REM1.3's phospho-status defines its plasma membrane nanodomain organization and activity in restricting PVX cell-to-cell movement
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.