Confocal Laser Scanning Microscopy

Imaging was performed using TSC-SP5 and TSC-SP8 laser scanning confocal microscopes (Leica, Bensheim, Germany) in 8-bit formats. For image generation, the HCX PL APO CS 40.0 × 0.70 dry and HC PL APO CS2 20.0 × 0.75 oil-immersion objective, sequential and bidirectional scan with a speed of 200 Hz were used. Images were acquired with 2×, 3×, and 2× line averages for the VENUS, EGFP, and mKATE2 channels, respectively. For fluorescence quantification purposes, the HCX PL APO CS 20.0 × 0.70 dry objective with 2× zoom factor, sequential and bidirectional scan with a speed of 400 Hz, a single line averaging, and a resolution of 512 × 512 pixels were used. Fluorescent Brightener 28 was excited using 405 nm UV diode laser. EGFP and VENUS were excited with the 488 and 514 nm lines of an argon ion laser, respectively, whereas mKATE2 was excited with a 594 nm HeNe laser. The emission spectra were set to 420–460 nm for Fluorescent Brightener 28, 492–510 nm for EGFP, 528–555 nm for VENUS, and 610–640 nm for mKATE2. Emitted fluorescence was configured to pass through pinhole aperture of 1 airy unit and detected with HyD detectors.

The 3rd leaf from hormone-treated seedlings or the 5th leaf from Hpa-inoculated plants was selected for microscopy. For the Hpa-inoculated samples, the adaxial surface was emerged in a drop of 10 μg/ml Fluorescent Brightener 28 (Sigma-Aldrich, Taufkirchen, Germany) for 30 s to stain the Hpa spores. The leaf midrib was excised, the leaf placed on 3 well-containing slide (Thermo Fisher Scientific, Braunschweig, Germany) and mounted in the Fluorescent Brightener 28. The EGFP and Fluorescent Brightener 28 channels were used to define individual sites of Hpa attack. The mKATE2 channel was used to assign the z-stack start and end planes for optimal coverage of the nuclei-localized fluorescence signals. The VENUS signal in the treated samples was used to set the threshold of fluorescence detection in comparison to mock. Saturation of fluorescence signals was avoided. The same imaging configurations were applied for mock and treatment. The maximum intensity projections, contrast and image merging were performed using Fjji (ImageJ v1.51).