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.
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