Using QS-responsive whole-cell bioreporters of Xcc and cabbage (Brassica oleracea) as a model system and confocal microscopy, we show a detailed chronology of QS-facilitated Xcc colonization in the host mesophyll region. We report the QS-enabled bacterial localization of parenchymal chloroplast within heterogeneously invaded host mesophyll tissue, leading triggered leaf-chlorosis and systemic infection.

Microscopy to observe the inoculated cabbage leaves

A confocal laser-scanning microscope (CLSM) (LSM700; Carl Zeiss, Germany) was used to visualize the bio-reporter cells and the chloroplast autofluorescence within inoculated cabbage leaves upto dpi (days post inoculation) 12. On each sampling dpi, the inoculated leaves were detached from the plant immediately prior to sectioning and sample preparation. For each inoculated leaf, after excising the diseased part, if present on specific day, multiple thin transverse sections were cut using a sterile razor blade with each sections approximately 100 to 150 µm thickness within 1cm² proximal green regions excluding the mid-rib from the clipped end. The leaf slices were mounted on a glass slide (IS-3099; Rohem Industries pvt. Ltd, India) in transverse orientation sequentially with a coverslip and scanned under a CLSM (with 100x / 1.4 oil DIC M27 objective) for green and red fluorescence. Dual colour images were acquired by sequentially scanning the multiple leaf sections with settings optimal for GFP (excitation: 488 nm and emissions: 505 to 550 nm band pass, with 518 nm emission maximum), and RFP/chlorophyll autofluorescence (excitation: 555 nm and emissions: 582 to 800 nm band pass, with 585 nm emission maximum). Cross talk between the individual channels in this setup was always monitored and was negligible in all cases. A bright field image was also acquired for each section using a photo-multiplier detector measuring the transmitted light. Microscope power settings were adjusted to optimize contrast for each individual image. Confocal images for GFP (green), RFP (red) and Differential Interference Contrast (DIC) were constructed simultaneously using a multitrack mode via Pigtail-coupled solid-state lasers. Multiple Z-section scans were acquired at 0.5 µm increments in each field (Samal and Chatterjee, 2019). At least five inoculated leaves per plant were examined to visualize bacterial fluorescence in case of each bioreporter strain and negative control plants (i.e. 1X PBS inoculated), with the experimental repeat for thrice independently.

Image Analysis and Statistical validation

Bacterial count for intra- and the inter-cellular host-mesophyll localization was performed by analyzing each Z-plane at a time in the CLSM Z-stack image using ZEN lite 2012 (Carl Zeiss) imaging software; where all constitutive-GFP expressing bioreporter cells were counted manually after minimizing the background host-autofluorescence under the green channel, that accounts for all the dual-reporter cells. Simultaneously, within each CLSM raw image, approximately 400 to 600 no. of Xcc derived dual bio-reporter cells per sample was analyzed for both GFP and RFP fluorescence patterns (approximately 70 to 100 cells per field were observed for 5 different fields) to calculate the percentage of bacterial QS induction (Samal and Chatterjee, 2019). Outlines of the individual bacterial cells as well as host parenchymal cell-walls were further confirmed under the DIC channel for each Z-plane.

The individual CLSM image panels were prepared using FIJI (Image J) software after analyzing their raw images using ZEN lite 2012 (Carl Zeiss) imaging software, and imported into Photoshop CS5 (Adobe) software for brightness and contrast adjustments and assembly of the composite figure for publication. Statistical comparisons towards significant difference levels were computed using the Student’s test (non-parametric, paired, two-tailed test), where a “p value” of less than 0.05 was considered significant.