Fluoview 1000 spectral based laser scanning confocal microscope

Leaf discs (~3 mm × 3 mm) were collected one hour after lights turned on in the growth chamber and were submerged in MES buffer (25mM MES-KOH pH6.15, 10mM KCl) with 100 μM SA (Sigma-Aldrich), 10 μM ABA (Sigma-Aldrich) or 0.1% DMSO (mock) for one hour. For measuring pathogen-induced stomatal closure, leaf discs were submerged in water or bacterial suspension (1x10⁸ cfu ml⁻¹Pst DC3118 in water) for two hours. Stomatal apertures were captured using Olympus FluoView 1000 Spectral-based Laser Scanning Confocal Microscope with excitation/emission at 405/460 nm. The length and width of the pore aperture were measured using ImageJ (https://imagej.nih.gov/ij/). Stomata from four different plants per genotype (1-2 leaf discs per plant, ~8 stomata per leaf disc) were imaged. At least 30 stomata per genotype were measured for each treatment and the ratio of width/length was graphed to represent the stomatal aperture status.

Doxicycline-inducible overexpression HEK293T cells were generated using PiggyBac transposition. The N-terminal (1-252 a.a.) middle (253 - 509 a.a.) and C-terminal (510 - 747 a.a.) parts of MPO were cloned into KA0717 vector via MluI/SpeI restriction sites. C-terminal part of MPO was further fragmented into 68 a.a, 67 a.a, 44 a.a, 58 a.a. long peptides. Following validation by Sanger sequencing, HEK293T cells were co-transfected together with transactivator and transposase-encoding vectors (KA0637 and SBI Biosciences #PB200PA-1, respectively) at a DNA mass ratio of 10:1:3 using Lipofectamine LTX (ThermoFisher) as per manufacturer’s instructions. Stable, transgene-positive HEK239T cells were selected using 250 μg/ml G418 (Sigma Aldrich). For imaging, transfected cells were grown on coverslips and overexpression was induced by 16h treatment with Doxycycline. After induction, the cells were fixed in 4% PFA/PBS for 15 min at room temperature, washed with PBS and permeabilized with 0.5% Triton-X/PBS for 5 min at room temperature. The coverslips were mounted in slides with ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher). Images were acquired on a Leica DMI8 Inverted Fluorescence Phase Contrast Microscope and on an Olympus Fluoview 1000 Spectral-based Laser Scanning Confocal Microscope.