Fig 7. Reduced cell-aggregation in QS mutants cannot be rescued by QS responders of Xcc in planta.

(A) Representative dpi 12 CLSM images for cell aggregate formation patterns of QS responder (i.e. Xcc 8004/gfp), QS null (i.e. Xcc ΔrpfF/mCherry) and QS blind (i.e. Xcc ΔrpfC/mCherry) cells for single as well as mixed infections as specified combinations (panels from left to right) in the in planta competition assay within proximal vascular regions of clip inoculated cabbage leaves under a CLSM upto dpi 12. The panels from top to bottom show gfp, mCherry, gfp-mCherry merged, DIC and gfp-mCherry-DIC merged images for both single and mixed infections respectively. Images were prepared using FIJI (image J) software. Scale bars on each panel, 10 μm. (B) Average no. of bacterial aggregates per 1 cm² of total leaf regions, and (C) Spatio-temporal distribution of no. of bacterial aggregates per 1 cm² proximal, middle and distal leaf regions respectively, for single and mixed infections on dpi 1, 6 and 12. WT; wild-type Xcc 8004, ΔF; Xcc ΔrpfF, and ΔC; Xcc ΔrpfC. On specified sampling dpi, multiple Z-stalks were acquired under a CLSM for each sample under green and red fluorescence along with DIC channel, maintaining 0.5 μm gap between two successive Z-planes. Bacterial aggregate size as well as no. were analysed by considering the X,Y and Z planes for each aggregate of a Z-stalk, where the bacterial aggregates present in all the Z-planes were counted manually and summed up to calculate their total no. in that region at a time. Total bacterial aggregate no. observed was normalized and the values are expressed per cm² leaf region. The no. of bacterial aggregates for each infection was determined by combining the analysed data for five sites per inoculated leaf, six leaves on each sampling day with experimental repeats for thrice. The characteristics of the total region of the leaf observed at each sampling time were slightly different. Data analysis [using FIJI (image J) software] was performed by taking six different confocal images as samples for each strain at a time with the experimental repeat of at least thrice and represented with Mean ± SD. P-values for significant difference level were determined by performing student’s T-test (two tailed, paired). ***; p < 0.001.