(A) Schematic representation of two root epidermal cells (cell #1 indicated as C1 and cell #2 indicated as C2) imaged at two different focal planes (cortical plane, close to the plasma membrane and designated as Zi and a median plane, in the middle of the cell designated as Zii). Note that the C1/C2 and Zi/Zii notations are used consistently according throughout the figures. (B) Confocal images of the subcortical part (i.e. Zi) of the Arabidopsis root epidermis expressing mCIT-SAC9, mCIT-SAC9∆CC mutated in the predicted coil-coiled domain, and mCIT-SAC9C459A mutated in its putative catalytic cysteine, under the control of SAC9 native promoter (SAC9prom). (C) Representative images of the fluorescent signal observe in sac9-3-/- mutant expressing mCIT-SAC9, TdTOM-SAC9, and mCIT-SAC9C459A. (D) Comparison of the number of labeled intracellular compartments per cell in sac9-3-/- root epidermis expressing mCIT-SAC9 and TdTOM-SAC9, mCIT-SAC9C459A. (E) Colocalization analysis on Ub10pro:TdTOM-SAC9 (magenta) and SAC9pro:CIT-SAC9C459A (green). (F) Confocal images of the subcortical part of the Arabidopsis root epidermis (upper panels, Zi) or the center of the cell (lower panels, Zii) expressing mCIT-SAC9 together with the PI(4,5)P2 biosensor 2mCH-2xPHPLC (note that the cells shown in Zi and Zii are the same, just on different focal plane). (G), Quantification of the number of endosomes labeled by mCIT-SAC9 observed per cell at two different focal planes (Zi and Zii). (H), Quantification of the number of endosomes labeled by FM4-64 observed per cell at two different focal planes (Zi and Zii). (I), Co-visualization of SAC9pro:mCIT-SAC9C459A (green) together with the plasma membrane marker Lti6b-2xmCH (magenta) in epidermal root cells taken in the Zi focal plane. The inset shows a magnification, with the yellow arrows indicating some of the intracellular compartments decorated by mCIT-SAC9C459A observed at the close vicinity to the labeled plasma membrane in cell C1. Scale bar: 10 µm in A-F; 5 µm in G (left panel) and 2 µm in the close-up of G (right panel). In the plot, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. Details of the statistical analysis could be found in Supplementary file 1D. The plane (Zi or Zii) in each image is mentioned, and the image display is representative for the plane used for the analysis. N=number of replicates; n=number of roots.