The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea
Journal of Experimental Botany
The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea
Julie Chong 2
Marie-Christine Piron 0 1
Sophie Meyer 0 1
Didier Merdinoglu 0 1
Christophe Bertsch 2
Pere Mestre 0 1
0 Université de Strasbourg, UMR 1131 Santé de la Vigne et Qualité du Vin , F-68000 Colmar , France
1 INRA, UMR 1131 Santé de la Vigne et Qualité du Vin , F-68000 Colmar , France
2 Laboratoire Vigne, Biotechnologies et Environnement (LVBE , EA3991) , Université de Haute Alsace , 33 rue de Herrlisheim, 68000 Colmar , France
During plant development, sugar export is determinant in multiple processes such as nectar production, pollen development and long-distance sucrose transport. The plant SWEET family of sugar transporters is a recently identified protein family of sugar uniporters. In rice, SWEET transporters are the target of extracellular bacteria, which have evolved sophisticated mechanisms to modify their expression and acquire sugars to sustain their growth. Here we report the characterization of the SWEET family of sugar transporters in Vitis vinifera. We identified 17 SWEET genes in the V. vinifera 40024 genome and show that they are differentially expressed in vegetative and reproductive organs. Inoculation with the biotrophic pathogens Erysiphe necator and Plasmopara viticola did not result in significant induction of VvSWEET gene expression. However, infection with the necrotroph Botrytis cinerea triggered a strong up-regulation of VvSWEET4 expression. Further characterization of VvSWEET4 revealed that it is a glucose transporter localized in the plasma membrane that is up-regulated by inducers of reactive oxygen species and virulence factors from necrotizing pathogens. Finally, Arabidopsis knockout mutants in the orthologous AtSWEET4 were found to be less susceptible to B. cinerea. We propose that stimulation of expression of a developmentally regulated glucose uniporter by reactive oxygen species production and extensive cell death after necrotrophic fungal infection could facilitate sugar acquisition from plant cells by the pathogen.
Botrytis cinerea; cell death; grapevine; pathogen resistance; sugar transport; SWEET transporter; Vitis vinifera
Sugar transport and partitioning play key roles in the
regulation of plant development and plant responses to biotic
and abiotic factors
(Lemoine et al., 2013)
. In plants, sugar
transport is mediated by proton-coupled sucrose
transporters (SUTs; belonging to the disaccharide transporter
family) and hexose and polyol transporters (MSTs; belonging to
the monosaccharide transporter family). These transporters
act as sugar/H+ symporters and belong to the major
facilitator superfamily predicted to share a common structure:
12 transmembrane domains connected by hydrophilic loops
(Doidy et al., 2012). Throughout plant development, sugar
efflux transporters from the SWEET family are also essential
for plant nectar production as well as plant seed and
(Chen et al., 2010)
. In addition, increasing
evidence shows that during plant–pathogen co-evolution,
micro-organisms have evolved sophisticated mechanisms to
highjack developmentally regulated sugar transport to
sustain their growth
(Baker et al., 2012)
. Competition for sugar
at the plant–microbe interface is controlled by the
abovementioned membrane transporters, whose regulation
determines the outcome of the interaction
(Doidy et al., 2012)
Several studies indicate that sugar transport and
partitioning are affected following infection with biotrophic fungal
and oomycete pathogens, acting as an additional sink within
the host tissue
(Lemoine et al., 2013)
. Increased invertase
activity and plant monosaccharide transporter expression
have indeed been observed in several plant–biotroph
(Fotopoulos et al., 2003; Doidy et al., 2012)
example, in the wheat–powdery mildew interaction, glucose is the
major carbon energy source transferred to the fungal
mycelium (Sutton et al., 1999), and both glucose transport and
invertase activity are enhanced after infection
et al., 2003; Sutton et al., 2007)
. During plant–fungus
interaction, enhanced sugar efflux from host cells and invertase
activity can also lead to sucrose and hexose accumulation into
the apoplast, which are taken up by fungal sugar transporters
(Lemoine et al., 2013)
rdingly, Wahl et al. (2010
shown that a high-affinity sucrose transporter is required for
virulence of the plant pathogen Ustilago maydis. Less results
are available concerning sugar transport modification after
infection with necrotrophic pathogens. However, Botrytis
cinerea challenge of Pinus cells enhances glucose transport
(Azevedo et al., 2006)
, and a higher invertase activity was also
measured after infection of Vitis vinifera with B. cinerea (Ruiz
and Ruffner, 2002).
The plant SWEET family of sugar transporters is a recently
identified protein family (PFAM code PF0383) consisting of
17 members in Arabidopsis and 21 in rice
(Chen et al., 2010)
SWEETs are integral membrane proteins, with seven
transmembrane domains, involved in sugar export and playing
important roles in nectar production as well as seed and
pollen development. SWEETs were identified in different
organisms (human, Caenorhabditis elegans and plants) based on
their capacity to transport glucose across a membrane along
a gradient concentration. AtSWEET1 was the first
characterized plant SWEET transporter and acts as a glucose
uniporter in different systems
(Chen et al., 2010)
and AtSWEET12 were subsequently identified as key players
in sucrose efflux from phloem parenchyma cells, a
prerequisite for phloem loading by import into the sieve
element–companion cell complex
(Chen et al., 2012)
. Later, it was shown
that SWEET17 exports fructose out of the vacuole and is a
major factor controlling fructose content in Arabidopsis
thaliana leaves and roots
(Chardon et al., 2013; Guo et al., 2014)
More recently, it was demonstrated that SWEET9, a
nectaryspecific transporter in eudicots, is an efflux transporter
essential for nectar production (Lin et al., 2014).
Interestingly, SWEET transporters are the target of
extracellular pathogens, which modify their expression to acquire
the sugar necessary to their growth. Indeed, induction of
SWEET genes upon pathogen infection has been reported
in rice and Arabidopsis. In Arabidopsis, SWEETs are
differentially regulated following infection with different types of
(Chen et al., 2010)
, suggesting that each
pathogen deploys a specific strategy to divert host carbohydrates
for its growth
. In rice, Xa13 is a SWEET
protein essential for pollen development that is also required
for Xanthomonas oryzae infection, Xa13-deficient plants
being resistant to this bacterial pathogen
(Chu et al., 2006)
The role of SWEET proteins as disease resistance
determinants has been further investigated in rice. Xa13 expression
is induced upon X. oryzae infection through the action of
PthXo1, a bacterial transcription activator-like (TAL)
effector. TAL effectors are targeted to the plant nucleus where they
act as transcription factors
(Bogdanove et al., 2010)
recessive Xa13 alleles conferring resistance to X. oryzae have
been identified and in all cases the mutations associated to the
resistance are found in the promoter region of the gene
et al., 2006; Römer et al., 2010)
. Failure of the bacterial TAL
effector to induce the expression of the plant SWEET protein
thus resulted in resistance. In rice, three out of 21 SWEET
genes are targeted by pathogenic X. oryzae pv. oryzae
et al., 2014)
. The results support the hypothesis that X. oryzae
pv. oryzae induces otherwise developmentally regulated host
genes, resulting in disease susceptibility. A similar mechanism
has been recently described for the citrus bacterial canker
disease. In this case, the citrus SWEET1 gene expression is
targeted by several TAL effectors from Xanthomonas citri
(Hu et al., 2014)
Sugar partitioning is highly important in the major fruit
crop grapevine (V. vinifera), since soluble sugar content in
berries is a major component of yield and economic value.
The accumulation of hexose sugars (i.e. glucose and
fructose) beginning at véraison (onset of ripening) and continuing
throughout the ripening process is one of the main hallmark
of berry development
(Lecourieux et al., 2013)
. Sucrose and
monosaccharide transporter genes from the
monosaccharide and sucrose transporter families have been identified
in V. vinifera. Several members of these families are
specifically expressed in different organs and regulated during berry
development (Afoufa-Bastien et al., 2010). Grapevine is
susceptible to diverse pathogens including oomycetes, fungi,
bacteria and viruses. However, regulation of carbon allocation
and sugar partitioning in the interaction between grapevine
and its different pathogens is poorly understood. A grapevine
member of the hexose transporter family, VvHT5, is highly
induced after downy and powdery mildew infection, together
with a cell wall invertase activity. VvHT5 expression could
participate in the source to sink transition after infection with
(Hayes et al., 2010)
Here we report the characterization of the SWEET family
of sugar transporters in V. vinifera. We identified 17 SWEET
genes in the V. vinifera 40024 genome and studied their
expression (i) in different vegetative and reproductive organs and (ii)
following grapevine infection with biotrophic (Erysiphe
necator, Plasmopara viticola) and necrotrophic (B. cinerea)
pathogens. Our results show differential regulation of SWEET gene
expression in plant organs as well as strong up-regulation of
VvSWEET4 upon infection with B. cinerea. Further study
of VvSWEET4 function revealed that it is up-regulated
following different treatments inducing plant cell death. Finally,
A. thaliana knockout mutants in the orthologous AtSWEET4
were found to be more resistant to B. cinerea. Overall, our
work supports the hypothesis that stimulation of expression
of a developmentally regulated glucose uniporter by
oxidative burst and extensive cell death after necrotrophic fungal
infection could facilitate sugar acquisition from plant cells by
Materials and methods
V. vinifera line 40024 and cultivars Syrah and Muscat Ottonel were
used in this study. V. vinifera 40024 grown in 80 l pots in a
greenhouse was used for organ tissue sampling for expression analysis.
Green cuttings from V. vinifera 40024 were used for downy and
powdery mildew inoculations. Cuttings were grown on potting soil in a
greenhouse with 22/19 °C (day/night) temperature and 16/8 h (light/
dark) photoperiod. V. vinifera 40024 and cv. Syrah in vitro
plantlets were used for chemical infiltration, bacterial infiltration and
transient expression experiments. Plantlets were grown in tubes on
supplemented with 0.7% bacto-agar in
a growth chamber at 21°C, under a 16/8 h (light/dark) photoperiod,
and multiplied every 3 months.
A. thaliana ecotype Col-0 plants and T-DNA insertion mutants
were grown in a growth chamber under a 12/12 h photoperiod and
a 20/16 °C day/night temperature regime. T-DNA insertion mutants
in AtSWEET4 were allele sweet4-1 (Salk_072225) from the Salk
(Alonso et al., 2003)
and allele sweet4-2 (GK 858G02) from
the GABI-KAT collection (Kleinboelting et al., 2012).
B. cinerea isolate B0510 was grown on potato dextrose agar
(Duchefa) at 22 °C. Fungal spores were prepared and quantified as
Broekaert et al. (1990
). Pseudomonas syringae pv. pisi
was grown and prepared according to Robert et al. (2001).
P. viticola isolate SC and E. necator isolate Chlo2b were maintained on
leaves from glasshouse-grown seedlings of V. vinifera cv. Muscat
Treatment of plants with chemicals and pathogens
P. viticola inoculation was performed as described in
et al. (2008
). In brief, detached leaves maintained in sealed Petri
dishes on humid Whatmann 3MM paper abaxial side up were
sprayed with freshly collected sporangia resuspended in water at
5 × 104 sporangia ml−1. Inoculated leaves were placed in a growth
chamber at 20 °C under a 16/8 h photoperiod for 6 days.
For E. necator infection, detached young growing leaves from
four independent plants (two or three per plant) were inoculated as
Miclot et al. (2012
). Inoculated leaves were incubated
for 6 days at 25 °C under a 16 h light photoperiod (50 μmol m−2 s−1).
After inoculation of detached leaves with E. necator and
P. viticola, visualization of sporulation at 72–96 h post-inoculation (pi)
indicated a successful infection.
For P. syringae pv. pisi inoculation, the bacterial suspension (109
cells ml−1 in 10 mM MgCl2) was vacuum infiltrated into detached
leaves from in vitro plantlets, as described in the Materials and
methods section Transient expression in grapevine leaves. Control leaves
were infiltrated with 10mM MgCl2.
For Arabidopsis and grapevine inoculation with B. cinerea, 5 µl
drops of a suspension of 5 × 105 ml−1 conidial spores in 15 g l−1 malt
extract and 0.1 M glucose were applied on leaves from young
V. vinifera 40024 plantlets or 6-week-old Arabidopsis plants. For B. cinerea
symptom scoring in Arabidopsis, the diameter of the lesions was
measured 2 and 3 days after inoculation.
Infection of Arabidopsis with P. syringae was performed on
6-week-old soil-grown plants. P. syringae pv. tomato was grown at
28 °C in NYGB medium (bactopeptone 5 g l−1, yeast extract 3 g l−1,
glycerol 20 g l−1) supplemented with rifampicin (100 µg ml−1) and
kanamycin (25 µg ml−1). Cultures were washed with 10 mM MgCl2
and leaves were infiltrated on the abaxial surface with a needleless
1 ml syringe. Growth of P. syringae pv. tomato in leaves was
determined as described in
Katagiri et al. (2002
For paraquat treatment, a 15 µM solution was vacuum-infiltrated
into detached leaves from in vitro-grown plantlets. Control leaves
were infiltrated with water.
In planta quantification of pathogen growth
B. cinerea growth in Arabidopsis leaves was determined by relative
quantification of fungal and plant DNA by means of
quantitative PCR analysis. Total fungal and plant DNA were extracted as
(Gachon and Saindrenan, 2004)
from 8-mm-diameter leaf
discs centred on the inoculation site at 3 days after pathogen
inoculation. Leaf discs were pooled from at least six individual plants. The
relative quantity of B. cinerea was calculated according to the
abundance of the fungal CUTINASE gene relative to the
Arabidopsisspecific ACTIN2 gene measured by quantitative PCR as described in
Berr et al. (2010
). Analyses were performed in triplicate.
Grapevine SWEET genes were identified by protein Blast of the 17
Arabidopsis SWEET proteins against the proteome of the
grapevine genome sequence
(Jaillon et al., 2007)
. The 17 V. vinifera
SWEET proteins identified (VvSWEET) were then used as a query
in a WU-Blast analysis against the A. thaliana proteome and each
VvSWEET was named from the AtSWEET protein producing the
best hit. In case of more than one VvSWEET showing the same
AtSWEET best hit, a suffix (a, b, c…) was added starting with the
protein showing the lowest E value. A. thaliana and Oryza sativa
SWEET proteins were named acco
rding to Chen et al. (2010
Sequence alignments were performed with Muscle implemented
(Gouy et al., 2010)
. Phylogenetic trees were constructed
using PhyML after alignment curation with Gblocks, with LG
model and 100 bootstrap repetitions, performed at www.phylogeny.
(Dereeper et al., 2008)
. OsSWEET proteins 7a, 7c, 7d and 7e,
which are highly similar to OsSWEET7b
(Chen et al., 2010)
identified as reducing the alignment’s quality following curation by
Gblocks. Those were removed from the alignment to improve the
quality of the tree.
The coding sequence of VvSWEET4 was amplified from cDNA
from V. vinifera 40024 leaves inoculated with B. cinerea using
primers with overhangs containing sites for restriction enzymes, digested
and cloned directionally into pBIN61 digested with the same
enzymes. pBIN61 is a pBIN19-derived vector containing the CaMV
35S expression cassette
(Bendahmane et al., 2002)
manipulations resulted in the pBIN61-VvSWEET4 clone.
To perform yeast transformation, VvSWEET4 was amplified
from pBIN61-VvSWEET4 using primers containing NotI restriction
sites, digested and inserted into NotI-digested pPMA1. Yeast vector
pPMA1 is a derivative of NEV-E, containing the PMA1 expression
cassette and harbouring the URA3 gene
(Sauer and Stolz, 1994)
Correct orientation of the construct was verified by PCR. These
manipulations resulted in the pPMA1-VvSWEET4 clone.
To perform subcellular localization experiments, VvSWEET4 was
amplified from pBIN61-VvSWEET4 using primers containing sites
for restriction enzymes, digested and cloned directionally into pGFP
digested with the same enzymes to produce a C-terminal green
fluorescent protein (GFP)-fused VvSWEET4. pGFP is a
pBIN61derived vector allowing cloning in-frame to the mGFP4 sequence.
This resulted in pGFP-VvSWEET4 clone.
Cloning of the B. cinerea necrosis- and ethylene-inducing protein
1 (NEP1)-like protein reported by Schouten et al. (2008) (GenBank
number DQ211824), hereafter called BcNEP1, was performed as
follows. To ensure that the protein was secreted after expression in
planta, the signal peptide from BcNEP1 was replaced by the signal
peptide from PR1 (pathogenesis-related protein 1) from V. vinifera.
To do so, we designed a primer consisting of the complete PR1 signal
peptide sequence followed by BcNEP1 sequence downstream of its
own signal peptide. This primer and a corresponding reverse primer,
both containing restriction sites, were used to amplify BcNEP1 from
cDNA from V. vinifera 40024 leaves inoculated with B. cinerea. The
PCR product was digested and cloned directionally into pBIN61 to
produce the pBIN61-BcNEP1pr1 clone.
All PCRs were performed with PHUSION High-Fidelity
polymerase (Finnzymes, Thermo Fisher Scientific, Villebon sur Yvette,
France) following the manufacturer’s instructions for annealing
temperatures and cycling. The identity of the clones was confirmed
by sequencing. Primers are listed in Supplementary Table S1.
Transient expression in grapevine leaves
Transient expression in grapevine leaves was realized as described
Santos-Rosa et al. (2008
) using Agrobacterium tumefaciens
C58C1 strain. Briefly, detached leaves from 8–10-week-old in
vitrogrown V. vinifera were submerged abaxial face down in cylindrical
flasks containing the bacterial culture and covered with a disc of
Miracloth. Flasks were placed into a desiccator and vacuum was
applied twice for 2 min. Leaves were then placed in sealed Petri
dishes on wet Whatmann paper.
Gene expression analysis by real-time quantitative RT-PCR
RNA extraction and DNase I treatment were performed as described
Chong et al. (2008
). Reverse transcription was performed on 1 µg
of RNA using the SuperScript II Reverse Transcriptase (Invitrogen,
Carlsbad, CA, USA) and oligodT priming as recommended by the
For real-time PCR, reactions were carried out on the CFX96
system (BioRad, Marnes-la-Coquette, France). PCR reactions
were carried out in triplicate in a reaction buffer containing 1X
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 µl. Thermal cycling conditions were 30 s at 95 °C followed by
40 cycles of 15 s at 94 °C, 30 s at 60 °C and 30 s at 72 °C. The
calibration curve for each gene was obtained by performing
realtime PCR with serial dilutions of the purified PCR product (from
102 to 108 cDNA copy number). The specificity of the individual
PCR amplification was checked using a heat dissociation curve
from 55 to 95 °C following the final cycle of the PCR and by
sequencing the final PCR products. The results obtained for each
gene of interest were normalized to the expression of two
reference genes (VvActin1 and VvEF1α) as described in
et al. (2002
) and relative expression (fold induction) compared
to appropriate controls (see figure legends) was calculated as
described by Pfaffl (2001). Mean values and standard deviations
were obtained from at least three technical and two biological
replicates. Primers used for real-time quantitative PCR are listed
in Supplementary Table S1.
Subcellular localization of VvSWEET4
pGFP-VvSWEET4 was mobilized into the C58C1 strain of
A. tumefaciens and used to transform A. thaliana Col-0 mutant by the floral
(Bechtold and Pelletier, 1998)
. Images from leaf sectors
were acquired with a LSM700 confocal laser microscope (Carl Zeiss,
Jena, Germany), using a 63×, 1.2 numerical aperture
water-immersion objective lens at 23 °C. Fluorescence of free GFP or GFP fusion
proteins was observed after excitation with a 488 nm laser line, using
a 505–550 band-pass emission filter.
Complementation of yeast EBY.VW4000
The pPMA1-VvSWEET4 construct was used to transform the
Saccharomyces cerevisiae EBY.VW4000 as described in
Chen et al.
). EBY.VW4000 strain is completely deficient in glucose uptake
due to multiple mutations in the hexose transporters
et al., 1999)
, but it can grow on a maltose medium. EBY.VW4000
strain was grown on 1% yeast extract/2% peptone medium
supplemented with 2% maltose. After transformation, cells were streaked on
0.17% yeast nitrogen base medium supplemented with 0.5%
ammonium sulphate, 2% maltose, and amino acids (leucine, histidine and
tryptophan at 50 mg l−1 each). For complementation growth assays,
cells were grown overnight in liquid yeast nitrogen base medium
supplemented with ammonium sulphate, maltose and amino acids to an
optical density at 600 nm (OD600) of 0.6, then OD600 was adjusted to
0.2 with water. Serial dilutions (×1, ×5, ×25 and ×125) were plated
on yeast nitrogen base/ammonium sulphate medium containing
either 2% maltose (as control) or 2% glucose plus leucine, histidine
and tryptophan (50 mg l−1). Plates were grown for 3 days at 30 °C.
Genotyping and transcript analysis of T-DNA mutants
Genomic DNA was extracted from A. thaliana Col-0 and from
sweet4 mutants (Salk_072225 and GK858G02 T-DNA insertions)
and was used as a template for PCR amplification of AtSWEET4
fragments. Primers specific to AtSWEET4 sequences flanking the
T-DNA insertions were designed. Sites of T-DNA insertion were
confirmed by sequencing junction fragments obtained by PCR
using oligonucleotides from the T-DNA left border and AtSWEET4
genomic sequence. After selfing of T3 progeny, genotyping by PCR
enabled the isolation of homozygous lines.
The expression level of AtSWEET4 was determined by qRT-PCR
in Col-0 and sweet4 leaves with primers specific to AtSWEET4 and
flanking the T-DNA. Total RNA extraction, cDNA synthesis and
PCRs were carried out as described in the Materials and methods
section Gene expression analysis by real-time quantitative RT-PCR.
Primers are listed in Supplementary Table S1.
Identification and phylogenetic analysis of SWEET genes in grapevine
To identify grapevine SWEET genes, the 17 Arabidopsis SWEET
proteins were blasted against the predicted proteome of the
grapevine genome sequence. Each Arabidopsis SWEET protein
produced significant hits (E values <10e−20) to a set of 16
V. vinifera proteins, with different E values depending on the Arabidopsis
protein used as a query. All putative V. vinifera SWEET proteins
presented the PFAM motif PF03083 and were all predicted to
have seven transmembrane domains characteristic of SWEET
proteins with the exception of GSVIVT01031172001, which
contained 14 transmembrane domains. This entry was manually
re-annotated to give two proteins containing seven
transmembrane domains and PF03083 motif. Thus, V. vinifera appears to
have 17 SWEET proteins, the same number as A. thaliana.
V. vinifera SWEET proteins, hereafter referred to as
VvSWEET, were named on the basis of their percentage of
identity to A. thaliana SWEET proteins (AtSWEET). The
names and identity of the 17 VvSWEET proteins are
presented in Supplementary Table S2.
We constructed a phylogenetic tree using the SWEET
proteins from Arabidopsis, grapevine and rice (Fig. 1). The
four previously reported plant clades of SWEET proteins
were clearly identified in the resulting tree. Proteins from
clade I were highly conserved among the three species, as
shown by the strong support of branches separating the three
protein groups, whereas in the other clades the
identification of orthologous genes was more difficult. VvSWEETs
appeared to be underrepresented in the group containing
AtSWEETs 10–15 and OsSWEETs 11–15 inside clade III.
leaves, and VvSWEET17a, which is highly expressed in leaves,
stems and tendrils (Fig. 2). Interestingly, VvSWEET12 seems
to be specifically expressed in roots.
Results from expression studies in different organs are in
agreement with a role for VvSWEETs in sugar transport in
sink tissues such as flowers and berries.
Expression profiles of VvSWEET genes in different
V. vinifera tissues
We analysed the expression of the VvSWEET gene family
in different organs of V. vinifera 40024 (leaves, stems,
tendrils, flowers, roots and berries at different stages) by using
quantitative RT-PCR with gene-specific primers (Fig. 2). We
were able to detect the expression of all VvSWEETs except
for VvSWEET9 and VvSWEET17b. The results show that
a number of VvSWEET genes are highly expressed in
flowers (VvSWEET3, VvSWEET4, VvSWEET5a, VvSWEET5b,
VvSWEET7, VvSWEET10, VvSWEET11) and in berries
after véraison (VvSWEET4, VvSWEET7, VvSWEET10,
VvSWEET11, VvSWEET15, VvSWEET17d). Overall,
VvSWEET gene expression tends to increase during berry
maturation, with expression being lower in small green
berries compared to ripe berries (Fig. 2). For most VvSWEETs,
basal expression is lower in vegetative organs, except for
VvSWEET1, which is mainly expressed in young and adult
Expression profiles of VvSWEET genes after challenge with biotrophic and necrotrophic pathogens
Regulation of SWEET gene expression following infection
with different types of pathogens has been observed in rice
and Arabidopsis. We hypothesized that a similar mechanism
may exist in the interaction between grapevine and pathogens,
especially biotrophs such as downy and powdery mildew.
We first studied the expression of VvSWEET genes in
compatible interactions between V. vinifera 40024 and E. necator or
P. viticola, the agents of powdery and downy mildew respectively. None
of the VvSWEET genes showed strong induction of expression
after inoculation with these two pathogens, at neither early (24h
pi) nor late (72h pi) time points (Fig. 3A and B). Inoculation with
E. necator resulted in a 3-fold induction of VvSWEET3,4 and 17d
expression after 72h (Fig. 3A), and VvSWEET2a was similarly
induced by P. viticola inoculation at 72h (Fig. 3B).
To get insight into VvSWEET gene expression after
challenge with a different type of pathogen, we inoculated
V. vinifera 40024 leaves with the necrotrophic fungus B. cinerea,
and monitored the expression of VvSWEETs (Fig. 3C).
Interestingly, VvSWEET4 expression was strongly induced
following B. cinerea inoculation, both at 72 and 96 h pi (Fig. 3C).
Expression of the other VvSWEETs was not affected by
B. cinerea inoculation, except VvSWEET2a and VvSWEET7,
whose expression was moderately induced (Fig. 3C).
In an independent experiment, we studied the expression of
VvSWEET4 along with two typical markers of B. cinerea
infection: VvSTS, a key enzyme in the synthesis of stilbene
phytoalexins, and VvHSR1, a cell death marker in grapevine
et al., 2002)
. We confirmed that B. cinerea inoculation triggered
a dramatic up-regulation of the VvSWEET4 gene (5000-fold
induction at 72 h and 3000-fold induction at 96 h pi compared to
control), both in V. vinifera 40024 (Fig. 3D) and in V. vinifera cv.
Chardonnay (data not shown). VvSWEET4 expression was
correlated with the expression of VvSTS and VvHSR1 (Fig. 3D).
VvSWEET4 is a plasma membrane protein and complements glucose-uptake deficiency in yeast EBY. VW4000
The predicted VvSWEET4 amino acid sequence
contains seven transmembrane domains characteristic of
SWEET transporters. To establish whether VvSWEET4 is a
membrane-localized protein, its coding sequence was fused
to GFP under the control of the 35S CaMV promoter and
the resulting construct was used to stably transform
A. thaliana. Control leaves expressing free GFP yielded a high
fluorescence predominantly visible in the cytoplasm and the
endoplasmic reticulum (Fig. 4A). The VvSWEET4-GFP
fusion protein was predominantly located at the periphery of
transformed epidermal cells and to a lesser extent in nuclei
(Fig. 4B). Fluorescence pattern and its association with the
cell periphery shows that VvSWEET4 is likely targeted to the
plasma membrane in planta.
To demonstrate the function of VvSWEET4 as a sugar
transporter, the gene was expressed in the hexose
transportimpaired S. cerevisiae mutant EBY.VW4000, which has a very
low rate of hexose uptake due to concurrent knockout of 20
endogenous transporter genes
(Wieczorke et al., 1999)
A full-length VvSWEET4 cDNA was cloned into the
yeast pPMA1 vector (pPMA1-VvSWEET4). As a control,
VvSWEET4 was cloned into pPMA1 in reverse orientation
relative to the PMA1 promoter. Both constructs were
introduced into EBY.VW4000 yeast strain, which is able to grow
on maltose, but not on glucose or fructose
(Wieczorke et al.,
. As expected, EBY.VW4000 transformed with both
constructs grew on maltose (Fig. 4C). As shown in Fig. 4D,
expression of pPMA1-VvSWEET4, restores EBY.VW4000
VvSWEET4 expression is strongly induced by infection with
B. cinerea, which is associated with induction of an
oxidative burst and plant cell death. To get further insight into
VvSWEET4 regulation, we analysed VvSWEET4
expression after generation of reactive oxygen species (ROS) and
after inoculation with P. syringae pv. pisi, a non-host
bacterium triggering a hypersensitive response (HR) in grapevine
(Robert et al., 2001)
. Finally, two virulence factors from
necrotizing pathogens were transiently expressed in grapevine
leaves, and the resulting VvSWEET4 expression was studied.
As shown in Fig. 5A, grapevine leaves infiltrated with
paraquat, a chemical generating toxic reactive oxygen
intermediates, developed necrotic areas within the infiltrated zone as
soon as 15 h after treatment. VvSWEET4 expression was
highly induced from 15 h after paraquat treatment (Fig. 5B).
The expression of VvHSR1 was also stimulated after
paraquat treatment, although to a lesser extent. VvSWEET4
and VvHSR1 were also up-regulated after inoculation with
P. syringae pv. pisi, a non-host pathogen triggering HR cell
death in grapevine (Fig. 5C).
In recent years, a class of proteins called NEP1-like proteins
that induce cell death in a variety of dicotyledonous plants
has been described in bacteria, fungi and oomycetes. We first
studied VvSWEET4 expression in V. vinifera leaves transiently
expressing PiNPP1.1, a NEP1-like protein from the oomycete
(Kanneganti et al., 2006)
. Even though
we could not observe a macroscopic HR, PiNPP
expression induced the expression of VvHSR and of VvSWEET4
in leaves (Fig. 5D). In contrast, transient expression of the
INF1 elicitin, which triggers the HR in Nicotiana species, did
not significantly stimulate VvSWEET4 and VvHSR
expression (Fig. 5D). Finally, we investigated VvSWEET4
expression after transient expression of BcNEP1, a NEP1-like
protein from the necrotrophic fungus B. cinerea
et al., 2008)
. Transient expression of BcNEP1 in leaves of
V. vinifera plantlets triggered cell necrosis, which could be
observed after 6 days (Fig. 5E). VvSWEET4 transcripts were
dramatically induced 4 days after BcNEP1 expression
(5000fold induction compared to control) and were maximal after
6 days (27 000-fold induction compared to control, Fig. 5F).
VvSWEET4 expression was correlated with the expression of
VvHSR. Expression of these two genes was higher in BcNEP
agroinfiltrated leaves compared to leaves expressing PiNPP
(Fig. 5D and F).
Overall, these results show that VvSWEET4 expression is
highly up-regulated by inducers of ROS and virulence factors
from necrotizing pathogens.
Knockout mutants in AtSWEET4 are less susceptible to infection with B. cinerea
T-DNA insertion mutants and Tilling collections are not
available in grapevine. Furthermore, extinction of gene
expression by RNAi is painstaking because grapevine
genetic transformation is time consuming and shows low
(Vidal et al., 2010)
. Therefore, to further
characterize the role of VvSWEET4 during B. cinerea infection, we
searched for knockout mutants in the closest SWEET
homologues in Arabidopsis. Sequence comparison by BLAST
analysis with the VvSWEET4 amino acid sequence revealed
that VvSWEET4 closest homologues are AtSWEET4 and
AtSWEET5 (60% identity/72% similarity to AtSWEET4;
57% identity/73% similarity to AtSWEET5). Interestingly,
previous studies have shown that AtSWEET4 expression
is induced after challenge with B. cinerea
(Ferrari et al.,
2007; Chen et al., 2010)
, whilst AtSWEET5 expression was
(Chen et al., 2010)
. Based on this
observation, we decided to study T-DNA knockout mutants in the
Search in available T-DNA collections enabled us to
identify two T-DNA insertions in AtSWEET4, one in the Salk
collection located at the beginning of the last exon
(sweet41) and one from the GABI-KAT (GK) collection
(sweet42) located in the fourth intron (Supplementary Fig. S1A).
Quantitative RT-PCR with primers spanning T-DNA
insertion confirmed that AtSWEET4 expression is enhanced by
B. cinerea infection in wild-type Col-0 and that its expression
is knocked out in both T-DNA homozygous insertion lines
(Supplementary Fig. S1B). Both mutant lines did not show
macroscopic phenotypic differences compared to Col-0.
Previous studies have shown that AtSWEET4 expression is
highly stimulated after inoculation with virulent P. syringae
(Chen et al., 2010)
. To get insight into the
potential role of AtSWEET4 in resistance to bacteria, we
measured bacterial growth in sweet4 mutants after local infection
with virulent P. syringae pv. tomato DC3000 (PstDC3000) or
avirulent P. syringae pv. tomato carrying the AvrRpt2 gene
(PstAvrRpt2). Bacterial growth was measured in wild-type
Col-0, sweet4-1 and sweet4-2 lines 48 and 72 h following
inoculation with P. syringae pv. tomato. Bacterial
multiplication was similar in sweet4 mutants and in Col-0, both for
virulent PstDC3000 (Supplementary Fig. S2A) and avirulent
PstAvrRpt2 strains (Supplementary Fig. S2B).
Sweet4 mutants were further tested for their
susceptibility to the necrotrophic fungus B. cinerea. Spores were
drop-inoculated on half leaves of Col-0 and sweet4-knockout
mutants and the lesion size was measured 2 and 3 days later.
As shown in Fig. 6A, both sweet4 mutant lines showed lesions
with decreased sizes compared to Col-0. Lesion size after
B. cinerea inoculation was measured 72 h pi. Analysis of
variance showed that mean lesion size in sweet4-1 and sweet4-2
lines was significantly different from mean lesion size in Col-0
(P<0.01, Fig 6B). Two additional independent experiments
confirmed the decreased size of B. cinerea lesions in sweet4
mutants 72 h pi (Fig. 6C). Decreased lesion size was
generally more pronounced in the sweet4-1 line compared with
the sweet4-2 line, where a very small residual expression of
AtSWEET4 could be measured (Supplementary Fig 1B).
Quantitative PCR analysis further confirmed lower levels
of fungal multiplication in both sweet4 lines compared with
Col-0, which correlates with decreased severity of disease
symptoms (Fig. 6D). Together, these results show that
knockout in AtSWEET4 increases resistance to the necrotrophic
fungus B. cinerea in Arabidopsis.
In this study we identified 17 putative SWEET transporters
in the grapevine genome and showed that their expression is
differentially regulated in grapevine vegetative and
reproductive organs. VvSWEET4 is strongly induced upon B. cinerea
infection and also following treatments inducing plant cell
death. Complementation of yeast EBY.VW4000 revealed
that VvSWEET4 acts as a glucose transporter and confocal
microscopy observations confirmed its subcellular
localization in the plasma membrane. Finally, mutants of A. thaliana
for the orthologous AtSWEET4 exhibited enhanced
resistance to B. cinerea.
The number of SWEET proteins found in the V. vinifera
genome is in agreement with a previous report
et al., 2013)
, because one of the 16 SWEET proteins
identified by Lecourieux et al. (2013) corresponds to two proteins
in our case. The fact that both studies reached the same
numbers using different search strategies (Blast and PFAM motif
searches) validates the report of the complete set of SWEET
transporters from V. vinifera.
We detected the expression of 15 out of 17 SWEET genes
in V. vinifera, and expression of each member shows a specific
pattern in different organs. VvSWEET17a is the gene whose
expression is the highest in vegetative organs (leaves, stem and
tendrils). In Arabidopsis, SWEET17, a fructose transporter, is
also highly expressed in leaves and in xylem tissues, in
accordance with a role in fructose partitioning in vegetative organs
(Chardon et al., 2013)
. The VvSWEET1 gene is also
preferentially expressed in leaves. VvSWEET1 shows 82% similarity
to AtSWEET1, which has been described as a glucose
(Chen et al., 2010)
. Interestingly, VvSWEET12
expression is highest in roots compared to other organs, and could
be involved in uptake and release of carbohydrates from
roots. The expression of VvSWEET9 and VvSWEET17b was
not detected in our study. Recently, a detailed transcriptomic
analysis of samples representing green and woody tissues and
organs at different developmental stages was realized in
(Fasoli et al., 2012)
. This analysis shows that expression
of VvSWEET9 and VvSWEET17b was only detected in
pollen and senescing leaves respectively, suggesting functions in
specialized tissues for these two transporters.
Several VvSWEETs show enhanced expression in
reproductive sink organs. Seven VvSWEETs (3, 4, 5a, 5b, 7, 10,
11) show enhanced expression in flowers and six (4, 7, 10, 11,
15, 17d) are up-regulated in berries post-véraison. In
grapevine, important accumulation of glucose and fructose in
berry mesocarp cells is a hallmark of the onset of ripening
(Lecourieux et al., 2013)
. In flowers, a lack of sugar
availability caused by various environmental or physiological
fluctuations may lead to drastic flower abortion
(Lebon et al., 2008)
High expression of VvSWEET transporters in flowers and
in berries after véraison highlights a putative important role
in sugar partitioning during flower and fruit development.
Further studies of grapevine SWEET transporters highly
expressed in berries should add important information for
the control of hexose accumulation in flesh cells.
The conservation of the SWEET sugar transporter
family among the plant kingdom (monocots and dicots, both
herbaceous and woody) suggests likely important functions
in grapevine development and responses to environmental
stresses. Proteins from clade I were highly conserved among
grapevine, rice and Arabidopsis and include the AtSWEET1
protein, which has been identified as a glucose uniporter in
(Chen et al., 2010)
. Interestingly, no
grapevine SWEET was found in the group from clade III
comprising AtSWEET11 and 12, two sucrose transporters involved
in sugar efflux from phloem parenchyma cells, a prerequisite
to phloem loading by H+/sucrose symporters in apoplastic
(Chen et al., 2012)
. Most woody plants, such
as grapevine, are considered to be symplastic phloem
‘loaders’ due to the presence of plasmodesmata connecting
mesophyll cells with phloem-associated cells
absence of AtSWEET11 and AtSWEET12 homologues in
grapevine is in agreement with sugar transport in the phloem
occurring in a symplastic way, which would not involve
Clade III also comprises OsSWEET11, OsSWEET13 and
OsSWEET14, three targets of X. oryzae effectors, which
trigger SWEET gene expression to acquire sugars from their
host. The absence of grapevine SWEET homologues close
to these rice SWEET transporters in the phylogenetic tree
may indicate that this virulence strategy is not exploited by
grapevine pathogens. Results obtained after infection with
the biotrophic grapevine pathogens P. viticola and E.
necator support this assumption. Indeed, our results show that
VvSWEETs expression is not strongly modified in
compatible interactions between grapevine and P. viticola or
E. necator. These results were confirmed for P. viticola by analysing
available RNAseq data (Perazzolli et al.,
et al., 2012
). Although we cannot rule out that constitutively
expressed VvSWEETs are involved in the interaction between
grapevine and biotrophic pathogens, these sugar
transporters may not be the target of biotrophic pathogen effectors.
It is possible that other classes of sugar transporters such
as hexose or H+/sucrose symporters are involved in carbon
partitioning during grapevine interaction with powdery and
downy mildew agents. Indeed, the VvHT5 transporter was
shown to be up-regulated by biotrophic pathogen infection
and wounding, suggesting a role in the provision of hexoses
to plant cells under stress conditions
(Hayes et al., 2010)
Interestingly, challenge with B. cinerea, a necrotrophic
pathogen, led to a dramatic up-regulation of the VvSWEET4
gene. VvSWEET4 expression was also induced after
infiltration with P. syringae pv. pisi, a bacterial pathogen
triggering an HR-like response when infiltrated in grapevine leaves
(Robert et al., 2001)
. B. cinerea kills plant cells by
activating an oxidative burst and producing phytotoxic
metabolites and proteins
(van Kan, 2006)
. Our results suggest that
VvSWEET4 induction is mediated by both ROS and
virulence protein factors from the fungus. Treatment of grapevine
leaves with paraquat, a chemical inducing the production of
ROS, or transient expression of two NEP-like proteins from
Phytophthora infestans or B. cinerea were found to strongly
up-regulate VvSWEET4 expression. VvSWEET4 expression
paralleled the expression of VvHSR1, a cell death marker in
(Bézier et al., 2002)
In contrast to biotrophic pathogens that establish an
intricate relation with their host, the strategy of plant cell
colonization by necrotrophic pathogens has long been considered as
much less subtle. Indeed, necrotrophic pathogens reportedly
kill host cells and feed on dead macerated tissues
. However, there is cytological and molecular genetic
evidence for B. cinerea developing appressoria
structures that differentiate on the surface and form a
penetration peg that breaches the cuticle; van Kan, 2006)
studies also showed that B. cinerea manipulates plant
physiological processes to colonize host tissue. Indeed, B. cinerea
is able to induce the expression of glutaredoxin and NPR1
genes, resulting in facilitated infection
(reviewed in Mengiste,
. Our results show that SWEET transporters could be
the target of a necrotrophic pathogen, and facilitate glucose
efflux from plant cells, resulting in a higher hexose
concentration in the apoplast, that would enhance fungal growth.
This hypothesis is in accordance with enhanced resistance to
B. cinerea infection of Arabidopsis sweet4 mutants,
characterized by a reduction in lesion size and pathogen growth
following inoculation. Interestingly, after infection of sunflower
cotyledons or tomato leaves with B. cinerea, a decrease in
plant hexoses (glucose, fructose) and sucrose was observed,
especially 48 and 72 h pi
(Berger et al., 2004; Dulermo et al.,
. Decrease in endogenous sugar levels paralleled an
increase in hexose transporter expression in the fungus
during the infection process (Dulermo et al., 2009).
An alternative explanation for the increased resistance to
B. cinerea found in sweet4 mutants is related to the
possible role of AtSWEET4 and VvSWEET4 in plant cell death.
Indeed, VvSWEET4 expression is triggered by treatments or
pathogens inducing cell death. It is known that infection by
B. cinerea is promoted by and requires an active cell death
programme in the host
(van Kan, 2006)
. Reduced cell death
induced by B. cinerea in atsweet4 mutants may thus result in
a reduced susceptibility.
A consequence of programmed cell death in plants can
be sugar redistribution, as it happens through senescence.
During the senescence program in the leaf, nutrients like
carbon, nitrogen, phosphate and potassium are exported
from the leaf to other plant organs such as developing fruits
and young leaves. In Arabidopsis, a monosaccharide
transporter (SFP1) belonging to the major facilitator
superfamily is induced during leaf senescence
(Quirino et al., 2001)
Several sugars such as galactose, fructose and glucose also
accumulate in senescent leaves
(Quirino et al., 2001)
Arabidopsis SAG29 gene is expressed primarily in
senescent tissues and was recently identified as AtSWEET15.
AtSWEET15-overexpressing plants exhibited an accelerated
senescence and were hypersensitive to salt stress
(Seo et al.,
. Interestingly, overexpression of OsSWEET5, which is
closely related to AtSWEET4, resulted in precocious
senescence in rice
(Zhou et al., 2014)
. In grapevine, transcriptomic
studies did not show expression of VvSWEET4 in senescent
(Fasoli et al., 2012)
. However, VvSWEET4 may be
involved in sugar redistribution during pathogen-induced
The precise role of AtSWEET4 and VvSWEET4 in plant
cell death remains to be elucidated. sweet4 mutants were still
able to mount a cell death response following B. cinerea
infection. Accordingly, transient overexpression of VvSWEET4 is
not sufficient to trigger cell death in grapevine or Nicotiana
benthamiana, although it caused a small increase in VvHSR1
expression in grapevine leaves (data not shown). Further
research should help unveiling the precise role played by
VvSWEET4 in plant cell death and the possible existence of
redundant functions in the SWEET family.
In conclusion, our work highlights a new role for SWEET
transporters in the interaction with necrotrophic fungi. It
was previously demonstrated that manipulation of SWEET
transporter expression is essential for virulence of
pathogenic bacteria. We show here that virulence factors from
B. cinerea (NEP protein), together with the induction of
an oxidative burst in plant cells trigger the expression of
a grapevine SWEET transporter that could enhance
hexose efflux and/or plant cell death, both being beneficial to
fungal growth. Finally, comprehension of the link between
SWEET transporters and pathogen susceptibility is
important for developing strategies improving grapevine resistance
to biotic stresses.
Supplementary material is available at JXB online.
Supplementary Fig. S1. Analysis of A. thaliana sweet4
Supplementary Fig. S2. Growth of Pseudomonas syringae
pv. tomato in A. thaliana Col-0 and sweet4 mutants.
Supplementary Table S1. Primers used in this study.
Supplementary Table S2. Grapevine SWEET genes.
We are grateful to Sabine Wiedemann-Merdinoglu (INRA, Colmar, France)
for providing P. viticola and E. necator isolates, Dominique Buffard (ISV,
Gif sur Yvette, France) for providing P. syringae pv. pisi, Yves Brygoo
(INRA, Versailles, France) for providing B. cinerea B0510 isolate, Sophien
Kamoun (The Sainsbury Laboratory, Norwich, UK) for INF1 and PiNPP
constructs and to Professor Eckhard Boles (IMB, JWGU, Frankfurt,
Germany) for providing yeast strain EBY.VW4000. We are grateful to
J. Mutterer (IBMP-CNRS, Strasbourg, France) for help with confocal
microscopy observations. Special thanks to Thierry Heitz (IBMP-CNRS,
Strasbourg) for P. syringae pv. tomato strains and for critical reading of
the manuscript. We are indebted to the experimental unit of INRA-Colmar
for their valuable technical support in the production and maintenance of
plants. We thank Camille Rustenholz and Gautier Arista (INRA, Colmar)
for analysis of publicly available RNAseq data.
Afoufa-Bastien D , Medici A , Jeauffre J , Coutos-Thévenot P , Lemoine R , Atanassova R , Laloi M. 2010 . The Vitis vinifera sugar transporter gene family: phylogenetic overview and macroarray expression profiling . BMC Plant Biology 10 , 245 .
Alonso JM , Stepanova AN , Leisse TJ , et al. 2003 . Genome-wide insertional mutagenesis of Arabidopsis thaliana . Science 301 , 653 - 657 .
Azevedo H , Conde C , Gerós H , Tavares RM . 2006 . The non-host pathogen Botrytis cinerea enhances glucose transport in Pinus pinaster suspension-cultured cells . Plant & Cell Physiology 47 , 290 - 298 .
Baker RF , Leach KA , Braun DM . 2012 . SWEET as sugar: new sucrose effluxers in plants . Molecular Plant 5 , 766 - 768 .
Bechtold N , Pelletier G. 1998 . In planta agrobacterium mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration .
Arabidopsis protocols . New York: Humana Press, 259 - 266 .
Bendahmane A , Farnham G , Moffett P , Baulcombe DC . 2002 .
Constitutive gain -of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato . The Plant Journal 32 , 195 - 204 .
Berger S , Papadopoulos M , Schreiber U , Kaiser W , Roitsch T. 2004 .
Complex regulation of gene expression, photosynthesis and sugar levels by pathogen infection in tomato . Physiologia Plantarum 122 , 419 - 428 .
Berr A , McCallum EJ , Alioua A , Heintz D , Heitz T , Shen W-H. 2010 .
Arabidopsis histone methyltransferase SET DOMAIN GROUP8 mediates induction of the jasmonate/ethylene pathway genes in plant defense response to necrotrophic fungi . Plant Physiology 154 , 1403 - 1414 .
Bézier A , Lambert B , Baillieul F. 2002 . Cloning of a grapevine Botrytisresponsive gene that has homology to the tobacco hypersensitivity-related hsr203J . Journal of Experimental Botany 53 , 2279 - 2280 .
Bogdanove AJ , Schornack S , Lahaye T. 2010 . TAL effectors: finding plant genes for disease and defense . Current Opinion in Plant Biology 13 , 394 - 401 .
Broekaert WF , Terras FRG , Cammue BPA , Vanderleyden J. 1990 .
An automated quantitative assay for fungal growth inhibition . FEMS Microbiology Letters 69 , 55 - 59 .
Chardon F , Bedu M , Calenge F , et al. 2013 . Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis . Current Biology 23 , 697 - 702 .
Chen D-C , Yang B-C , Kuo T-T. 1992 . One-step transformation of yeast in stationary phase . Current Genetics 21 , 83 - 84 .
Chen L-Q , Hou B-H , Lalonde S , et al. 2010 . Sugar transporters for intercellular exchange and nutrition of pathogens . Nature 468 , 527 - 532 .
Chen L-Q , Qu X-Q , Hou B-H , Sosso D , Osorio S , Fernie AR , Frommer WB . 2012 . Sucrose efflux mediated by SWEET proteins as a key step for phloem transport . Science 335 , 207 - 211 .
Chong J , Le Henanff G , Bertsch C , Walter B. 2008 . Identification, expression analysis and characterization of defense and signaling genes in Vitis vinifera . Plant Physiology and Biochemistry 46 , 469 - 481 .
Chu Z , Yuan M , Yao J , et al. 2006 . Promoter mutations of an essential gene for pollen development result in disease resistance in rice . Genes and Development 20 , 1250 - 1255 .
Dereeper A , Guignon V , Blanc G , et al. 2008 . Phylogeny.fr: robust phylogenetic analysis for the non-specialist . Nucleic Acids Research 36 , W465 - W469 .
Doidy J , Grace E , Kühn C , Simon-Plas F , Casieri L , Wipf D. 2012 .
Sugar transporters in plants and in their interactions with fungi . Trends in Plant Science 17 , 413 - 422 .
2009. Dynamic carbon transfer during pathogenesis of sunflower by the necrotrophic fungus Botrytis cinerea: from plant hexoses to mannitol . The New Phytologist 183 , 1149 - 1162 .
Fasoli M , Dal Santo S , Zenoni S , et al. 2012 . The grapevine expression atlas reveals a deep transcriptome shift driving the entire plant into a maturation program . The Plant Cell 24 , 3489 - 3505 .
Ferrari S , Galletti R , Denoux C , De Lorenzo G , Ausubel FM , Dewdney J. 2007 . Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3 . Plant Physiology 144 , 367 - 379 .
Fotopoulos V , Gilbert MJ , Pittman JK , Marvier AC , Buchanan AJ , Sauer N , Hall JL , Williams LE . 2003 . The monosaccharide transporter gene, AtSTP4, and the cell-wall invertase, Atbetafruct1, are induced in Arabidopsis during infection with the fungal biotroph Erysiphe cichoracearum . Plant Physiology 132 , 821 - 829 .
Gachon C , Saindrenan P. 2004 . Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea . Plant Physiology and Biochemistry 42 , 367 - 371 .
Galzy R. 1964 . Technique de thermotherapie des viroses de la vigne.
Annales des Epiphyties 15 , 245 -256 Gamalei Y. 1989 . Structure and function of leaf minor veins in trees and herbs . Trees 3 , 96 - 110 .
Gouy M , Guindon S , Gascuel O. 2010 . SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building . Molecular Biology and Evolution 27 , 221 - 224 .
Guo WJ , Nagy R , Chen HY , Pfrunder S , Yu YC , Santelia D , Frommer WB , Martinoia E. 2014 . SWEET17, a facilitative transporter, mediates fructose transport across the tonoplast of Arabidopsis roots and leaves .
Plant Physiology 164 , 777 - 789 .
Hayes MA , Feechan A , Dry IB . 2010 . Involvement of abscisic acid in the coordinated regulation of a stress-inducible hexose transporter (VvHT5) and a cell wall invertase in grapevine in response to biotrophic fungal infection . Plant Physiology 153 , 211 - 221 .
Hu Y , Zhang J , Jia H , Sosso D , Li T , Frommer WB , Yang B , White FF , Wang N , Jones JB . 2014 . Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease . Proceedings of the National Academy of Sciences, USA 111 , E521 - 529 .
Jaillon O , Aury JM , Noel B , et al. 2007 . The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla .
Nature 449 , 463 - 468 .
Kanneganti T-D , Huitema E , Cakir C , Kamoun S. 2006 . Synergistic interactions of the plant cell death pathways induced by Phytophthora infestans Nepl-like protein PiNPP1.1 and INF1 elicitin . Molecular PlantMicrobe Interactions 19 , 854 - 863 .
Katagiri F , Thilmony R , He SY . 2002 . The Arabidopsis thalianaPseudomonas syringae interaction . The Arabidopsis Book/American Society of Plant Biologists 1 , e0039 .
2012. GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database . Nucleic Acids Research 40 , D1211 - D1215 .
Lebon G , Wojnarowiez G , Holzapfel B , Fontaine F , Vaillant-Gaveau N , Clément C. 2008 . Sugars and flowering in the grapevine (Vitis vinifera L.) . Journal of Experimental Botany 59 , 2565 - 2578 .
Lecourieux F , Kappel C , Lecourieux D , Serrano A , Torres E , ArceJohnson P , Delrot S. 2013 . An update on sugar transport and signalling in grapevine . Journal of Experimental Botany 65 , 821 - 832 .
Lemoine R , La Camera S , Atanassova R , et al. 2013 . Source-to-sink transport of sugar and regulation by environmental factors . Frontiers in Plant Science 4 , 272 .
Lin IW , Sosso D , Chen L-Q , et al. 2014 . Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9 . Nature 508 , 546 - 549 .
Mengiste T. 2012 . Plant immunity to necrotrophs . Annual Review of Phytopathology 50 , 267 - 294 .
Miclot A-S , Wiedemann-Merdinoglu S , Duchêne E , Merdinoglu D , Mestre P. 2012 . A standardised method for the quantitative analysis of resistance to grapevine powdery mildew . European Journal of Plant Pathology 133 , 483 - 495 .
Perazzolli M , Moretto M , Fontana P , Ferrarini A , Velasco R , Moser C , Delledonne M , Pertot I. 2012 . Downy mildew resistance induced by Trichoderma harzianum T39 in susceptible grapevines partially mimics transcriptional changes of resistant genotypes . BMC Genomics 13 , 660 .
Schouten A , van Baarlen P, van Kan JAL. 2008 . Phytotoxic Nep1-like proteins from the necrotrophic fungus Botrytis cinerea associate with membranes and the nucleus of plant cells . The New Phytologist 177 , 493 - 505 .
Seo PJ , Park J-M , Kang SK , Kim S-G , Park C-M. 2011 . An Arabidopsis senescence-associated protein SAG29 regulates cell viability under high salinity . Planta 233 , 189 - 200 .
Slewinski TL . 2011 . Diverse functional roles of monosaccharide transporters and their homologs in vascular plants: a physiological perspective . Molecular Plant 4 , 641 - 662 .
Sutton PN , Gilbert MJ , Williams LE , Hall JL . 2007 . Powdery mildew infection of wheat leaves changes host solute transport and invertase activity . Physiologia Plantarum 129 , 787 - 795 .
Sutton PN , Henry MJ , Hall JL . 1999 . Glucose, and not sucrose, is transported from wheat to wheat powdery mildew . Planta 208 , 426 - 430 .
Vandesompele J , De Preter K , Pattyn F , Poppe B , Van Roy N , De Paepe R , Speleman F. 2002 . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes . Genome Biology 3 , research0034 . 1 - 0034 .11 Pfaffl MW . 2001 . A new mathematical model for relative quantification in real-time RT-PCR . Nucleic Acids Research 29 , e45 .
Van Kan JAL. 2006 . Licensed to kill: the lifestyle of a necrotrophic plant pathogen . Trends in Plant Science 11 , 247 - 253 .
Quirino BF , Reiter WD , Amasino RD . 2001 . One of two tandem Arabidopsis genes homologous to monosaccharide transporters is senescence-associated . Plant Molecular Biology 46 , 447 - 457 .
Robert N , Ferran J , Breda C , Coutos-Thévenot P , Boulay M , Buffard D , Esnault R. 2001 . Molecular characterization of the incompatible interaction of Vitis vinifera leaves with Pseudomonas syringae pv. pisi: expression of genes coding for stilbene synthase and class 10 PR protein .
European Journal of Plant Pathology 107 , 249 - 261 .
Römer P , Recht S , Strauss T , Elsaesser J , Schornack S , Boch J , Wang S , Lahaye T. 2010 . Promoter elements of rice susceptibility genes are bound and activated by specific TAL effectors from the bacterial blight pathogen, Xanthomonas oryzae pv . oryzae. The New Phytologist 187 , 1048 - 1057 .
Ruiz E , Ruffner HP . 2002 . Immunodetection of Botrytis-specific invertase in infected grapes . Journal of Phytopathology 150 , 76 - 85 .
Santos-Rosa M , Poutaraud A , Merdinoglu D , Mestre P. 2008 .
Development of a transient expression system in grapevine via agroinfiltration . Plant Cell Reports 27 , 1053 - 1063 .
Sauer N , Stolz J. 1994 . SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein . The Plant Journal 6 , 67 - 77 .
Vannozzi A , Dry IB , Fasoli M , Zenoni S , Lucchin M. 2012 . Genomewide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses . BMC Plant Biology 12 , 130 .
Vidal JR , Gomez C , Cutanda MC , Shrestha BR , Bouquet A , Thomas MR , Torregrosa L. 2010 . Use of gene transfer technology for functional studies in grapevine . Australian Journal of Grape and Wine Research 16 , 138 - 151 .
Wahl R , Wippel K , Goos S , Kämper J , Sauer N. 2010 . A novel highaffinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis . PLoS Biology 8 , e1000303 .
Wieczorke R , Krampe S , Weierstall T , Freidel K , Hollenberg CP , Boles E. 1999 . Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae . FEBS Letters 464 , 123 - 128 .
Yuan M , Zhao J , Huang R , Li X , Xiao J , Wang S. 2014 . Rice MtN3/ saliva/SWEET gene family: evolution, expression profiling, and sugar transport . Journal of Integrative Plant Biology 56 , 559 - 570 .
Zhou Y , Liu L , Huang W , Yuan M , Zhou F , Li X , Lin Y. 2014 .
Overexpression of OsSWEET5 in rice causes growth retardation and precocious senescence . PLoS One 9 , e94210 .