Extended Data Fig. 3: Successful inducible expression and subcellular targeting of MAP-mCH-dOCRL and negative controls.

a, Inducible system used in this study. We performed site-directed D468G mutation in dOCRL catalytic domain, similarly to D523G mutation already described in human OCRL1 that abolishes its phosphatase activity. We could therefore use MAP-mCH-dOCRL_168-509^D468G (hereafter named MAP-mCH-dOCRL^dead) as negative control, together with a MAP-3xmCH recombinant protein. UBQ10 promoter is responsible for mild/strong and ubiquitous expression of GVG (GAL4 VP16 GR) synthetic gene. GR domain binds dexamethasone (dex) and subsequently induces GVG homodimerization and nuclear import. There, GAL4 domain binds UAS DNA elements and VP16 strongly activates the expression of downstream gene: MAP-mCH-dOCRL_168-509, or MAP-mCH-dOCRL_168-509^D468G (MAP-mCH-dOCRL^dead), or MAP-3xmCH. MAP is myristoylation and palmytoylation sequence, responsible for plasma membrane targeting. The mCH corresponds to monomeric CHERRY fluorescent protein. dOCRL^D468G_168-509 is an inactive phosphatase domain (later on called dOCRL^dead). 3xmCH correspond to three fused mCHERRY. b, We addressed the timing of expression and the localization of each construct in Arabidopsis lines stably transformed with UBQ10pro:GVG:MAP-mCH-dOCRL, or UBQ10pro:GVG:MAP-mCH-dOCRL^dead, or UBQ10pro:GVG:MAP-3xmCH. In root meristematic epidermal cells, without dex treatment, none of the recombinant-proteins were detected using confocal microscopy. A 16 h treatment with 0.5 µM dex led to the detection of mCH fluorescence, indicating that induction of the genetic construct had occurred. However, we observed a mosaic induction and a significant number of roots had no cells expressing the fluorescent reporter to detectable levels. To overcome these issues, we optimized the treatment to a 16 h induction with 5 µM dex. Using this set up, we robustly observed red fluorescence using confocal microscopy where MAP-mCH-dOCRL, MAP-mCH-dOCRL^dead and MAP-3xmCH. Each of the synthetic protein was efficiently targeted to membranes, including the plasma membrane, where PI(4,5)P₂ accumulates, and intracellular compartments. Therefore, a 16 h 5 µM dex treatment is sufficient for an effective expression of dOCRL in Arabidopsis stable transgenic lines. For all three inducible constructs we monitored mCH fluorescence without dex treatment or after a 16h-treatment with either 0.5 µM or 5 µM dex. Signal intensity is color-coded (green fire blue scale); 2 Replicates. Scale bars, 20 µm.