An ancestral allele of grapevine transcription factor MYB14 promotes plant defence
Journal of Experimental Botany
An ancestral allele of grapevine transcription factor MYB14 promotes plant defence
Dong Duan 1 2
Sabine Fischer 0
Patrick Merz 4
Jochen Bogs 3 4
Michael Riemann 2
Peter Nick 2
Qiao Zhao, Tsinghua University
0 Institute of Molecular Genetics, Johannes Gutenberg-University Mainz , J.-J.-Becherweg 32, D-55128 Mainz , Germany
1 College of Life Sciences, Northwest University , Xi'an 710069 , China
2 Molecular Cell Biology, Botanical Institute 1, Karlsruhe Institute of Technology , Kaiserstr. 2, D-76131 Karlsruhe , Germany
3 Fachhochschule Bingen , D-55411 Bingen am Rhein , Germany
4 Dienstleistungszentrum Ländlicher Raum Rheinpfalz, Breitenweg 71, Viticulture and Enology Group , D-67435 Neustadt , Germany
Stilbene synthase is a key enzyme for the production of the phytoalexin resveratrol. Some clones of Vitis sylvestris, a wild European grapevine species which is almost extinct, have been shown to accumulate more resveratrol in response to different forms of stress. In the current study, we asked whether the induction of stilbene synthase transcripts in Hoe29, one of the V. sylvestris clones with elevated stilbene inducibility, might result from the elevated induction of the transcription factor MYB14. The MYB14 promoter of Hoe29 and of Ke83 (a second stilbene-inducible genotype) harboured distinct regions and were applied to a promoter-reporter system. We show that stilbene synthase inducibility correlates with differences in the induction of MYB14 transcripts for these two genotypes. Both alleles were induced by UV in a promoter-reporter assay, but only the MYB14 promoter from Hoe29 was induced by flg22, consistent with the stilbene synthase expression of the donor genotypes, where both respond to UV but only Hoe29 is responsive to Plasmopara viticola during defence. We mapped upstream signals and found that a RboHdependent oxidative burst, calcium influx, a MAPK cascade, and jasmonate activated the MYB14 promoter, whereas salicylic acid was ineffective. Our data suggest that the Hoe29 allele of the MYB14 promoter has potential as a candidate target for resistance breeding.
flg22; grapevine (V; sylvestris); MYB14; Plasmopara viticola; stilbene synthase; UV
Grapevine is an economically important crop, which is highly
susceptible to various biotic diseases, and, therefore, requiring
extensive plant protection measures. Viticulture accounts for
70% of the European fungicide consumption. In order to get the
‘pure wine’ with fewer chemicals, more environmentally friendly
approaches are warranted. An important element for
sustainable viticulture is resistance breeding using resistance factors
originating from North American grapes (Gessler et al., 2011).
These resistance factors have to be understood in context
with the complex evolution of plant immunity: The plant
© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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innate immune system is composed of two distinct layers
(Jones and Dangl, 2006). The first layer exploits ubiquitous
molecules in pathogenic microorganisms, termed
pathogenassociated molecular patterns (PAMPs), to recognize
pathogen attack by receptors at the plasma membrane and to
activate a basal, so-called PAMP triggered immunity, PTI
(Jones and Dangl, 2006; Boller and He, 2009). Most of these
PAMPs are essential for the life cycle of the invader, such that
the PAMPs are preserved. Pathogens that have undergone
coevolution with their hosts, have usually developed an
alternative strategy: They can quell PTI by injecting chemical
signals, so-called effectors, into the host cell and thus reinstate
pathogenicity, which is termed effector-triggered immunity,
ETI (Takken and Tameling, 2009). This ETI has evolved
during a long battle between the pathogen and the host plant.
The molecular mechanisms underlying these two layers of
plant immunity differ, but can also overlap (Thomma et al.,
2011), which contributes to the complexity of the
The economically important grapevine pathogen Downy
Mildew (Plasmopara viticola) has co-evolved over millions
of years together with wild American grapes, such that these
wild grapes had enough time to evolve ETI and, therefore,
can cope with these pathogens. The recent discovery of
Plasmopara viticola strains that can infect specific grapevine
genotypes (Rouxel et al., 2013; Gómez-Zeledón et al., 2013)
support the hypothesis that the resistance of these American
grapes is based upon a canonical ETI. However, these wild
grapes are not suited for winery, due to their unpleasant
‘foxy taste’ (off-flavour). As a strategy to separate the desired
immunity from the undesired flavour, those wild grapes have
been crossed with cultivated grape varieties. This strategy has
been successful and has culminated in economically
important new varieties with good resistance against downy mildew
(P. viticola). However, the rising success of these new
varieties is expected to initiate the next round of the
evolutionary warfare. In fact, the resistance conferred by the genetic
factor ‘Resistance to P. viticola 3 (Rpv3)’ which forms the
base of most current disease-resistant grapevine varieties,
has already been observed to become eroded by new strains
of Plasmopara (Peressotti et al., 2010; Gessler et al., 2011;
Gómez-Zeledón et al., 2013).
As a strategy to render the success of resistance breeding
more sustainable, new sources of resistance are required. In
this context, it might also be rewarding to exploit factors
stimulating the basal immunity (PTI). The ancestor of
cultivated grapevine, V. vinifera L. ssp. vinifera, the European
Wild Grape (V. vinifera L. ssp. sylvestris Hegi) lacks a history
of coevolution with these pathogens and, therefore, should
not harbour any ETI-like defence against downy mildew.
Nevertheless, many genotypes of the European Wild Grape
show good tolerance against several grapevine diseases (Tisch
et al., 2014), such as downy mildew (P. viticola), powdery
mildew (Erysiphe necator), and black rot (Guignardia bidwelli),
which means that some genotypes of V. sylvestris might
command a more efficient basal immunity. In fact, during
previous work, we have identified several genotypes of V. sylvestris
with a strong induction of antimicrobial stilbenes in response
to either a short pulse of UV irradiation or to infection with
P. viticola (Duan et al., 2015) indicating that early signals
shared between these two stress factors must be involved.
These genotypes were also endowed with a partial resistance
to downy mildew and, therefore, have potential as new
breeding resources to be exploited for sustainable viticulture in the
Stilbenes, as important phytoalexins, are a central factor
for the basal immunity of grapevine. Transcription of the key
enzyme stilbene synthase (StSy) can be activated by the PAMP
flg22 (Chang and Nick, 2012) as part of PTI, but also by the
bacterial trigger Harpin (mimicking an ETI-like pattern of
defence). Although both immunity responses culminate in an
accumulation of stilbene synthase transcripts and share a part
of early signalling events, they differ in perception, the role of
oxidative burst, and integration into a qualitatively different
output of stilbene metabolites. The transcriptional
regulation of the stilbene biosynthetic pathway is mediated by two
R2R3-MYB-type transcription factors, MYB14 and MYB15
that were shown to activate the stilbene synthase promoter
(Höll et al., 2013). In our previous work (Duan et al., 2015),
the V. sylvestris genotypes Hoe29 and Ke83 were found to
be endowed with an elevated stilbene inducibility in response
to UV light, which was correlated with a strong induction
of stilbene synthase transcripts. Interestingly, in response to
inoculation with P. viticola, stilbene synthase transcripts were
elevated for Hoe29, but not for Ke83.
In the current study, we test the idea whether the strong
inducibility of stilbene synthase transcripts in Hoe29 might
result from the elevated induction of MYB14 and MYB15.
Specific differences were discovered by Illumina-based
nextgeneration sequencing and confirmed by cloning for the
MYB14 promoter of Hoe29, whereas MYB15 did not reveal
obvious changes. Both sylvestris genotypes shared certain
MYB14 promoter domains (whereas they differ in others),
which were absent from the promoter of the cultivated
variety Augster Weiss (that is a weak stilbene producer). Using a
promoter–reporter assay in grapevine suspension cells (Höll
et al., 2013), we show that differences in the inducibility of the
MYB14 promoter can account for the stress-specific
differences in the expression of stilbene synthase observed between
the three genotypes (Hoe29, Ke83, and Augster Weiss).
Although both sylvestris genotypes show good UV
inducibility of MYB14, only the Hoe29 allele of this promoter is
competent for induction by PTI (triggered by flg22). We also
mapped known signalling events for PTI, such as dependence
on NADPH oxidase (RboH) or induction by jasmonic acid,
and calcium influx. Our data suggest that a particular region
in the promoter of a specific sylvestris allele of MYB14
harbours potential as a candidate target for resistance breeding.
Materials and methods
The Vitis vinifera ssp. sylvestris genotypes ‘Ke83’ and ‘Hoe29’, as well
as the V. vinifera cultivar Augster Weiss used in this study, have already
been described in Ledesma-Krist et al. (2014) and Duan et al. (2015).
All accessions are maintained as living specimens in the grapevine
collection of the Botanical Garden of the Karlsruhe Institute of
Technology (Olmo, 1976). For DNA and RNA extraction, the leaves
were harvested from greenhouse-grown plants cultivated at a day/
night temperature of 22/18 °C and a 14/10 h light/dark photoperiod.
Preparation of leaf samples
The fully expanded leaves were excised and subjected to UV-C stress as
described in Duan et al. (2015). The leaves of the different genotypes were
harvested at different time points after the treatment: C (control fresh
leaf, without UV-C treatment), 0.5h, and 6h, respectively, immediately
frozen in liquid nitrogen, and stored at −80 °C until RNA extraction.
Downy mildew (P. viticola) infection was carried out as described
previously (Genet et al., 1997; Kortekamp, 2006; Duan et al., 2015).
Three experimental situations were used: fresh leaf without
inoculation (C), control leaf incubated in the absence of P. viticola but
kept under the same conditions (120 h-C), and controlled
inoculation with a suspension of P. viticola and incubation for 120 h (120
h-S), respectively. The leaf material was immediately frozen in liquid
nitrogen and stored at −80 °C until RNA extraction.
cDNA synthesis and quantitative Real-Time RT-PCR
Total RNA was isolated using the SpectrumTM Plant Total RNA
Kit (Sigma, Deisenhofen) according to the protocol of the
manufacturer. The extracted RNA was treated with a DNA-free DNase
(Qiagen, Hilden, Germany) to remove any potential contamination
by genomic DNA. The mRNA was transcribed into cDNA using the
M-MuLV cDNA Synthesis Kit (New England Biolabs; Frankfurt
am Main, Germany) according to the instructions of the
manufacturer. The RNase inhibitor (New England Biolabs; Frankfurt am
Main, Germany) was used to protect the RNA from degradation.
The amount of RNA template was 1 μg.
Quantitative real-time RT-PCR was performed on an Opticon 2
system (Bio-Rad, München) as described by Svyatyna et al. (2014).
To compare the mRNA expression levels between different samples,
the Ct values from each sample were normalized to the value for the
EF-1α internal standard obtained from the same sample. For each
triplicate, these normalized Ct values were averaged. The difference
between the Ct values of the target gene X and those for the EF-1α
reference R were calculated as follows: △Ct(X)=Ct(X)–Ct(R). The
final result was expressed as 2–△Ct(X). Each experiment was repeated
with three biological replicates.
Illumina next generation sequencing and data analysis
Genomic DNA was extracted from young leaves of Hoe29 using
the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following
the instructions of the manufacturer. For Illumina Next Generation
Sequencing, 1.5 μg genomic DNA were sheared using Covaris
(Woburn, Massachusetts), and the subsequent library preparation
was carried out with the TruSeq DNA LT Sample Prep Kit (Illumina
Inc., San Diego, CA). The resulting library was sequenced in a 100 bp
paired-end run on an Illumina HiSeq2000 generating approximately
200 million reads (IMSB, Mainz, Germany). The subsequent analysis
of NGS data was conducted with the CLC bio Genomics Workbench
(Aarhus, Denmark). Raw reads were filtered for quality (P=0.01,
no ambiguity nucleotides) and adapter trimmed. De novo assembly
of remaining trimmed reads (approximately 187 million reads) was
conducted using standard parameters. The resulting contigs served
as the database for BLASTn searches using the MYB14 sequence of
PN40024 (NW_003724037.1) as a query. Trimmed reads were mapped
to cloned promoter sequences of Hoe29, Ke83, and Augster Weiss with
varying lengths and similarity fractions: 1.0/1.0; 0.98/0.98; 0.96/0.96.
Cloning of the MYB14 promoter
In order to conduct the transient expression assay, the MYB14
fragments of genomic DNA – Hoe29, Ke83, and Augster Weiss were
amplified with the Phusion DNA polymerase (NEB, Frankfort,
Germany), including the promoter sequences of Hoe29 (1351 bp),
Ke83 (1245 bp), and Augster Weiss (1347 bp). The primers MYB14-F
(5′-CTACTGACGTGCACTAGCCT-3′) and MYB14-R (5′-GCAG
AGTGAAAGTGCAACACG-3′) were designed according to the
reference sequence in GenBank (NW_003724037.1). The isolated
sequences were compared with the database sequence using the
multiple sequence alignment program ClustalW2 (http://www.ebi.
ac.uk/Tools/msa/clustalw2/). After the alignments, specific primers
for GATEWAY cloning (see Supplementary Table S1 at JXB online)
were designed to amplify the chosen promoter sequence of Hoe29,
Ke83, and Augster Weiss using the GATEWAY®-Cloning
technology (Invitrogen Corporation, Paisley, UK). The promoter regions
of these three genotypes were ligated into the luciferase vector pLuc
(Horstmann et al., 2004) and verified by DNA sequencing (GATC
Biotech, Cologne, Germany).
Transient transfection and dual-luciferase assay
A transient assay using a cell suspension culture of a ‘Chardonnay’
petiole callus maintained on Grape Cormier (GC) medium (Bao
Do and Cormier 1991), was performed as described previously
(Czemmel et al., 2009; Merz et al., 2014) with minor modifications
as follows: after a transfection (48 h), the cells were harvested after
a treatment with different stresses at specified time points, and then
lysed by grinding on ice in 200 μl of 2× passive lysis buffer (PLB,
Promega, Madison, Wl) for 1.5 min using a pestle and mortar. After
centrifugation of the lysates for 1 min at 1 000 g, luciferase activities
were measured with the dual-luciferase reporter assay system (PJK,
Kleinblittersdorf, Germany), by the sequential addition of 50 μl
Beetle Juice and Renilla Glow Juice to 20 μl of the individual lysate
supernatants. Light emission was measured with a lumat LB9507
Luminometer (Berthold Technologies, Bad Wildbad, Germany).
The specific promoter linked to a firefly luciferase gene was
cobombarded with the Renilla luciferase plasmid pRluc as an internal
standard and the relative luciferase activity was calculated as the
ratio between the firefly and Renilla (control) luciferase activity. All
transfection experiments were performed in triplicate with similar
relative ratios to the respective control.
Treatment of the cells for transient promoter assays
For the UV-C experiment, the cells were treated for 2 min at a
distance of 12.5 cm by UV-C (15 W, OSRAMHNS, OFR) and
then harvested at 3 h (Duan et al., 2015). The bacterial peptide
flg22 QRLSTGSRINSAKDDAAGLQIA (Felix et al., 1999), a
22-amino-acid peptide, was purchased from a commercial
producer (Antikörper online, Aachen, Germany) and diluted in sterile
H2O as a stock solution of 1 mM. Diphenyleneiodonium chloride
(DPI) (Sigma-Aldrich, Germany) was dissolved in dimethyl
sulphoxide (DMSO) as a stock solution of 10 mM. The calcium
ionophore A23187 (Sigma-Aldrich, Germany) was diluted in DMSO as
a stock solution of 50 mM. Gadolinium chloride (GdCl3)
(SigmaAldrich, Germany) was used as an inhibitor of
mechanosensitive calcium channels and diluted with DMSO to a 20 mM stock
solution. PD98059, a chemical inhibitor for the mitogen-activated
protein kinase (MAPK) cascade (Sigma-Aldrich, Deisenhofen,
Germany), was dissolved and sterilized into a 100 mM stock
solution in DMSO. Salicylic acid (SA) (Sigma-Aldrich, Germany)
and (±)-jasmonic acid (JA) (Sigma-Aldrich, Germany) were
dissolved in ethanol (EtOH) to obtain stock solutions of 50 mM
and 500 mM, respectively. The inhibitor 1-phenylpyrazolidinone
(phenidone) (Sigma-Aldrich, Germany) was dissolved in DMSO
to obtain a 2 M stock solution. Polyoxyethylen-20-sorbitan
monolaurate (Tween® 20), required in a low concentration (1 ‰) to
keep phenidone soluble, was obtained from Carl Roth in Germany.
All treatments were accompanied by appropriate solvent controls,
and the maximal concentration of solvent used in the test samples
did not exceed 1‰.
Specific alleles of the MYB14 promoters in sylvestris
During the comparison of the sylvestris genotype Hoe29,
which has high stilbene-inducibility (Duan et al., 2015),
significant differences in the region of the MYB14 promoter
were discovered with respect to the reference genome
(established for the vinifera variety ‘Pinot Noir’) by next-generation
sequencing. This region was, therefore, cloned from the two
sylvestris genotypes Hoe29, and Ke83 (a second sylvestris
genotype with high stilbene-inducibility), together with the
ancient vinifera variety ‘Augster Weiss’, which is male-sterile
and, therefore, often used for breeding. The alignment of
these promoters showed significant differences that were then
analysed with respect to predicted promoter motives using
the PlantCARE algorithm (Lescot et al., 2002).
In Fig. 1, the significant differential cis-elements are
summarized for the MYB14 promoters of Hoe29 and Ke83,
compared with Augster Weiss (the full alignment with the details
on these cis-elements are given in Supplementary Fig. S1 at
JXB online). The Hoe29 promoter harboured a specific 5′- Differential activation of MYB14 promoters from
UTR Py-rich stretch, which was absent in the alleles from Ke83 different genotypes
and Augster Weiss due to a deletion of 10 bp. This cis-acting
element has been reported to confer high transcriptional lev- Using a promoter–reporter assay in grapevine suspension
els (Daraselia et al., 1996; Wang et al., 2013). Furthermore, a cells, we mapped known early signalling events such as the
19-bp-long AT-rich insertion was found in Hoe29 and Ke83 dependence on NADPH oxidase (RboH), or induction by
(ATTTATTAAATTTATTTTT) which has been found to act as jasmonates and calcium influx to investigate whether the
an enhancer (Bustos et al., 1989; Sandhu et al., 1998). In addi- differences in the inducibility of the MYB14 promoter can
tion, several cis-elements linked to light responsiveness have account for the stress-specific differences in the expression
also been found, such as a 3-AF1 binding site and a GATA- of stilbene synthase observed between the three genotypes
motif specific for Hoe29; and a MRE and an as-2-box in Ke83. (Hoe29, Ke83, and Augster Weiss).
Most notably, there was a big deletion of 107 bp in Ke83
removing two putative CAAT boxes present in Hoe29. These putative
enhancer elements were also absent in Augster Weiss (although
this genotype did not carry the 107 bp deletion). In addition, the
promoter in Hoe29 displayed a TCA element shown to confer
salicylic-acid responsiveness (Goldsborough et al., 1993).
phenylpropanoid pathway is activated, we followed the
transcript levels of MYB14 in Hoe29, Ke83, and Augster
Weiss in response to UV-C and P. viticola by quantitative
As shown in Fig. 2A, for all the genotypes tested, no
significant transcript levels can be detected in the controls. However,
as early as 0.5 h after a UV pulse, these transcripts had been
clearly induced, with the response of Hoe29 and Ke83 being
much stronger than that of Augster Weiss. This difference was
magnified at 6h, when the induction in Hoe29 was 16-fold
compared with the control and more than two times
compared with Augster Weiss; also in Ke83, this induction was
nearly three times higher compared with Augster Weiss.
For infection with downy mildew, the expression of
MYB14 in Hoe29 was strongly induced by 30-fold compared
with the control (Fig. 2B), which was more than nine times
higher compared with Augster Weiss. The induction in Ke83,
although also higher than in Augster Weiss, was not
comparable with that in Hoe29.
Activation of MYB14 by UV-C requires an oxidative burst
The rapid generation of reactive oxygen species (ROS),
termed an oxidative burst, is an early inducible plant response
during pathogen invasion or after treatment with elicitors
(Wojtaszek, 1997). To test whether the induction of MYB14
by UV-C requires an oxidative burst, the NADPH oxidase
inhibitor DPI was used to quell the increase in ROS
abundance following challenge with UV irradiation.
As shown in Fig. 3, the promoters were activated in all
genotypes after UV irradiation, but were stronger in Hoe29
Responses of MYB14 to UV-C and P. viticola
To verify whether the transcription factor MYB14 is
induced under conditions where the stilbene branch of the
Fig. 1. Comparison of MYB14 promoters in the two sylvestris genotypes Hoe29 and Ke83, along with the cultivated variety Augster Weiss. 3-AF1
binding site (11 bp): a light-responsive element. circadian (10 bp): cis-acting regulatory element involved in circadian control. 5′UTR Py-rich stretch
(10 bp): cis-acting element conferring high transcriptional levels. GATA-motif (7 bp): part of a light-responsive element. TCA-element (10 bp):
cisacting element involved in salicyclic acid responsiveness. MRE (7 bp): MYB binding site involved in light responsiveness. Deletion (107 bp) in Ke83
compared with Hoe29 and Augster Weiss. as-2-box (10 bp): involved in shoot-specific expression and light responsiveness. AT rich regions (19 bp)
in Hoe29 and Ke83.
Fig. 2. Expression of MYB14 in response to UV-C and downy mildew.
(A) UV-C irradiation for 10 min. (B) Downy mildew infection for 120 h.
Quantification of transcripts by quantitative real-time PCR normalized to
the expression of elongation factor EF1-α. * indicate differences that are
statistically significant on the P <0.05 level and ** indicate P <0.01 level.
Data represent mean values from three independent experimental series,
error bars represent standard errors.
and Ke83 (around 3-fold) compared with Augster Weiss (only
around 1.5-fold). Pretreatment with DPI nearly abolished the
induction by UV; in Hoe29, the induction was even
Impact of JA and SA on the activation of MYB14 promoters
Since jasmonic acid (JA) signalling is activated in response
to herbivores, necrotrophic pathogens, and to wounding, we
tested whether JA signalling was involved in the activation of
the MYB14 promoters at the early signalling stage, by
applying exogenous jasmonates. As shown in Fig. 4A, this induced
the promoter activity by around 4-fold in Hoe29, whereas
there was hardly any induction in Ke83 and Augster Weiss.
The salicylic acid (SA) pathway acts antagonistically to JA
signalling, and is triggered during both PTI and ETI. Since
a putative SA-response element had been found in the
promoter from Hoe29 (Fig. 1), we therefore tested the effect of
Fig. 3. Effect of DPI on promoter activity of MYB14 in response to UV-C.
Values give fold induction levels of promoter activity in the presence
of UV-C and UV-C with NADPH oxidase inhibitor (DPI) relative to the
respective control (C) (promoter activity without any treatments).
+UVC: fold induction of promoter activity for 3 h after the UV-C irradiation
for 2 min; +UV-C+DPI: fold induction of promoter activity for 3 h after
the addition of DPI (10 μM) and UV-C for 2 min. Values were calibrated
against the co-bombarded Renilla luciferase plasmid pRluc as an internal
standard. * indicate differences that are statistically significant on the P
<0.05 level and ** indicate P <0.01 level. Mean values and standard errors
from three independent experimental series.
exogenous SA. As shown in Fig. 4B, the induction in Hoe29
and Ke83, although significantly higher than in Augster
Weiss, was very weak (around 30%) and thus not comparable
with the JA induction in Hoe29 (Fig. 4A). This means that
while jasmonate can effectively activate the Hoe29 allele of
the MYB14 promoter, SA seems to be fairly marginal.
The MYB14 promoters are activated in response to a
The influx of Ca2+ is considered to be the earliest signalling
event in basal immunity and we therefore used the antibiotic
ionophore A23187 as a probe which can permeate through
the cell membrane and release Ca2+ in the cytoplasm. This
allows calcium to be triggered in the absence of an external
stimulus which provides an important tool for functional
analysis. To test whether the influx of Ca2+ is sufficient for the
activation of the MYB14 promoters, the calcium ionophore
was applied. As shown in Fig. 5, this treatment induced
promoter activity by around 80% for Hoe29, whereas the
induction (although significant) was much weaker (around 20%) in
Ke83 and Augster Weiss.
The PAMP flg22 can induce the MYB14 promoter in
Hoe29 but not in Ke83
In order to test whether the stronger induction of MYB14
by the calcium ionophore in Hoe29 was linked with a higher
sensitivity of this allele to basal immunity, the responses to
the PAMP flg22 were monitored (Fig. 6A). Treatment with
this PAMP induced the MYB14 promoter activity in Hoe29
by a similar factor (+70%) as treatment with the calcium
ionophore. Again, Ke83 and Augster Weiss did not show
this induction. Since this pattern suggested that the Hoe29
Fig. 4. Activities of MYB14 promoters in response to (±)-jasmonic acid
(JA) and salicylic acid (SA). Values show promoter activities relative to
the untreated control after the treatments of 50 μM (±)-jasmonic acid
(JA) for 4 h (A) and 50 μM salicylic acid (SA) for 4 h (B), respectively. *
indicate differences that are statistically significant on the P <0.05 level
and ** indicate P <0.01 level. Mean values and standard errors from three
independent experimental series.
allele of the MYB14 promoter was activated by PTI, whereas
the Ke83 allele was not, we decided to map known upstream
events involved in flg22-triggered basal immunity for Hoe29.
RboH-dependent oxidative bursts are necessary. In order
to test whether a RboH-dependent oxidative burst was
involved in the activation of the Hoe29 allele of the MYB14
promoter, DPI was used which almost eliminated the
activation in response to flg22 (Fig. 6B). A negative control showed
that application of DPI alone did not cause a significant
change of activity.
Calcium influx is necessary. Since the activity of a calcium
influx channel is essential for the activation of early defence,
inhibition of this influx by GdCl3, an inhibitor of
mechanosensitive calcium channels should therefore suppress the
activation of PTI by flg22 (Chang and Nick, 2012). Since a
Fig. 5. Activities of MYB14 promoters in response to the calcium
ionophore A23187. Values show promoter activities relative to the
untreated control after treatment with 50 μM of A23187 for 4 h. * indicate
differences that are statistically significant on the P <0.05 level. Mean
values and standard errors from three independent experimental series.
calcium ionophore was able specifically to activate the Hoe29
allele of the MYB14 promoter of Hoe29 (Fig. 5), we tested
whether the specific activation of the Hoe29 allele in response
to flg22 required a calcium influx. As shown in Fig. 6C, this
was the case. This finding suggests the Ca2+ influx is essential
for the flg22-induced activation of the MYB14 promoter.
MAPK signalling is necessary. A mitogen-activated
protein kinase (MAPK) cascade is implied in the activation of
defence gene expression which can be inhibited in grapevine
cell cultures by the specific inhibitor PD98059 (Chang and
Nick, 2012). As shown in Fig. 6D, this inhibitor can
efficiently suppress the flg22-induced activation of the MYB14
promoter, demonstrating that MAPK signalling is necessary
for this activation.
JA synthesis is necessary. Since exogenous JA could
efficiently activate the Hoe29 allele of the MYB14 promoter
(Fig. 4A), we tested whether endogenous jasmonate was
necessary for the activation of MYB14 promoter in response to
flg22. Since jasmonate synthesis initiates from a peroxidation
of lipids, phenidone, an inhibitor of lipoxygenases, can block
the synthesis of JA. As shown in Fig. 6E, a pretreatment with
phenidone can efficiently inhibit the flg22-triggered induction
of the Hoe29 allele of the MYB14 promoter. Thus, JA
synthesis is necessary for activation.
In the current study, to understand the functional relevance
of these specific promoters and to map the upstream
signalling, we employed a promoter–reporter assay (Höll et al.,
2013). We show that both sylvestris promoters (but not the
promoter from the weak stilbene producer Augster Weiss) are
induced by UV light in cell culture and that this induction
requires the activity of a NADPH oxidase. However, only the
Fig. 6. Activities of the MYB14 promoter from Hoe29 in response to flg22 and the effect of inhibitors on this activity. Values show promoter activities
relative to the untreated control after treatment with 1 μM flg22 for 4 h (A), the modulation of this activity by pretreatment with: 10 μM of the NADPH
oxidase inhibitor DPI (B), 20 μM of the calcium-channel blocker GdCl3 (C), 100 μM of the MAPK cascade inhibitor PD98059 (D), and 2 mM of the
lipoxygenase inhibitor phenidone (E). Activities were measured 4 h after the addition of flg22; pretreatments were for30 min. In the phenidone experiment
(E), 2 mM phenidone containing 0.1% Tween® 20 was used, control 1 was pretreated with 0.1% Tween® 20 for 30 min alone and then kept for 4 h without
flg22, control 2 was pretreated with 0.1% Tween® 20 for 30 min and then incubated with 1 μM flg22 for 4 h. * indicate differences that are statistically
significant on the P <0.05 level. ** indicate differences that are statistically significant on the P <0.01 level. Mean values and standard errors from three
independent experimental series.
MYB14 promoter from Hoe29 was also induced by jasmonic
acid and flg22 (again dependent on the activity of a NADPH
oxidase) indicating that this allele of MYB14 was, in addition,
a target of the signalling driving basal immunity (PTI). By
contrast, the MYB14 promoter from Ke83, although
inducible by UV light, was not induced in the context of PTI. This
is consistent with our previous findings (Duan et al., 2015),
where StSy transcripts were strongly induced by UV light in
both genotypes whereas, for infection by P. viticola, induction
was only observed in Hoe29, but not in Ke83. We will present
a signature model of immunity signalling that can explain
most, if not all of our observations (Fig. 7).
The earliest known cellular response of basal immunity is
the activation of a rapid influx of Ca2+ and H+ (Nürnberger,
1999). In the case of the PAMP flg22, perception is brought
about by the receptor FLS2 (for reviews see Boller and
Fig. 7. The signature model of immunity signalling. Details are explained in the discussion. Binding of flg22 to the receptor will therefore result in
a substantial increase of cytosolic calcium within a few minutes. The stimulation of the membrane-bound NADPH oxidase RboH through specific
calcium-dependent protein kinases, leading to an apoplastic oxidative burst generating superoxide anions. Hydrogen peroxide is generated in the
peroxisomes from superoxide. Calcium reaching the plastid will activate specific lipoxygenases there as the first committed step of the oxylipin
pathway generating jasmonates. MYB14 transcription factor. Stilbenes.
Wasternack and Hause, 2013) as the first committed step of
the oxylipin pathway generating jasmonates. In Arabidopsis,
mutants affected in vacuolar calcium channels (Bonaventure
et al., 2007) fail to activate AtLOX2, the lipoxygenase, which
is the central trigger for the oxylipin pathway. The molecular
mechanism is not clear but might be linked with the binding
of lipoxygenase to the membrane due to a conserved calcium
binding loop interspersed between two β-sheets (Tatulian
et al., 1998). This will alter the specificity of lipoxygenase –
whereas the free enzyme converts linoleic acid to conjugated
dienes, however, upon binding to the membrane, it
preferentially forms a conjugated ketodiene. In consequence, within
a few minutes, cis-(+)-12-oxophytodienoic acid (OPDA) is
exported from the plastid and converted to jasmonic acid
(JA) and its potent conjugate JA-Ile.
We therefore tested, whether exogenous jasmonic acid
could activate MYB14 in the absence of flg22. This is, in fact,
what we can observe (Fig. 4A), whereby this activation only
works with the Hoe29 allele, whereas the alleles from Ke83,
and Augster Weiss are not responsive to jasmonic acid. To
test, whether induction of (endogenous) jasmonic acid is
necessary for this activation, we treated the cells with phenidone,
an inhibitor of jasmonate synthesis targeted to lipoxygenases
(Ismail et al., 2012), and we found that phenidone can block
the flg22-induced activation of MYB14. Thus, jasmonate is
necessary and sufficient to convey the activation of flg22 to
the Hoe29 allele.
This points to a scenario where flg22 activates the MAPK
cascade, as well as jasmonate signalling, that converge on a
target on the sylvestris MYB14 promoter that is present in
Hoe29, but not in Ke83. Nevertheless, RboH seems to be
necessary as well and this effect of RboH seems to be different
from that in the UV-activation of MYB14 (which was similar
in both sylvestris alleles of this promoter). This apparent
discrepancy can be resolved when RboH dependent signalling
converges with jasmonate synthesis. This point of
convergence might again be the lipoxygenase that is not only
activated by calcium, but requires hydrogen peroxide (Fig. 7, ).
Hydrogen peroxide is generated in the peroxisomes from
superoxide and is then further converted to water by catalase
(Fig. 7, ). It has been known for a long time that the
activity of catalase can be inhibited by the important stress factor
salicylic acid, leading to elevated levels of hydrogen peroxide
(Durner and Klessig, 1996).
Our model would therefore predict that salicylic acid, by
blocking the reduction of hydrogen peroxide, should promote
the activation of lipoxygenase and, therefore, the activation
of the Hoe29 allele of MYB14 by flg22 should also depend on
RboH. This prediction was tested experimentally and it was
found that DPI can block the activation (Fig. 6B), consistent
with the prediction. We have further found that salicylic acid
can activate the Hoe29 allele of MYB14 (Fig. 4B). However,
the activation was observed for both sylvestris alleles pointing
to additional targets of salicylic acids (that are different from
jasmonate). But, since this activation was also very weak
(although significant), the impact of salicylic acid alone (i.e.
without synergy with flg22) seems to be fairly marginal.
Open questions and outlook
The current work proposes a mechanism to explain the
observed phenotype (Duan et al., 2015) of a specific sylvestris
genotype, Hoe29, and draws a link between specific regions
in the promoter of the transcription factor MYB14, elevated
inducibility of this promoter by the signalling activated
during basal immunity, and the observed strong accumulation of
resveratrol and viniferins correlated with the improved
tolerance of these genotypes against downy mildew. Although we
can reproduce in the promoter–reporter system the response
patterns observed in the plant, for instance the differential
activation of stilbene synthase transcription in the Hoe29
versus the Ke83 genotypes, the general activation of the
promoter is lower than the observed accumulation of transcripts
in planta. This indicates that differentiated grapevine cells
harbour enhancing factors that are not present in
non-differentiated suspension cells. A similar phenomenon with similar
ratios is observed for stilbene synthase when the induction of
transcripts in planta is compared with the inductions observed
in the promoter–reporter system (Höll et al., 2013). Whether
these factors are of epigenetic nature or are simply additional
signalling factors remains to be elucidated. In addition, the
role of the MYB15 factor should be addressed as well as the
MYB14-independent direct signalling to the stilbene
synthase promoter. These aspects are currently being analysed
and are expected to complement and refine the proposed
working model. As proof of the concept for the approach,
the Hoe29 allele of MYB14 will also be transformed into a
vinifera host with poor stilbene performance to see whether
up-regulation of StSy transcription is sufficient to produce
high levels of bioactive stilbenes. The target of this research
is to define targets for molecular breeding of grapevine
varieties with elevated basal immunity due to enhanced MYB14
Supplementary data can be found at JXB online.
Figure S1. The alignment and the significant differential
cis-elements for the MYB14 promoters of Hoe29 and Ke83,
compared with Augster Weiss and the reference genome
(from the vinifera cultivar Pinot Noir).
Table S1. Sequence of oligonucleotide primers for
GATEWAY® cloning of the MYB14 constructs for the
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 by a fellowship
from the Chinese Scholarship Council to Dong Duan. We gratefully
acknowledge Joachim Daumann and Kerstin Huber (Karlsruhe Institute
of Technology) for taking care of plants in the Botanical garden, and
Claudia Vogel (Dienstleistungszentrum Laendlicher Raum Rheinpfalz,
Neustadt) for kind assistance in the preparation of the promoter–reporter
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