The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea

Journal of Experimental Botany, Nov 2014

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.

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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 Introduction 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 pollen development (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 interactions (Fotopoulos et al., 2003; Doidy et al., 2012) . For 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 (Fotopoulos 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) . Acco rdingly, Wahl et al. (2010 ) have 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) . AtSWEET11 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 pathogens (Chen et  al., 2010) , suggesting that each pathogen deploys a specific strategy to divert host carbohydrates for its growth (Slewinski, 2011) . 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) . Several 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 (Chu 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 (Yuan 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 subspecies 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 biotrophic pathogens (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 the pathogen. Materials and methods Biological material 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 Galzy medium (Galzy, 1964) 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 collection (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 described in 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 Ottonel. Treatment of plants with chemicals and pathogens P.  viticola inoculation was performed as described in Santos-Rosa 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 described in 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 described (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. Phylogenetic analysis 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 in Seaview (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. fr (Dereeper et  al., 2008) . OsSWEET proteins 7a, 7c, 7d and 7e, which are highly similar to OsSWEET7b (Chen et al., 2010) , were identified as reducing the alignment’s quality following curation by Gblocks. Those were removed from the alignment to improve the quality of the tree. Plasmid constructions 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) . These 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 in 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 in 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 supplier. 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 Vandesompele 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 dip method (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. (1992 ). EBY.VW4000 strain is completely deficient in glucose uptake due to multiple mutations in the hexose transporters (Wieczorke 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. Results 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 (Bézier 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., 1999) . 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 Phytophthora infestans (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 (Schouten 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 efficiency (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 not detected (Chen et  al., 2010) . Based on this observation, we decided to study T-DNA knockout mutants in the AtSWEET4 gene. 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 pv. tomato (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. Discussion 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 (Lecourieux 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 uniporter (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 V. vinifera (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 different systems (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 sugar transport (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 (Gamalei, 1989) . The 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 SWEET transporters. 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., 2012; Vannozzi 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 grapevine (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 (van Kan, 2006) . However, there is cytological and molecular genetic evidence for B.  cinerea developing appressoria (infection structures that differentiate on the surface and form a penetration peg that breaches the cuticle; van Kan, 2006) . Several 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, 2012) . 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., 2009) . 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) . The 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., 2011) . 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 leaves (Fasoli et  al., 2012) . However, VvSWEET4 may be involved in sugar redistribution during pathogen-induced cell death. 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 Supplementary material is available at JXB online. Supplementary Fig. S1. Analysis of A.  thaliana sweet4 mutants. 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. Acknowledgements 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. 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Julie Chong, Marie-Christine Piron, Sophie Meyer, Didier Merdinoglu, Christophe Bertsch, Pere Mestre. The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea, Journal of Experimental Botany, 2014, 6589-6601, DOI: 10.1093/jxb/eru375