Electrostatic interactions between NLD and negatively charged membranes are necessary for correct targeting. (A–H) Pharmacological approach showing the importance of membrane electronegativity in targeting NLD to PM of root cells. Confocal imaging of root cells from lines expressing NLD_full:mCitrine (A and B), the PI4P biosensor mCitrine:1XPH^FAPPI (C and D), the biosensor of surface membrane charge mCitrine:FARN⁶⁺ (E and F) or transmembrane GFP:LTI6b (G and H) after 30-min treatment with 60 µM PAO, an inhibitor of PI4Ks, or mock treatment (DMSO; A, C, E, and G). (I and J) Quantification of drug treatment effect in root cells on NLD subcellular localization (I) and GFP:LTI6b (J) using the ratio of the fluorescence intensity at PM compared with the fluorescence intensity in cytosol (PM/cyto fluorescence ratio; mean ± SD). (K–N) Targeted mutagenesis of the nine positively charged amino acids located in the NLD C-terminus impaired NLD subcellular localization. Confocal imaging of root cells and mature pollen from lines NLD_9R→9K:Citrine (9 R residues mutated to K residues; K and L), or NLD_9R→9Q:Citrine (9 R residues mutated to Q residues; M and N). (O and P) Quantification of mutagenesis effect on localization using the ratio of the fluorescence intensity at PM compared with the fluorescence intensity in cytosol in root cells (PM/cyto fluorescence ratio; O) and in pollen grain (endo-PM/cyto fluorescence ratio; mean ± SD; P). ***, significant difference as compared with the mock treatment (I) or as compared with NLD_full:mCitrine (O and P; P < 0.001; Wilcoxon test, n = cell number analyzed). Scale bars = 20 µm.