Videoconfocal vico system

For apoptosis detection, the Terminal transferase-mediated dUTP-biotin Nick End-Labeling (TUNEL) assay was conducted, using the DeadEnd kit (Promega, Madison, WI, USA). Nuclei were stained with Hoechst 33258. Cells undergoing apoptotic cell death were imaged using an Eclipse 80i Nikon Fluorescence Microscope equipped with a VideoConfocal (ViCo) system (Nikon Instruments, Amsterdam, The Netherlands). Apoptosis was expressed as percentage of TUNEL-positive nuclei cells over total cells (at least 500 cells for each coverslip).

GFP-expressing cells were observed using an Eclipse 80i Fluorescence Microscope equipped with a VideoConfocal (ViCo) system (Nikon Corporation, Tokyo, Japan). For this purpose, five-day-old cell cultures of X. fastidiosa mutant strain DDgfp were scraped from BCYE agar plates and resuspended in sterile phosphate-buffered saline (PBS) solution. Fluorescent images were captured through a bandpass ET/GFP cube filter (Nikon Corporation, Japan) (excitation at 440–470 nm, emission at 525–550 nm) and analyzed with Image-Pro Plus 6.0 software (Media 11 Cybernetics, Rockville, MD, USA). Adobe Photoshop CS6 software (Adobe Systems, Inc., San Jose, CA, USA) was used to assemble and label the final figures.