REM1.3's phospho-status defines its plasma membrane nanodomain organization and activity in restricting PVX cell-to-cell movement
Fig 3
Mutational analysis reveals three critical phospho-residues required for REM1.3 regulation of PVX-GFP propagation and PD conductance.
(A) In silico analysis of REM1.3 protein sequence. Prediction of putative phosphorylation sites was performed by Diphos, DEPP and NETPHOS coupled with published MS data. Predictions highlight three residues S74, T86 and S91 with high probability to be phosphorylated. Disordered prediction was performed by pDONR VL XT. Numbers indicate amino acid position. (B) In vitro kinase assay on recombinant affinity purified 6His-REM1.3 or 6His-REM1.3DDD by incubation with [γ-33P]-ATP and microsomal extracts of PVX-infected N. benthamiana leaves, as described in Fig 2. Phosphorylated proteins were detected by autoradiography and total proteins by silver staining. Asterisk * indicates phosphorylation of a N. benthamiana protein of close molecular weight not detected by silver staining. (C) Graph represents the relative quantifications from 4 independent reactions, using WT signal as a reference. (D) Left, Representative epifluorescence microscopy images of PVX-GFP infection foci on N. benthamiana leaf epidermal cells at 5 DAI. Graph represents the mean relative PVX-GFP foci area in cells transiently expressing RFP alone, wild-type RFP-REM1.3 or carrying single serine /threonine mutations to alanine. Co-infiltration of PVX-GFP with an empty A. tumefaciens strain served as mock control. Approximately 160 foci per condition from 3 independent biological repeats were measured. Letters indicate significant differences revealed by Dunn’s multiple comparisons test p<0.001. Right, Graph represents the mean relative PVX-GFP foci area in cells transiently expressing wild-type RFP-REM1.3 or triple RFP-REM1.3 phosphodead and phosphomimetic mutants compared to mock control (co-infiltration of PVX-GFP with an empty A. tumefaciens strain). Approximately 250 foci per condition from 5 independent biological repeats were measured Letters indicate significant differences revealed by Dunn’s multiple comparisons test p<0.001. Epifluorescence microscopy images show representative PVX-GFP infection foci on N. benthamiana leaf epidermal cells at 5 DAI. (E) GFP diffusion to neighbor cells was estimated by epifluorescence microscopy at 5 DAI in N. benthamiana cells transiently expressing RFP-REM1.3 or phosphomutants. Measurements from 3 independent biological repeats were normalized to mock control (co-infiltration with an empty A. tumefaciens strain). Letters indicate significant differences determined by Dunn’s multiple comparisons test p<0.001.