Tomato (Solanum lycopersicum) and Nicotiana benthamiana (lab strain) plants were used in this study. Two different cultivars of tomato plants were used: ‘Rio Grande’ containing the Pto resistance gene (gently provided by Dr. Selena Giménez, Centro Nacional de Biotecnología, Madrid, Spain), and ‘Moneymaker’ (a gift from Prof. Jonathan Jones, The Sainsbury Laboratory, Norwich, UK). Plants were grown under standard greenhouse conditions (temperature from 20-25 °C, 16-hour day/8-hour night photoperiod, relative humidity from 50-70%). Pots were subirrigated once a day with nutrient Hoagland solution .
The tomato plant treatments were carried out by stem-feeding . Tomato 4-week old plants were excised with a scalpel just above cotyledons, and stems were immersed in the different compounds. After 30 minutes, all the stems were transferred to water and leaf tissue was collected at the indicated times. For the SA treatments, 2 mM SA were used, and samples were taken at 0, 0.5, 1, 8 and 24 h post-treatment. A sample was collected before starting to perform the experiment. For the scopoletin, umbelliferone or esculetin treatments, tomato plants were also stem-fed for 30 minutes, but this time stems were immersed in 0.5 mM of the corresponding phenolic compounds, and samples were collected at 0, 1, 6 and 24 h post-treatment. All the treatments were performed in a growth chamber at a constant temperature of 24 °C and a photoperiod of 16 h of light (300 µmol/m2/s) and 8 h of darkness. Both the third and fourth leaves from explants were harvested at the indicated times.
Bacterial growth conditions, inoculum preparation and inoculation
The bacterial strains herein used were Pseudomonas syringae pv. tomato DC3000 (Pst DC3000 AvrPto) and Pst DC3000, which contains deletions in genes AvrPto and AvrPtoB (Pst DC3000 ∆AvrPto) . The inoculum preparation and infection was carried out according to López-Gresa and collaborators . The third and fourth leaves, from bottom to top, were harvested at 0, 10, 18, 24, 36 and 48 h post-inoculation. Three biological replicates were analysed for each time and tomato-bacteria interaction.
Tomato plants were inoculated with TSWV according to Soler and collaborators . Briefly, one gram of the TSWV-infected leaves was homogenised in 20 mL of phosphate buffer (3 mM monosodium phosphate, 75 mM disodium phosphate, pH 7.4) containing 0.15 M NaCl, 1% polyvinylpolypyrrolidone, 0.02% mercaptoethanol, 1% carborundum and 1% active carbon. The 4-week old plants were inoculated on the uppermost leaflet from the third and fourth leaves (leaf position numbered from the base to the apex) using 50-100 μL of the viral extract per leaflet. Plants were dusted with carborundum, and were then inoculated by rubbing with a cotton swab soaked in virus suspension or were mock-inoculated with buffer only. One week later, the fifth and sixth leaves were inoculated. The fifth and sixth leaves were collected for all the analytical measurements at the indicated time points of infection. Plants were inspected visually for symptom evaluation, and disease severity was scored at the indicated time points. Samples corresponding to 3 replicates for the time course RNA analysis were collected and frozen at -80 ºC.
To determine the metabolite content, new sets of experiments were performed using 10 tomato plants of each genotype (Moneymaker, RNAi Twi1 1.1 and RNAi Twi1 28.3) and sampling at 6 dpi.
RNA extraction and quantitative RT-PCR analysis
The total RNA of the tomato tissues was isolated using TRIzol reagent (Invitrogen) following the manufacturer's protocol. A quantitative RT-PCR analysis was performed as previously described in the work by Campos and collaborators . A house-keeping gene transcript, Elongation Factor 1 alpha (eEF1α), was used as an endogenous reference. The PCR primers used to amplify Twi1 were 5’-GGATGCGAAGAGCTATGGAG-3’ as the forward primer and 5’-CGGACCAATAGCCCAATTTT-3’ as the reverse primer. For eEF1α amplification, 5’-CCACCTCGAGATCCTAATGG-3’ and 5’-ACCCTCACGTATGCTTCCAG-3’ were used as the forward and the reverse primer, respectively.
The full-length cDNA (1412 bp) of Tomato wound-induced gene (Twi1)  was amplified by RT-PCR from the Moneymaker tomato leaves infected with the bacterial pathogen Pseudomonas syringae pv. tomato using 5’-ATGGGTCAGCTACATTTTTTC-3’ as the forward primer and 5’-TTAACGATATGAAGTTATGTC-3’ as the reverse primer. The resulting PCR product was cloned into the pCR8/GW/TOPO entry vector (Invitrogen), following the manufacturer's protocol, and was sequenced. Then Twi1 was subcloned in the pGWB8 Gateway binary vector . In order to generate the Twi1-silenced transgenic tomato plants, the method described by Helliwell and Waterhouse was followed . Briefly, a selected 341 bp sequence of Twi1 was amplified from the full-length cDNA clone using the forward primer 5’-GGCTCGAGTCTAGAGAAATCAAGTTCCATTGTTTAT-3’, which introduced restriction sites XhoI and XbaI, and the reverse primer 5’-CCGAATTCGGATCCACTTCTCATTGAAAAAC-3’, which added restriction sites BamHI and EcoRI. The PCR product was first cloned in the pGEM T Easy vector (Promega) and sequenced. After digestion with the appropriate restriction enzymes and purification, the two Twi1 fragments were subcloned into the pHANNIBAL vector in both the sense and antisense orientations. Finally, the constructs made in pHANNIBAL were subcloned as a NotI flanked fragment into binary vector pART27 to produce highly effective intron-containing ‘‘hairpin’’ RNA silencing constructs. This vector carries the neomycin phosphotransferase gene (NPT II) as a transgenic selectable marker.
N. benthamiana agroinfiltration and tomato transformation
The pGWB8-Twi1 construction and the pGWB8 empty vector were transformed into the Agrobacterium tumefaciens C58 strain, while the pART27-Twi1 construction was transformed into A. tumefaciens LBA4404. The leaves of the 4-week-old N. benthamiana plants were infiltrated with the A. tumefaciens C58 carrying pGWB8-Twi1 or the empty vector, and with the C58 strain carrying the p19 plasmid (1:1), which encodes silencing suppressing protein p19 .
The transformed LBA4404 A. tumefaciens carrying pART27-Twi1 was co-cultured with the tomato Moneymaker cotyledons to generate the RNAi Twi1-silenced transgenic tomato plants (RNAi Twi1). The explant preparation, selection and regeneration methods followed those published by Ellul and co-workers . The tomato transformants were selected in kanamycin-containing medium and propagated in soil. The Moneymaker tomato wild-type plants regenerated in vitro from cotyledons under the same conditions as the transgenic lines were used as controls in subsequent analyses. The transgenic plants generated in this study have been identified and characterised in our laboratory and are to be used exclusively for research purposes.
Metabolite extraction procedure
Extraction of the methanol-soluble compounds from tomato leaves was performed according to López-Gresa and collaborators . Tissue (0.5 g fresh weight) was ground to powder in a mortar using liquid nitrogen, and then homogenized in 1.5 mL 100% methanol. The extracts were sonicated for 10 min and centrifuged for 15 min at 10000 x g to remove cellular debris. The supernatant corresponding to each sample was divided in two equal portions and dried at 40 ºC with a flow of nitrogen. One half of the dried residue was resuspended in 900 μL of 50 mM sodium acetate (pH 4.5) and 100 μL of water containing 10 U of almond beta-glycosidase (EC 184.108.40.206) (14.3 U/mg, Fluka) to analyse total (free + conjugated) forms. The other half was resuspended in 900 μL of 50 mM sodium acetate (pH 4.5) and 100 μL of water to analyze free form. The reactions were incubated overnight at 37 ºC and stopped by adding 75 μL of 70% perchloric acid to the incubation mixtures (5% (v/v) final concentration). After centrifugation at 14000 x g for 15 min to remove polymers, the supernatants were extracted with 2.5 mL of cyclopentane/ethyl acetate (1:1, v/v). The organic upper phase was collected and dried at 40 ºC under a flow of nitrogen. The residue was resuspended in 100 μL of methanol and filtered through 13-mm nylon 0.45 μm Minispike filters (Waters) prior to analysis.
The HPLC-fluorescence analysis was performed following Yalpani and coworkers , slightly modified by Campos et al . Compounds (SA, 2,4-dihydroxybenzoic acid (DHBA), scopoletin, esculetin, and umbelliferone) were detected by a 470 Waters fluorescence detector (λ excitation 313 nm; λ emission 405 nm), and quantified with the Waters Empower software using commercial compounds as a standard. Data were corrected for losses in the extraction procedure, and the recovery of metabolites ranged between 50% and 80%.
For the in vitro assay test, a 20 μL aliquot from the final 200 μL volume reaction was injected into HPLC-UV to detect SA, 2,4-DHBA, scopoletin, esculetin and umbelliferone. Commercial standards were used to quantify the results.
UPLC-PDA-Micromass Q-ToF analysis
A 5 μL aliquot from the final 100 μL sample extraction was analysed by UPLC-MS by an ACQUITY UPLC-PDA system coupled to a Q-ToF Micromass spectrometer (Waters) according to Campos and co-workers . All the data were acquired with the Masslynx NT4.1 software (Waters Corp. Mildford, MA, USA). For the untargeted analysis of the hydrolysed polar and semi-polar compounds, a metabolomic study of the total forms was performed using the negative ESI-MS spectra.
For the in vitro assay test, a 5 μL aliquot from the final 200 μL volume reaction was injected into UPLC-PDA-MS to detect and quantify with standards the phenylpropanoids cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, o-coumaric acid and chlorogenic acid, the flavonoids quercetin, kaempferol, naringenin and apigenin, and the simple phenolics benzoic acid, 4-hydroxybenzoic acid and 2,4,6-trihydroxybenzoic acid (THBA).
Twi1 recombinant protein puriﬁcation
Four grams of frozen N. benthamiana leaves infiltrated with recombinant A. tumefaciens C58, carrying either pGWB8-Twi1 or the pGWB8 empty vector (control), were ground to a ﬁne dust in liquid nitrogen and resuspended in 8 mL of extraction buffer (20 mM sodium phosphate, 0.5 M NaCl, 40 mM imidazole, pH 7.4, containing 80 μL of PMSF 1 mM and 16 μL 2-mercaptoethanol). The plant material was homogenised, filtered through Miracloth (Calbiochem), and tissue debris was removed by two successive centrifugations at 10,000 x g and 4 °C for 15 min. The sample was then filtered through 4-mm and 0.45-μm pore diameter nylon membranes (Waters). Protein samples were subjected to FPLC (Fast Protein Liquid Chromatography) with a nickel-loaded HisTrap HP 1 mL column (GE Healthcare Life Sciences). The column was washed with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 40 mM imidazole, pH 7.4), and the retained proteins were eluted with a 40-500 mM linear imidazole gradient in the same buffer at a ﬂow rate of 1 mL/min by measuring A280 at the column outlet and collecting 500 μL fractions by a RediFrac-1 automatic collector (Amersham Pharmacia Biotech).
Fractions were pooled in threes and analysed by SDS-PAGE following the method described by Conejero and Semancik . Proteins were stained with Coomassie Brilliant Blue R-250. The recombinant Twi1 protein appeared only in the leaf tissues infiltrated with the pGWB8-Twi1 construction. The fractions containing the recombinant Twi1 protein were desalted using a PD-10 column, eluted with Tris buffer (Tris-HCl 50 mM, pH 7.5), and assayed for activity in vitro. The equivalent fractions from the control sample were also tested.
In vitro assay of recombinant Twi1 activity
The reaction mixture used to perform the standard assay for glycosyltransferase activity contained: 200 μL of the purified protein in Tris buffer (Tris-HCl 50 mM, pH 7.5), 0.1 mM of the ﬁnal concentration of the sugar acceptor and 2 mM UDP-glucose (Fluka) or UDP-xylose (CarboSource Services, Complex Carbohydrate Research Centre, University of Georgia, USA) in a 206,5 microL final volume reaction. All the compounds used for Twi1 glycosyltransferase activity test were purchased from Sigma. Reactions were incubated overnight at 37 °C and stopped by adding one volume of 100% methanol. Then samples were filtered through a 4-mm nylon membrane (0.45 μm pore, Waters) and were analysed by high performance liquid chromatography (HPLC, Waters) according to the Yalpani  and Bellés  indications, or by a Q-ToF-MS analysis.
To detect the conjugated products of SA, GA, 2,4-DHBA, scopoletin, esculetin and umbelliferone, HPLC-fluorescence was employed as described before. The conjugated forms of the other simple phenolics and of the phenylpropanoids were detected by Q-ToF-PDA-MS, as reported above.
Bioinformatics and statistical analyses
The symptomatology of each plant, which was monitored at the indicated time points and scored according to symptom severity, was statistically analysed by a Kruskal-Wallis test. Different letters indicate significant differences (p-value < 0.05) between the RNAi Twi1 transgenic and wt infected plants.
The “Infectivity Index” consists of the total number of days that each plant presents symptoms . The data from a representative experiment of three independent assays were used to perform the statistical analysis by the Mann-Whitney nonparametric test. A p-value of < 0.05 was considered statistically significant. The IBM SPSS v.19 package was used for all the statistical analyses.
For the untargeted analysis of the hydrolysed polar and semi-polar profiles, the UPLC-MS data were processed with XCMS online resources (https://xcmsonline.scripps.edu) with the appropriate script for the alignment of chromatograms and the quantification of each MS feature . The resulting dataset was submitted to a Partial Least Square (PLS) study by the SIMCA-P software (v. 11.0, Umetrics, Umeå, Sweden) using unit variance (UV) scaling.
The phylogenetic tree analysis was generated with the MegAlign software bundled in the DNASTAR Lasergene package. The glycosyltransferases considered in the alignment were: IS5a (GB: AAB36653), Togt1 (GB: AAK28303), Togt2 (GB: AAK28304), IS10a (GB: AAB36652), SA-GTase (GB: AAF61647) and JIGT (GB: BAA19155) from Nicotiana tabacum; Scopoletin GT (NCBI: XP_015062099) from Solanum pennellii; Sgt1 (GB: AAB48444) and Scopoletin GT (NCBI: XP_006346388) from Solanum tuberosum; Scopoletin GT (NCBI: XP_016539537) from Capsicum annuum, UGT73B3 (NCBI: NP_567953) and UGT73B5 (NCBI: NP_179150) from Arabidopsis thaliana, as well as Twi1 (GB: CAA59450) and GAGT (GB: CAI62049) from Solanum lycopersicum.