B5 vitamins

Seeds of tomato (Lycopersicum esculentum cv. Moneymaker) were surface sterilized by immersion in 70% (v/v) ethanol for 5 min followed by incubation in 2.8% NaClO for 10 min and subsequently washed with sterile distilled water for 30 min. The treated seeds were placed onto Murashige & Skoog (MS) agar medium including B5 vitamins (Duchefa Biochemie B.V, Haarlem, Netherlands) and germinated in the dark at 22 °C. After seven days seedlings were exposed to light and after 10 days uniform seedlings were selected and inoculated with bacteria. The seedlings were dipped into bacterial suspensions of either live or heat killed bacteria and were subsequently transferred to a 300 ml pot containing 60 g of hot vapor treated garden soil (150–600 mg l^–1 N, 150–600 mg l^–1 P₂O₅, 200–900 mg l^–1 K₂O, 150 mg l^–1 Mg, pH = 5–6.5) (Franz Kranzinger GmbH, Straßwalchen, Austria). All pots were randomly placed in a growth chamber for 60 days at 22 °C, a photoperiod of 16 h (100–130 PPFD, μmol m^–2 s^–1 at the ground level), and relative humidity of 65%. After 25 and 45 days one third of all pots was fertilized with 20 mL 0.1% WUXAL Super (Hauert Manna Düngerwerke GmbH, Austria) consisting of 8% N (2.3% NO₃^–, 3.7% NH₄⁺, 2% Urea), 8% P₂O₅, 6% K₂O and trace elements in distilled water (conductivity 0.51 mS).

The grape (Vitis vinifera) cell suspension originated from disk (1-2 mm) of berries at veraison (1 cm) of the variety Gamay Red, as described in Gollop et al. (2002 (link)). Liquid wt cultures were grown on Gamborg salts (pH 5.9) with B5 vitamins (Duchefa, The Netherlands), supplemented with 250 mg/l casein hydrosylaze, 100 mg/L myo-inosytol, 0.2 mg l-1 kinetin and 0.1 mg l-1 NAA, 20 g l-1 sucrose and 0.8% (w/v) agar (Gollop et al., 2002 (link)). Cells were cultured in liquid medium with the same nutrient and hormone composition and maintained at 25 ± 1°C with constant shaking at ±100 rpm under constant light conditions (25 μmol m⁻²s⁻¹). A 50-ml stock of suspended cells was maintained by sub-culturing 2.5 gr of cell to fresh medium once a week.

N. benthamiana seeds were sterilised for 5 minutes in 70% ethanol, followed by 5 minutes in 2% sodium hypochlorite solution, and washed 5 times with sterile water. The seeds were germinated on half-strength Murashige & Skoog medium including B5 vitamins (Duchefa Biochemie, Haarlem, the Netherlands) with 1% bactoagar, pH 5.7, and allowed to grow for 17 days at 25°C under a 16-hour photoperiod. Roots of 17-day-old seedlings were inoculated with 10 μL zoospore solution from P. palmivora LILI [63 (link)], which had been transformed anew with a pTOR-tdTomato fluorescent reporter provided by Dr Stephen Whisson (The James Hutton Institute, Dundee, UK) following the protocol described by Evangelisti and colleagues [64 (link)]. Zoospores were harvested as previously described [65 (link)] and diluted to the concentration of 20,000 zoospores/mL (200 zoospores/seedling). Control (mock) plants were inoculated with 10 μL of sterile water. Inoculated seedlings were grown at 25°C under a 24 hours photoperiod for 9 days. Inoculated seedlings were imaged using Leica M165FC microscope for visualising the extent of infection by P. palmivora and an Epson Perfection Flatbed Scanner (Epson UK, Hemel Hempstead, UK) using default settings and a resolution of 600 dots per inch for full-colour images to record betalain pigmentation.