Epidermal cells live imaging and quantification

Live imaging was performed using a Leica SP5 confocal laser scanning microscopy system (Leica, Wetzlar, Germany) equipped with Argon, DPSS and He-Ne lasers and hybrid detectors. N. benthamiana leaf samples were gently transferred between a glass slide and a cover slip in a drop of water. YFP and mCitrine (cYFP) fluorescence were observed with similar settings (i.e. excitation wavelengths of 488 nm and emission wavelengths of 490 to 550 nm). In order to obtain quantitative data, experiments were performed using strictly identical confocal acquisition parameters (e.g. laser power, gain, zoom factor, resolution, and emission wavelengths reception), with detector settings optimized for low background and no pixel saturation. Pseudo-colored images were obtained using the ‘Red hot’ look-up-table (LUT) of Fiji software (http://www.fiji.sc/). All quantifications were performed on raw images for at least min of 10 cells, at least two plants by condition with at least three independent replicates.

For quantification of the PM Spatial Clustering Index (SCI), which reveals the degree of segregation of fluorescence signal on the surface plane of the PM (Figure 1—figure supplement 4), fluorescence intensity was plotted with a 10 µm line on raw images of cells PM surface view, three line plots were randomly recorded per cell and at least 15 cells per experiments were analyzed. For each plot, the Spatial Clustering Index was calculated by dividing the mean of the 5% highest values by the mean of 5% lowest values. For fluorescence intensities quantification, the mean grey value was recorded using a region of interest (ROI) of 5 µm x 5 µm on PM surface view raw images.