Fig 1.
REM1.3 modulates plasmodesmata callose accumulation and displays altered PM organization and dynamic following PVX infection.
(A) Representative confocal images of aniline blue stained N. benthamiana leaf epidermal cells transiently expressing YFP-REM1.3 in the absence (mock is infiltration with empty A. tumefaciens) or the presence of PVX at 2 days after infiltration (DAI). Color-coding indicates fluorescence intensity. (B) Left, Pit field aniline blue fluorescence intensity was quantified by ImageJ as described in S1 Fig and expressed as the percentage of the mock control. Right, Quantification of the PD residency of YFP-REM1.3 in the absence (mock) and in the presence of PVX using the PD index [28] as described in S1 Fig. Graphs represent quantifications from 3 independent biological experiments. At least 15 cells per condition were analysed per experiment. Significant differences were determined by Mann-Whitney comparisons test *** p<0.001. (C) Super-resolved trajectories of EOS-REM1.3 molecules (illustrated by different colours) in the PM plane in the absence (Mock) and presence of PVX obtained by high-resolution microscopy spt-PALM. EOS-REM1.3 was transiently expressed in N. benthamiana (D) Diffusion coefficients (D) of EOS-REM1.3 expressed as log(D) in the absence (Mock) and presence of PVX. Statistical significances were assessed by Mann-Whitney test *** p<0.001 using data collected over two independents experiments. (E) Mean Square Displacement (MSD) over time for the global trajectories of EOS-REM1.3 followed during at least 600 ms reflecting two independent experiments. (F) Live PALM analysis of EOS-REM1.3 localization in the absence (mock) and presence of PVX by tessellation-based automatic segmentation of super-resolution images. (G) Computation of EOS-REM1.3 single molecule organization features based on tessellation-based automatic segmentation images. For REM1.3 nanodomain size distribution for the indicated conditions, the Gaussian fits in absence (mock) and presence of PVX are indicated by lines. Total nanodomain area is expressed as percentage of the total PM surface. Percentage of EOS-REM1.3 molecules localizing into nanodomains, relative to all molecules observed. Localization density refers to the number of molecules observed per μm2 per second. Statistics were performed on at least 10 data sets per condition, from two independent experiments. Significant differences were determined by Mann-Whitney test * p<0.05, *** p<0.001.
Fig 2.
PVX and viral proteins induce REM1.3 phosphorylation in its N-terminal domain.
(A, B) In vitro protein phosphorylation assays were performed by incubation of recombinant affinity-purified 6His-REM1.3 and N. benthamiana extracts with [γ-33P]-ATP. The samples were run on SDS-PAGE gels and developed by autoradiography. Soluble (Sol) or microsomal (μ) extracts of healthy leaves in (A), or microsomal and PM extracts from healthy and PVX-infected plants in (B) were used. (C) In vitro phosphorylation of 6His-REM1.3N by leaf microsomal extracts of healthy or PVX-infected N. benthamiana leaves. Bars show the quantification of phosphorylated 6His-REM1.3N bands from 5 independent repeats. (D) In vitro phosphorylation of 6His-REM1.3 by leaf microsomal extracts in the presence of total RNA extracts from PVX-infected leaves. (E) Experimental flow-chart to study the role virus protein in membrane-bound kinase activation. (F) 6His-REM1.3N phosphorylation by microsomal extracts infected with PVX-GFP or expressing the indicated viral proteins at 4 DAI. Leaves expressing GFP alone, infiltrated with water or with A. tumefaciens strain GV3101 alone served as controls. Expression of the viral proteins is presented in S3 Fig. (G) Graph represents the quantification of 6His-REM1.3N bands from three independent repeats (n = 3), as a percentage of the activity induced by A. tumefaciens strain GV3101 alone. Error bars show SE, and significance is assessed by Dunnett’s multiple comparison test to water control (*, P < 0.1; **, P < 0.05; ***, P < 0.001). Phosphorylated proteins were detected by autoradiography and total proteins by stain free procedure. In all experiments 10μg of total protein extracts and 1μg of affinity purified 6His-REM1.3 or 6His-REM1.3N were loaded per lane.
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.
Fig 4.
REM1.3's dynamic localization in PD and PM nanodomains is regulated by its phospho-status.
(A) Representative confocal mages showing aniline blue staining of callose deposition at the PD pitfields in N. benthamiana leaf epidermal cells transiently expressing YFP-REM1.3 or phosphomutants. Color-coding indicates fluorescence intensity. (B) Graphs show aniline blue fluorescence intensities in cells transiently expressing YFP-REM1.3 and phosphomutants relative to control cells expressing YFP alone. Three independent biological experiments were performed and at least 15 cells per condition and per experiment were analyzed. Letter indicate significant differences revealed by Dunn’s multiple comparisons test p<0.001. (C) PD index of YFP-REM1.3 phosphomutants was calculated as described in S1 Fig. Graphs present quantifications from 3 independent biological experiments. Letter indicate significant differences revealed by Dunn’s multiple comparisons test p<0.002. (D) Super-resolved trajectories (illustrated by different colours) of transiently expressed EOS-REM1.3, and phosphomutants, transiently expressed in N. benthamiana cells, observed by spt-PALM. Scale bars, 2 μm. (E) Distribution of diffusion coefficients (D) represented as log(D) of the different fusion proteins. Mean Square Displacement (MSD) over time for the global trajectories of each EOS-REM1.3 construct followed during at least 600ms. 27 cells for EOS-REM1.3, 15 cells for EOS-REM1.3AAA and 17 cells for EOS-REM1.3DDD were analyzed in 3 independent experiments. Statistical analysis was performed by Mann-Whitney test * p<0.05 ** p<0.01. (F) Live PALM analysis of EOS-REM1.3 phosphomutants by tessellation-based automatic segmentation of super-resolution images. (G) Computation of EOS-REM1.3 and phosphomutants single molecule organization features based on tessellation-based automatic segmentation images. For REM1.3 and phosphomutants nanodomain size distribution and the Gaussian fits are indicated. Total nanodomain area is expressed as percentage of the total PM surface. Percentage of EOS-REM1.3 molecules localizing into nanodomains, relative to all molecules observed. Localization density refers to the number of molecules observed per μm2 per second. Statistics were performed on at least 13 data sets per condition extracted from 3 independent experiments. Statistical differences determined by Mann-Whitney test * p<0.05, ** p<0.01.
Fig 5.
AtCPK3 phosphorylates REM1.3 in a calcium-dependent manner.
(A, B) In vitro phosphorylation of purified 6His:REM1.3N by kinase(s) from different cellular fractions of N. benthamiana leaves, CEs, leaf crude extracts; Sol, Soluble fraction; μ, microsomal fraction; PM, Plasma Membrane; C-PM: “Control-PM” is PM fraction not treated by TX100, but submitted to sucrose gradient; DRM, Detergent resistant membranes [62]. The graph represents the relative quantification of 3 independent experiments normalized to the activity in the PM fraction +/- SEM. (C) Quantification of the calcium dose response of kinase activity on 6His-REM1.3N phosphorylation by N. benthamiana microsomal extracts from healthy and PVX infected leaves. (D, E, F) Autoradiography gels show in vitro phosphorylation of 6His-REM1.3, 6His-REM1.3N and 6His-REM1.3C, 6His:REM1.3DDD or 6His:AtREM1.2 by affinity purified GST-AtCPK3 in the presence or the absence of Ca2+. Bands corresponding to autophosphorylation of AtCPK3-GST and transphosphorylation of 6His-tagged group 1 REM variants are indicated. Gels were stained by coomassie blue to visualize protein loading. Asterisk* indicates a non-specific band present in both 6His-REM1.3C and 6His-REM1.3N preparation.
Fig 6.
AtCPK3 physically interacts in vivo with group 1b REMs and impairs PVX cell-to-cell movement in a REM-dependent manner.
(A) Primary sequence of AtCPK3. EF-hands are helix E-loop-helix F structural domains able to bind calcium. Ai: Autoinhibitory domain. The position of the DLK motif (Aspartic acid-Leucine-Lysine) at the catalytic domain conserved in all CPKs is indicated. (B) Confocal images showing AtCPK3-YFP and AtCPK3CA-YFP localization in N. benthamiana epidermal cells. Scale bar shows 10 μm. Western blot against GFP showing AtCPK3-YFP and AtCPK3CA-YFP expression in the microsomal fraction (μ) of N. benthamiana leaves. (C) In planta Bimolecular Fluorescence Complementation (BiFC) analysis showing interaction of AtCPK3 with Group 1 REMs. REM1.3-YFPN/REM1.3-YFPC was used as a positive control, and AtCPK3CA-nYFP/ AtCPK3CA-cYFP as a negative control. Mean fluorescence intensity at the cell boundary level was recorded using ImageJ. Statistical differences were determined by Mann-Whitney test compared to AtCPK3CA +AtCPK3CA.*** p = 0.0002, **** p <0.0001. All scale bars indicate 20μm. (D) PVX-GFP spreading in N. benthamiana cells expressing RFP-REM1.3 or AtCPK3FL-RFP, AtCPK3CA-RFP, AtCPK3CAD202A-RFP Graph represents the area of PVX-GFP infection foci relative to the mock control (co-infiltration of PVX-GFP with empty A. tumefaciens). At least 200 PVX-GFP infection foci from at least 3 independent repeats were imaged at 5DAI. Letters indicate significant differences revealed by Dunn’s multiple comparisons test p<0.001. (E) Effect of AtCPK3CA on PVX-GFP cell-to-cell movement in WT N. benthamiana or in transgenic lines constitutively expressing hairpin REM (hpREM) constructs. At least 200 PVX-GFP infection foci from at least 3 independent repeats were imaged at 5DAI. For each N. benthamiana line the effect of AtCPK3CA is expressed as a percentage of the corresponding mock control (empty Agrobacteria). Absolute values of the average foci area for each mock control are indicated.