Axio imager upright fluorescence microscope

Proximity ligation assay (PLA) was performed using Duolink® In Situ starter kit Mouse/Rabbit (Sigma-Aldrich, DUO92101) following manufacturer’s instructions, to assess co-localisation between HA-FGFR2b and pULK1_S638. Briefly, T47D cells were transfected with FGFR2b_HA and 10,000 cells were seeded on the IBIDI slide. Cells were serum-starved for 24 h and placed on ice for 30 min. anti-HA was incubated for 40 min on ice, removed and replaced with serum-free media containing 100 ng/mL FGF10 for 40 min at 37 C. Cells were then fixed with 4% formaldehyde and permeabilised in 0.02% saponin. pULK1 S638 antibody was incubated in Duolink® antibody diluent for 1 h at RT, washed and incubated with Duolink® PLUS/MINUS probes for 1 h at 37 °C. Cells were then washed and incubated with Duolink® ligase solution for 30 min at 37 °C, washed and incubated with polymerase for 100 min at 37 °C. Cells were washed a final time, then mounted using DuoLink® In Situ mounting media containing dapi. Cells were imaged using a Zeiss Axio Imager upright fluorescence microscope and images analysed using ImageJ.

Imaging was performed on an Axio Imager upright fluorescence microscope (Zeiss) and Metafer VSlide Scanner (Metasystems) using Zeiss Axio Imager Z2. All measurements and analyses were performed using Imaris 7.2.3 software (Bitplane).

At 6–8 dpi, fourth or fifth leaf samples were collected and submerged in 1M KOH and incubated for 48 h at 37°C with gentle agitation. The KOH solution was replaced with fresh 1M KOH solution after 18–24 h. The KOH solution was discarded, and the leaf material was washed gently 2–3 times with 50 mm Tris‐HCl, allowing material to incubate in the Tris‐HCl solution for 10–20 min per wash. 1–2 mL 50 mm Tris‐HCl and 10–20 μL 1 mg/mL wheat germ agglutinin conjugated to fluorescein isothiocyanate (WGA‐FITC, Sigma‐Aldrich, Castle Hill, NSW, Australia) were added, and samples incubated at ambient temperature for an hour or kept at 4°C before mounting on microscope slides. Stained leaf samples were mounted on slides using a few drops of 40% glycerol before covering with cover slips. GFP3 fluorescence filters were used on a Leica MZFLIII fluorescence dissecting microscope or a Zeiss Axioimager upright fluorescence microscope to score the presence of P. purpurea infection sites in the sampled leaves.