Time-lapse Fluorescence Microscopy of Xanthomonas citri

Time-lapse fluorescence microscopy assays were carried out as previously described (Oka et al, 2022 (link)). Briefly, after the cultivation of the X. citri inocula in 2xTY supplemented with the appropriate antibiotics, cells were harvested by centrifugation (5000 rpm, 5 min), washed in water twice and the OD_600nm was normalized to 0.5. Then, 1 µL of each X. citri strain were pipetted onto a thin LB-agarose support supplemented with propidium iodide (1 µg/mL), appropriate antibiotic and observed with a Nikon Eclipse Ti microscope equipped with filters for GFP (GFP-3035B-000-ZERO, Semrock) and propidium iodide (TxRed-4040B, Semrock) and a Nikon Plan APO 100x objective. Images were collected every 10 min. Image processing and quantitative analysis of the number of cells having a damaged cell envelope and cell counting related to Movies EV1–5 were performed manually using Fiji software (Schindelin et al, 2012 (link)) multipoint tool. Time-lapse microscopy was also performed for bacterial competition experiments between X. citri wild-type transformed with pBBR-RFP and X. citri Δ8Δ2609-GFP transformed with pBBR-GFP (Movie EV6). Cells were grown and washed as above and the cultures were mixed at a 1:1 ratio and microscopy was performed as described above, in the absence of propidium iodide.