et al.
(2007). After expression for 48 h, the transgenic suspension cells were
subjected to the various treatments before assaying the luciferase
activity. All transfection experiments were carried out in triplicate
and each set of promoter experiments was repeated at least twice.
For the induction treatments, the bacterial peptide flg22, the
bacterial elicitor Harpin, SA, and MeJA were prepared as described
previously (Duan et al., 2016). The UV-C induction was performed
as described by Duan et al. (2015). For inhibition treatments, the
transgenic suspension cells were pre-treated with the respective
inhibitors for 30 min before SA was administered.
2-(2-Amino-3methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), diphenylene
iodonium chloride (DPI), gadolinium chloride (GdCl3), and
phenidone were prepared as described by Duan et al. (2016). All
experiments were accompanied by controls, where each inhibitor was
added without SA to assess the impact of inhibitors on cells. All
treatments were accompanied by solvent controls, with the maximal
concentration of the solvent not exceeding 0.1%.
After the transgenic suspension cells had been treated for 6 h at
22 °C in the dark, the harvested cells were collected to measure
luciferase activities using the dual-luciferase reporter assay system
as described previously (Czemmel et al., 2009; Höll et al., 2013). The
relative luciferase activity was calculated as mean values of firefly
and Renilla luciferase ratios after subtraction of the cell background
(cells that had not been bombarded).
Isolation and analysis of two STS alleles and their
To verify the different patterns of expression and
hormonal regulation observed for the STSs from the Chinese wild
V. pseudoreticulata accession Baihe-35-1 versus V. vinifera cv.
‘Carigane’, genomic fragments containing the STS coding
region and its native promoter were amplified by PCR using
genomic DNA isolated from the two genotypes. The
PCRamplified fragment of V. pseudoreticulata (VpSTS) was 3802 bp
in length, of which the promoter accounted for 2264 bp, while
the fragment amplified from V. vinifera cv. ‘Carigane’ (VvSTS)
was significantly shorter (3559bp), due to a shorter (2021 bp)
promoter. Using the Genoscope Genome Browser, both
amplified fragments could be assigned to the same locus in
chromosome 16 (Fig. 1B). The alignment revealed a highly conserved
coding region with 98.63% identity at the nucleotide level
and 99.49% identity at the amino acid level (Fig. 1A, C, D).
In contrast, the promoter sequences, despite some degree of
congruence, displayed specific differences. In particular, the
promoter of the wild allele (VpSTS) harboured two stretches
(of 13 bp and 244 bp, respectively) that were absent in the
VvSTS allele, whereas the VpSTS allele lacked a stretch of
18 bp found in VvSTS. Comparison of the two promoters with
respect to predicted cis-regulatory elements revealed that both
promoter alleles shared somef putative stress-responsive
elements: WBOXATNPR1 was reported to confer a response to
SA (Yu et al., 2001); WBOXNTERF3 has been shown to drive
response to wounding (Nishiuchi et al., 2004); CCAATBOX1 is
linked to responsiveness to heat (Rieping et al., 1992); CBFHV
has been shown to be the binding site for CBF1 during the
response to dehydration (Xue et al., 2002); MYBCORE was
found to be involved in the regulation of flavonoid biosynthesis
(Solano et al., 1995); and GT1GMSCAM4 has been linked to
the response to salt and biotic stress (Park et al., 2004).
The two STS promoter alleles respond differentially to
To test whether the structural differences between the VpSTS
and VvSTS promoters are of functional relevance, the 2264 bp
(VpSTS) and the 2021 bp (VvSTS) promoter fragments were
inserted into SacI and NcoI sites of pCAMBIA1301, replacing
the Cauliflower mosaic virus (CaMV) 35S promoter region and
placed upstream of a GUS reporter, then transformed in a stable
manner into Arabidopsis as the heterologous system using the
floral dipping method (Clough and Bent, 1998). After selection
by 25 mg l–1 hygromycin, and verification of the transgene by
genomic PCR, the basal expression was assessed (Fig. 2). In
the next step, the response of the two promoters to a
compatible strain of powdery mildew UCSC1 (G. cichoracearum)
was analysed (Fig. 3). A CaMV-35S::GUS construct, used
as a positive control (Fig. 2A), showed, as expected, a strong
expression in all tested tissues including seedling, root, stem,
leaf, bud, and silique. In contrast, the two STS promoters were
active at a much weaker level, with VpSTS being active in all
tissues, whereas the activity of the VvSTS promoter could only
be measured in leaves and even here its activity was
considerably lower than that of the VpSTS promoter (Fig. 2B).
To assess whether the VpSTS and VvSTS promoters can be
induced by powdery mildew, GUS expression was followed by
X-Gluc staining (Fig. 3A), qRT-PCR analysis of GUS
transcripts (Fig. 3B), and quantification of GUS enzymatic
activity (Fig. 3C) in leaves after inoculation with the compatible
powdery mildew G. cichoracearum strain UCSC1 compared
with a mock inoculation with aseptic water. The CaMV-35S
promoter used as positive control yielded a constitutively
strong GUS expression and did not reveal any significant
difference between pathogen inoculation and water treatment.
In contrast, the VpSTS promoter was significantly induced in
response to G. cichoracearum (Fig. 3A). Quantification of GUS
transcripts (Fig. 3B) and enzymatic activity (Fig. 3C) revealed
a strong but transient induction detectable from 6 hours
postinoculation (hpi), and reaching its peak at 12 hpi, followed by a
sharp decline approaching the low expression seen in the mock
control. The maximal activity reached at 12 hpi was
comparable with that achieved by the constitutive CaMV-35S promoter.
In the mock control, there was a slight increase as well, but
this was not statistically significant. In contrast to VpSTS, the
VvSTS promoter produced only a slight induction. Again, this
induction remained below the threshold for significance. These
findings show that the VpSTS promoter not only confers a
higher basal expression, but also is sufficient to produce
inducibility by the compatible powdery mildew G. cichoracearum in
the heterologous Arabidopsis system.
To test whether these differences in the promoter responses
to infection by the biotrophic pathogen correlate with a
differential response to SA and MeJA, respectively, we
followed the accumulation of GUS transcripts after treatment
with 1 mM of either exogenous SA or exogenous MeJA. We
observed that the VpSTS promoter was strongly and rapidly
induced by SA: already at the first time point (which, due to
the time required for excision, was ~2 min after application
of SA), the transcript was found to be slightly elevated; after
4 h an almost 5-fold induction had been reached; and this
high level of expression persisted over the entire 12 h of this
experiment. In contrast, the VvSTS promoter was induced
to a much lower extent and more slowly: even after 8 h the
induction was only ~2-fold. For MeJA, both promoters
responded more slowly compared with SA: here, the VpSTS
promoter was induced weakly, but significantly, after 8h, and
the VvSTS promoter after 12 h (Supplementary Fig. S1).
The full-length VpSTS allele is sufficient to confer
resistance to powdery mildew in a heterologous system
To test whether the full-length VpSTS allele is sufficient to
confer pathogen resistance, the two STS alleles (consisting of
the full-length coding sequence under control of the
respective native promoter) derived from V. pseudoreticulata and
V. vinifera were inserted into the pART-CAM-S vector (Xu
et al., 2014) after restriction by XhoI and SacI, and then
transformed into Arabidopsis as the heterologous system (Fig. 4).
Transformants verified by resistance to the hygromycin marker
and genotyping by PCR were propagated, and the T3
generation was used to follow colonization by G. cichoracearum
strain UCSC1 (Fig. 4) in parallel with the response of the STS
transgene (Fig. 5) over 1 week. Whereas leaves of the wild type
(WT) were covered with abundant sporangiophores at 7 days
post-inoculation (dpi), lines expressing the complete alleles
of Vitis STS exhibited lower sporulation densities, which was
most pronounced for the VpSTS allele (Fig. 4B).
When colonization was followed over time by
histology, fungal spores were found to have attached to the
leaf surface successfully at 1 dpi in all three genotypes.
Likewise, germination and appressoria formation (from 2
dpi) seemed to proceed normally. However, the progress of
colonization was impaired from 4 dpi in the lines
expressing the allele from V. pseudoreticulata, and from 5 dpi in
the lines expressing the allele from V. vinifera. In contrast,
G. cichoracearum continued colonization in the WT Col-0
and, from 5 dpi, the mycelium had almost covered the whole
infected surface. The inhibition of colonization was
preceded by accumulation of STS transcripts and formation
of the stilbene glycoside α-piceid (Fig. 5B, C), particularly
in the allele driven by the VpSTS promoter. In summary,
the phenotype of the transgenic Arabidopsis lines showed
that expression of full-length alleles of STS was correlated
with arrested colonization of the compatible powdery
mildew G. cichoracearum, and that this arrest was more
pronounced for the allele derived from the wild Chinese species
The VpSTS promoter is more efficiently activated by
The STS allele from V. pseudoreticulata had been found to
confer pathogen resistance in the heterologous Arabidopsis
system (Fig. 4), namely in a situation where no
co-evolutionary history between pathogen and gene activation can
definitely be expected. Likewise, a co-evolutionary context
cannot account for the resistance of this wild Chinese Vitis
species to powdery mildew of grapevine (Erysiphe
necator), since this pathogen is native to North America and
came to China only a few decades ago (Wang, 1993; Wang
et al., 1995). These considerations stimulated the question
of whether the STS allele of V. pseudoreticulata is
activated in the context of basal immunity (PTI), or whether it
is linked rather to a cell death-related, ETI-like immunity.
To dissect the signalling upstream of STS, we employed a
dual-luciferase promoter reporter system in a vinifera cell
culture (cv. ‘Pinot Noir’) as the homologous expression
system (Höll et al., 2013) using biolistic transformation.
We used this genotype because ‘Pinot Noir’ under normal
conditions cannot efficiently deploy a programmed cell
death response that would overlay the effect of the
introduced transgene. The transgenic suspension cells were
treated either with 10 µg l–1 flg22, a potent inducer of
basal immunity, or with 9 µg ml–1 of the bacterial elicitor
Harpin, triggering a cell death-related ETI-like response in
these cells (Chang and Nick, 2012). Alternatively, a short
pulse (2 min) of UV-C was used as an abiotic inducer of
stilbene metabolism (Duan et al., 2015). After induction
by these three treatments, the cells were allowed to express
the luciferase reporter for 6 h in the dark before measuring
the activity. Using this system, we observed that the VpSTS
promoter was significantly (P<0.05) activated by the
bacterial PAMP flg22 by ~1.9-fold, while this activation was not
found for the VvSTS promoter. In contrast to flg22, neither
the elicitor Harpin nor a pulse of UV-C (a powerful
abiotic activator of stilbene accumulation in Vitis) was able to
stimulate luciferase activity over the values observed in the
controls (Fig. 6B, C).
The VpSTS and VvSTS promoters are differentially
induced by SA
As pointed out in the Introduction, defence against
biotrophic pathogens has been associated with SA, whereas
Fig. 5. Time course for the expression of the STS reporter upon heterologous expression of VpSTS::STS compared with VvSTS::STS in
response to inoculation with G. cichoracearum. (A) Chemical structures and biosynthetic pathway of stilbenes. Both mock-inoculated and
pathogen-inoculated leaves were sampled at the indicated time points for the quantification of STS transcripts by qRT-PCR (B), and the
resveratrol glucoside α-piceid (C). The housekeeping gene AtGAPDH (AT1G13440) was used as the internal standard for the transcript
measurements (B). Values represent means and SEs of three biological replicates. * and ** indicate statistical significance of the difference
from mock controls by a one-sided paired t-test with confidence levels of P<0.05 or P<0.01, respectively. (This figure is available in colour at
jasmonates have been shown to act in the defence against
necrotrophic pathogens. We therefore tested the response of
the two STS promoters to 50 µM of either SA or MeJA for
6 h using the luciferase assay described above (Fig. 7). We
found that SA activated the VpSTS promoter by 3.7-fold
(P<0.01) compared with a solvent control (Fig. 7A), whereas
the induction of the VvSTS promoter was significantly
weaker (2.5-fold). In contrast, both promoters responded in
a similar way to MeJA treatment (Fig. 7B). Here, the
induction of the VpSTS promoter was only 2.3-fold, and that of
Fig. 6. Activation of VpSTS and VvSTS promoters by flg22 in a grapevine suspension cell line derived from the vinifera cultivar ‘Pinot Noir’. Induction of
promoter activity was measured either 6 h after addition of 10 μg l–1 flg22 (A) or of 9 μg ml–1 Harpin (B), or after irradiation with UV-C for 2 min (C). Data
represent mean values and SEs from three biological replicates. * indicates statistical significance of the difference from controls by a one-sided paired
t-test with a confidence level of P<0.05.
the VvSTS promoter was 2.2-fold. Thus, the VpSTS
promoter was found to be specifically responsive to SA, whereas
both promoter alleles were not different with respect to their
responses to MeJA.
Induction of VpSTS and VvSTS promoters by SA
depends on calcium influx and NADPH oxidase activity
Previous work on grapevine suspension cells had shown that
the transcripts of STS were induced by the elicitor Harpin
dependent on a calcium influx channel and an apoplastic
oxidative burst triggered by an NADPH oxidase (Chang et al.,
2011). We therefore tested the effect of GdCl3 (an inhibitor
of calcium influx) as well as the NADPH oxidase inhbitor
DPI on the activation of STS promoters by SA (Fig. 8).
Whereas 20 µM GdCl3 by itself did not cause any modulation
of promoter activity, this pre-treatment almost completely
abolished the activation of the two STS promoters by SA
(Fig. 8A). When the transformed cells were pre-treated with
GdCl3 for 30 min before the SA treatment, the induction of
the VpSTS promoter was decreased from 3.7-fold to 1.5-fold,
and the activity of the VvSTS promoter was decreased from
2.5-fold to 1.4-fold. Inhibition of NADPH oxidase by DPI
had a similar but milder effect on SA inducibility of VpSTS
promoter activity (Fig. 8B). Here, pre-treatment of the
transgenic cells with DPI for 30 min decreased the SA induction of
VpSTS promoter activity from 3.7-fold to 2.3-fold, and from
2.5-fold to 2.0-fold for the VvSTS promoter, but the decrease
remained below the threshold for significance. Again, the
inhibitor alone did not produce any significant modulation.
These results show that calcium influx is necessary for the
induction of VpSTS and VvSTS promoters by SA, whereas
apoplastic reactive oxidative species (ROS) generated by
the NADPH oxidase act as positive modulators of the
SA-dependent signalling significantly activating the VpSTS
promoter. This positive modulation is different for the two
promoter alleles, since the VpSTS promoter responded to
SA more strongly than the VvSTS promoter, but both alleles
show the same residual activation observed under elimination
of NADPH oxidase.
To understand whether the signal generated by
apoplastic ROS amplifies with SA-dependent activation of STS
promoter activity at an early or a late state of signalling,
a time course experiment was conducted where DPI was
administered at different time points with respect to the
inductive SA treatment (Supplementary Fig. S2). We observed that
if the transgenic suspension cells were pre-treated with DPI
for 30 min before the SA treatment, or treated with SA and
DPI at the same time, the induction of the VpSTS promoter
by SA was obviously suppressed. The VvSTS promoter
activity decreased as well, but, again, this decrease remained
below the significance threshold. However, when the
transgenic cells were treated with DPI after SA had been acting for
30 min, the activation of both promoters was also decreased,
and when administered 1 h after SA treatment, DPI had little
effect on the activation of STS promoters by SA. This
indicates that ROS are effective up to 30 min after application of
SA, showing that they are not part of an early induction step
but modulate a downstream event.
Induction of VpSTS and VvSTS promoters by SA is
modulated by the MAPK pathway and jasmonate
The MAPK signalling cascade has been reported to act both
upstream and downstream of the SA signalling pathway
(Zhang and Liu, 2001; Zhang et al., 2007), and was found to
be necessary for induction of STS transcripts in grapevine
(Chang and Nick, 2012). To explore whether the activation of
STS promoters by SA is dependent on the MAPK pathway,
the specific inhibitor PD98059 was used to suppress MAPK
signalling in the SA treatment. When the transgenic
suspension cells were pre-treated with PD98059 for 30 min before
the SA treatment, we found that the SA induction of both
the VpSTS and VvSTS promoters was clearly, but partially,
suppressed by the inhibitor of MAPK pathway (Fig. 9A).
Compared with the induction by SA alone, the inhibitor
reduced the activation of the VpSTS promoter from
3.7fold to 1.7-fold, while the activation of the VvSTS promoter
decreased from 2.5-fold to 1.8-fold (which was not
significant). In the absence of SA, the MAPK inhibitor did not
affect the promoter activities (Fig. 9A). Similarly to DPI, the
inhibition by PD98059 remained partial, indicating that the
MAPK cascade acts as a positive modulator of SA signalling
to the STS promoters.
Since we had found that MeJA can induce the two STS
promoter alleles to a similar extent (Fig. 7B), we probed for the
contribution of JA to the promoter induction by SA, using
phenidone, an inhibitor of lipoxygenases, because the
inhibition of lipoxygenases quells the entire oxylipin metabolism
including the synthesis of JA. In fact, we observed that the
activation of both STS promoters by SA was affected after
pre-treatment with 2 mM phenidone for 30 min (Fig. 9B):
VpSTS promoter activation was decreased significantly from
3.7-fold to 2.0-fold, and VvSTS promoter activation from
2.5fold to 1.9-fold, which is not significant. This finding indicates
that synthesis of JA is a positive modulator of SA signalling
to the STS promoters. As already found for DPI (Fig. 8B),
the differential activation of the two promoter alleles by SA is
not completely eliminated when MAPK activation (Fig. 9A)
or JA synthesis (Fig. 9B) is inhibited. This means that these
factors promote the SA effect by a second independent
Wild relatives of crop plants are often valuable genetic
resources for breeding, because they have preserved resilience
factors that have been lost during domestication due to biased
selection for fast growth and high yield. For grapevine,
resistance factors against biotrophic pathogens such as downy or
powdery mildew have been successfully introgressed from
wild American species (Eibach et al., 2007; Gessler et al.,
2011). These resistance factors are generally interpreted in
the context of ETI. Since both downy and powdery mildew
of grapevine were introduced into Europe only in the 19th
century and from there to Central and East Asia even later,
ETI is not expected either in Non-American wild grapevine
species or in vinifera. Nevertheless, several resistance factors
have been identified in those grapevines that lack a
co-evolutionary history with these pathogens: for downy mildew,
the factor Rpv10 from V. amurensis is already exploited in
commercial vinifera varieties in Germany (Schwander et al.,
2012). As pointed out in detail in the Introduction, also for
powdery mildew, several valuable resistance factors have been
discovered in different wild Chinese grapevines as well as in
the Uzbek vinifera variety ‘Kishmish Vatkana’. These factors
also indicate that ETI may not be the only mechanism that
can be exploited for resistance breeding.
The Chinese wild V. pseudoreticulata accession Baihe-35-1
was identified as a genotype with high resistance to
powdery mildew (Wang et al., 1995). Similar to the European
Wild grapevine, V. sylvestris, an ETI-like mechanism is not
very likely, since powdery mildew of grapevine is not native
to China, but was introduced from either Europe or North
America, with the first incidences in China reported in the
1950s (Wang, 1993; Wang et al., 1995). However, since the
accumulation of stilbenes resulting from induction of STS
genes represents a crucial factor in the resistance to powdery
mildew (Schnee et al., 2008), we therefore addressed the role
of a powdery mildew-responsive STS allele from the
resistant Chinese wild Vitis species and used the susceptible
vinifera cultivar ‘Carigane’ as reference. Grapevine STS genes are
organized in a large family and can be activated by various
abiotic and biotic factors (Parage et al., 2012). Some members
have been reported to respond to pathogen attack (Xu et al.,
2011; Dai et al., 2012). The differential responsiveness of the
two STS alleles is correlated with distinct differences in their
promoter sequences, whereas the transcripts are fairly similar.
Using transformation of STS promoter constructs driving a
GUS reporter in Arabidopsis as the heterologous model, we
can show that the specific differences in the VpSTS promoter
are necessary and sufficient to confer high responsiveness to
a compatible strain of powdery mildew, G. cichoracearum
UCSC1, and that this elevated responsiveness is correlated
with an elevated responsiveness to exogenous SA, whereas
the response to MeJA is not altered. Expression of the
fulllength STS alleles in the same system shows that they confer
accumulation of stilbenes in Arabidopsis accompanied by a
partial resistance against the compatible strain of powdery
mildew. Arabidopsis lacks the molecular and cellular
machinery to accumulate stilbenes, which may be the reason why the
stilbenes formed by the transformants are glycosylated. It
remains to be elucidated to what extent they are cleaved in
response to pathogen attack.
A function for SA in basal immunity? Lessons from
To understand the signalling upstream of STS and to
integrate the data into the type of defence response (PTI versus
ETI-like cell death-related immunity), we introduced the two
promoter alleles into a promoter–reporter system (Höll et al.,
2013) based on a grapevine cell line derived from the
vinifera cultivar ‘Pinot Noir’. In this cell line, the bacterial PAMP
flg22 activates PTI, whereas the bacterial elicitor Harpin
activates a cell death-related ETI-like response (Chang and Nick,
We found that flg22, but not Harpin, was able to activate
VpSTS in this system, whereas VvSTS was not responsive.
This places VpSTS in the context of basal immunity,
consistent with the responsiveness of this promoter to MeJA (albeit
that both alleles were responsive to the same degree). This
finding is consistent with the observation that in the cell line
used as host for the VpSTS promoter, flg22 causes the
accumulation of JA and its bioactive conjugate JA-Ile, whereas
Harpin fails to do so (Chang et al., 2016). These observations
are consistent with a model where STS is activated by basal
immunity (possibly via activation of jasmonate signalling),
rather than by cell death-related, ETI-like immunity.
On the other hand, the VpSTS promoter was significantly
responsive to SA, and this responsiveness was much more
pronounced compared with the VvSTS promoter, a pattern
that had already been observed upon heterologous
expression in Arabidopsis. Activation of the SA pathway is
traditionally discussed in the context of cell death-related defence
(Tenhaken and Rübel, 1997). Our results showed that
induction of the VpSTS promoter by SA required calcium influx,
NADPH oxidase activity, the MAPK pathway, and jasmonate
synthesis, namely events that are typical for basal immunity.
The molecular events underlying early signalling seem to
be mostly shared between PTI and ETI, including calcium
influx, oxidative burst, or activation of MAPK cascades.
On the other hand, flg22 and Harpin, while both activating
STS, can generate a qualitatively different defence response
in the grapevine cell line used for the promoter–reporter assay
(Chang and Nick, 2012), leading to the question of at what
point the bifurcation of basal and cell death-related
immunity is generated.
Interestingly, we find that SA specifically activates the
VpSTS promoter, as does flg22. Moreover, SA uses the
same signalling events (calcium influx, NADPH
oxidasegenerated ROS, MAPK signalling, and jasmonate) that
have already been found to activate flg22-induced
accumulation of STS transcripts (Chang and Nick, 2012) as well as
activation of MYB14, a transcription factor gene activating
STS (Duan et al., 2016). Addition of exogenous SA to
tobacco suspension cells can induce a transient calcium
influx (Kawano et al., 1998), and MAPK signalling is
activated by SA in tobacco (Zhang and Liu, 2001), whereas the
link between SA and apoplastic oxidative burst seems not
to be that straightforward (see, for example, Pogány et al.,
2009). Here, we found that both calcium and ROS
signalling act downstream of SA (Fig. 8), because exogenous SA
would over-ride the effect of Gd3+ and of DPI if SA were
acting downstream of calcium and ROS signalling. Using a
time course experiment, where the NADPH oxidase
inhibitor DPI is administered at different time points with respect
to the inducing SA (Supplementary Fig. S2), we see that
the apoplastic oxidative burst is needed up to 60 min after
induction by SA (i.e. relatively late). It should be mentioned
here that also for flg22-triggered basal defence, the
apoplastic burst occurs later than calcium influx, whereas for
Harpin-triggered cell death-related defence this sequence
is reversed (Chang and Nick, 2012). These coincidences
indicate a pattern whereby SA acts in the context of basal
immunity. This conclusion is further supported by the
fact that activation of the VpSTS promoter by SA can be
blocked by the inhibitor PD98059 (Fig. 9A), which can
block phosphorylation and activation of MAPKs (English
and Cobb, 2002), as well as by phenidone, an inhibitor of
JA synthesis (Fig. 9B).
We therefore arrive at a model where SA can activate the
STS promoter through the same signalling machinery that
is also activated by the PAMP flg22. The promoter allele
of the wild Chinese grapevine can recruit this signalling
more efficiently as compared with the allele found in the
vinifera cultivar ‘Carigane’, and this efficient recruitment
underlies the more efficient activation of stilbene
accumulation in response to powdery mildew, which may be the
reason why the investigated accession of the wild Chinese
grapevine V. pseudoreticulata is resilient against powdery
mildew. The reason for this superior recruitment of
signalling for STS induction remains to be elucidated;
however, a scenario where a member of the phenylpropanoid
pathway (cinnamyl alcohol dehydrogenase) is modulated
by an NB-LRR protein to culminate in powdery mildew
resistance is also suggested by the analysis of the Ren1
factor from the Central Asiatic seedless variety ‘Kishmish
Vatkana’ (Coleman et al., 2009).
Outlook: convergence of JA and SA signalling on the
It has been shown that two R2R3-MYB-type transcription
factor genes, MYB14 and MYB15, can regulate the
expression of STSs in grapevine (Höll et al., 2013). Further
analysis of a MYB14 allele from V. sylvestris that was found to
be correlated with high stilbene inducibility revealed that
this promoter was activated by MeJA (as well as by UV
light and flg22-triggered basal immunity), whereas SA was
not effective (Duan et al., 2016). Thus, there exist two
pathways culminating in activation of the STS promoter: one
pathway acts through activation of MYB14 (presumably in
concert with MYB15) in the context of basal immunity and
is mostly independent of SA. The second pathway activates
the STS promoter in a manner strongly dependent on SA
(but also requires the same events transducing basal
immunity as the MYB14/MYB15-dependent pathway). Therefore,
we conjecture that additional transcription factors different
from MYB14/MYB15 possibly participate in the regulation
of STS. Promising candidates are the WRKY transcription
factors that also act as regulators of SA-dependent defence
responses (Wang et al., 2006), and often are induced by SA.
For instance, expression of VvWRKY1 is regulated by SA in
grapevine (Marchive et al., 2007). Some WRKY
transcription factor genes, such as rice WRKY13 (Qiu et al., 2007) or
Arabidopsis WRKY70 (Li et al., 2004), can stimulate SA
signalling to the cost of JA signalling. Recently, co-expression
of specific STS members whose promoters contain a large
number of W-box motifs with specific WRKY transcription
factor genes has been reported for a drought-tolerant
genotype of vinifera (Corso et al., 2015). Since both STS
promoters addressed in our study are rich in predicted W-box
motifs (Fig. 1B) that are thought to act as binding domains
for WRKY proteins, we will address the role of WRKY for
the SA-dependent induction of VpSTS.
The classical dichotomy of a cell death-free, basal
immunity (PTI), and a cell death-related, evolutionarily advanced
immunity (ETI) has been questioned by transitional
situations, suggesting that there is far more flexibility available
than thought previously (Thomma et al., 2011). Our
finding that SA, which classically has been associated with cell
death-related defence, can be recruited for basal immunity is
consistent with this ‘blurred dichotomy’. We wonder whether
the signalling function of SA as a molecule depends on the
state to which (JA-dependent) basal immunity has progressed
when SA is formed, an idea which we can address by
modulating relative temporal patterns of early signals.
Independently of the molecular mechanism that will
emerge from such studies, the current work shows that
resistance against biotrophic pathogens such as powdery mildew
can be achieved by multiple strategies: introgression of
ETIlike HR-linked defence from North American wild grapes is
certainly one of these strategies (Feechan et al., 2013a), but
boosting basal immunity to culminate in a swift and strong
stilbene response might be an alternative strategy worth
Supplementary data are available at JXB online.
Figure S1. Time course for the accumulation of the GUS
reporter upon heterologous expression of VpSTS::GUS
compared with VvSTS::GUS in transgenic Arabidopsis leaf
tissues in response to treatment with 1 mM SA or MeJA.
Figure S2. Response of two stilbene synthase promoters to
SA with the time course of DPI quelling the increase of ROS
Table S1. Primers used for gene cloning and qRT-PCR for
This work was supported by the BACCHUS Interreg IV Upper Rhine
project co-financed by the European Union/European Regional Development
Fund (ERDF), the German Federal Agency for Agriculture (Programme for
Sustainable Agriculture, BÖLN), and the Key Laboratory of Horticultural
Plant Biology and Germplasm Innovation in Northwest China.
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