DOI: 10.5061/dryad.pc866t1nr
title: Bacterial QS expands the Xanthomonas campestris pv. campestris invasion of host tissue to trigger host-chlorophagy and maximize disease symptom

description: 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.
description: <b>Microscopy to observe the inoculated cabbage
 leaves</b> 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
 1cm<sup>2</sup> 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<i> </i>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
 <i>Z</i>-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. <b>Image
 Analysis and Statistical validation</b> 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 <i>Xcc</i> 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.