Arabidopsis thaliana calcium-dependent protein kinase 3 (CPK3) is specifically involved in the restriction of PlAMV cell-to-cell movement.

A. Representative images of PlAMV-GFP infection foci at 5 dpi in the different mutant backgrounds. Scale bar = 500 µm

B. Box plots of the mean area of PlAMV-GFP infection foci 5 days after infection in CPK multiple mutant lines, normalized to the mean area measured in Col-0. Three independent biological repeats were performed, with at least 47 foci per experiment and per genotype. Significant differences were revealed using a One-Way ANOVA followed by a Tukey’s multiple comparison test. Letters are used to discriminate between statistically different conditions (p<0.05).

C. Box plots of the mean area of PlAMV-GFP infection foci in cpk3-1 and cpk3-2 single mutants and in CPK3 over-expressing lines (Pro35S:CPK3-HA #8.2 and Pro35S:CPK3-HA #16.2), normalized to the mean area measured in Col-0. Three independent biological repeats were performed, with at least 56 foci per experiment and per genotype. Significant differences were revealed using a One-Way ANOVA followed by a Tukey’s multiple comparison test. Letters are used to discriminate between statistically different conditions (p<0.05).

D. Representative images of A. thaliana plants infected with PlAMV-GFP and imaged with a CCD Camera from 10 to 17 dpi. The region of interest used for measurement of pixel intensity is circled with a white dotted line. Multicolored scale is used to enhance contrast and ranges from blue (low intensity) to red (high intensity). Scale bar = 4 cm.

E. Box plots of the mean cumulated intensity measured in infected leaves in Col-0, cpk3-2 and Pro35S:CPK3-HA #16.2 during systemic viral propagation. Two independent experiments were conducted. Statistical differences could be observed between the genotypes and time-points using a Kruskal-Wallis followed by a Dunn’s multiple comparison test (p<0.05). For clarity, only the results of the statistical test of the comparison of the different time-points (10, 14 and 17 dpi) within a genotype are displayed and are color-coded depending on the genotype.

F. Kinase activity of CPK3 dead variant. Recombinant proteins GST-CPK3 WT and K107M were incubated with REM1.21–118–6His in kinase reaction buffer in the presence of EGTA (−) or 100 µM free Ca2+ (+). Radioactivity is detected on dried gel (upper panel). The protein amount is monitored by Coomassie staining (lower panel).

G. Box plots of the mean area of PlAMV-GFP infection foci in cpk3-2 complemented lines cpk3-2/Pro35S:CPK3-myc and cpk3-2/ Pro35S:CPK3K107M-myc. Three independent biological repeats were performed, with at least 51 foci per experiment and per genotype. Significant differences were revealed using a One-Way ANOVA followed by a Tukey’s multiple comparison test. Letters are used to discriminate between statistically different conditions (p<0.0001)

CPK3 diffusion decreases upon PlAMV infection in Arabidopsis thaliana

A. Confocal images of the secant view of A. thaliana epidermal cells of the cpk3-2/CPK3-mRFP1.2 or cpk3-2/ProUbi10:CPK3G2A-mRFP1.2. Scale bar = 5µm

B. Box plots of the mean area of PlAMV-GFP infection foci in cpk3-2 and complemented lines cpk3-2/ProUbi10:CPK3-mRFP1.2 and cpk3-2/ProUbi10:CPK3G2A-mRFP1.2. Three independent biological repeats were performed, with at least 32 foci per experiment and per genotype. Significant differences were revealed using a One-Way ANOVA followed by a Tukey’s multiple comparison test. Letters are used to discriminate between statistically different conditions (p<0.0001).

C. Confocal images of the surface view of A. thaliana epidermal cells of the Col-0/ProUbi10:CPK3-mRFP1.2, infiltrated either with free GFP (“mock”) or PlAMV-GFP. Scale bar = 5µm

D. Representative trajectories of Col-0/CPK3-mEOS3.2 infiltrated either with free GFP (“mock”) or PlAMV-GFP; Scale bar = 2µm

E. Distribution of the diffusion coefficient (D), represented as log(D) for Col-0/ProUbi10:CPK3-mEOS3.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP. Data were acquired from at least 16 cells over the course of three independent experiments.

F. Box plot of the mean peak value extracted from the Gaussian fit of log(D) distribution. Significant difference was revealed using a Mann-Whitney test. *: p<0.05.

G. Mean square displacement (MSD) over time of Col-0/ProUbi10:CPK3-mEOS3.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP. Representative trajectories extracted from Figure 2D illustrate each curve. Scale bar = 1µm. Data were acquired from at least 16 cells over the course of three independent experiments.

H. Voronoi tessellation illustration of Col-0/ProUbi10:CPK3-mEOS3.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP. ND are circled in red. Scale bar = 2µm

I. Distribution of the ND diameter of Col-0/ProUbi10:CPK3-mEOS3.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP.

J. Box plot representing the mean peak value of ND diameter extracted from the Gaussian fit of Figure 2I. No significant differences were revealed using a Mann-Whitney test.

K. Boxplot of the proportion of Col-0/ProUbi10:CPK3-mEOS3.2 detections found in ND five days after infiltration with free GFP (“mock”) or PlAMV-GFP. No significant differences were revealed using a Mann-Whitney test.

PlAMV-induced activation of CPK3 in N. benthamiana induces its confinement and clustering in PM domains

A. Confocal images of the surface view of N. benthamiana epidermal cells transiently expressing ProUbi10:CPK3-mRFP1.2, ProUbi10:CPK3-mRFP1.2 + PlAMV-GFP or ProUbi10:CPK3CA-mRFP1.2. Scale bar = 5µm

B. Representative trajectories of ProUbi10:CPK3-mEOS3.2, ProUbi10:CPK3.2-mEOS3.2 + PlAMV and ProUbi10:CPK3CA-mEOS3.2. Scale bar = 2µm.

C. Distribution of the diffusion coefficient (D), represented as log(D) for ProUbi10:CPK3-mEOS3.2, ProUbi10:CPK3-mEOS3.2 + PlAMV-GFP and ProUbi10:CPK3CA-mEOS3.2. Data were acquired from at least 15 cells over the course of three independent experiments.

D. Box plots of the fraction of immobile proteins (log(D)<-2). Significant differences were revealed using a One-Way ANOVA followed by a Tukey’s multiple comparison test. Letters are used to discriminate between statistically different conditions (p<0.005).

E. Mean square displacement (MSD) over time of fast (circle) or slow (triangle) diffusing ProUbi10:CPK3-mEOS3.2, ProUbi10:CPK3-mEOS3.2 + PlAMV-GFP, ProUbi10:CPK3CA-mEOS3.2. Standard error is displayed from three independent experiments.

F. Voronoï tessellation illustration of ProUbi10:CPK3-mEOS3.2, ProUbi10:CPK3-mEOS3.2 + PlAMV-GFP and ProUbi10:CPK3CA-mEOS3.2. ND are circled in red. Scale bar = 2µm

G. Distribution of the ND diameter of ProUbi10:CPK3-mEOS3.2, ProUbi10:CPK3-mEOS3.2 + PlAMV-GFP and ProUbi10:CPK3CA-mEOS3.2

H. Box plot representing the mean peak value of ND diameter extracted from the Gaussian fit of Figure 3G. No significant differences were revealed using a Kruskal-Wallis followed by a Dunn’s multiple comparison test.

I. Boxplot of the proportion of detections found in ND of ProUbi10:CPK3-mEOS3.2, ProUbi10:CPK3-mEOS3.2 + PlAMV-GFP and ProUbi10:CPK3CA-mEOS3.2. Significant differences were revealed using a Kruskal-Wallis followed by a Dunn’s multiple comparison test. Letters are used to discriminate between statistically different conditions (p<0.005).

Group 1 REM hampers PlAMV-GFP cell-to-cell propagation and REM1.2 diffusion increases upon infection

A. Box plots of the mean area of PlAMV-GFP infection foci in rem1.2, rem1.3, rem1.4 single mutants along with rem1.2 rem1.3 double mutant and rem1.2 rem1.3 rem1.4 triple mutant. Three independent biological repeats were performed, with at least 36 foci per experiment and per genotype. Significant differences were revealed using a One-Way ANOVA followed by a Tukey’s multiple comparison test. Letters are used to discriminate between statistically different conditions (p<0.05).

B. Box plots of the mean cumulated intensity measured in infected leaves in Col-0 and rem1.2 rem1.3 rem1.4 during systemic viral propagation. Two independent experiments were conducted. Statistical significance of the difference between Col-0 and rem1.2 rem1.3 rem1.4 at each time point was assessed using a Mann-Whitney test. *: p<0.05, **: p<0.01

C. Representative images of A. thaliana plants infected with PlAMV-GFP and imaged with a CCD Camera from 10 to 17 dpi. Systemic leaves are circled with a white dotted line. Multicolored scale is used to enhance contrast and ranges from blue (low intensity) to red (high intensity). Scale bar = 4 cm.

D. Confocal images of the surface view of A. thaliana epidermal cells of the Col-0/ProUbi10:mRFP1.2-REM1.2, infiltrated either with free GFP (“mock”) or PlAMV-GFP. Scale bar = 5µm

E. Representative trajectories of Col-0/ProUbi10:mEOS3.2-REM1.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP. Scale bar = 2µm

F. Distribution of the diffusion coefficient (D), represented as log(D) for Col-0/ProUbi10:mEOS3.2-REM1.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP. Data were acquired from at least 16 cells over the course of three independent experiments.

G. Box plot of the mean peak value extracted from the Gaussian fit of log(D) distribution. Significant difference was revealed using a Mann-Whitney test. *: p<0.05.

H. Mean square displacement (MSD) over time of Col-0/ProUbi10:mEOS3.2-REM1.2 infiltrated either with free GFP (“mock”) or PlAMV-GFP. Representative trajectories extracted from Figure 4E illustrate each curve. Scale bar=1µm.

I. Voronoi tessellation illustration of Col-0/ProUbi10:mEOS3.2-REM1.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP. ND are circled in red. Scale bar = 2µm

J. Distribution of the ND diameter of Col-0/ProUbi10:mEOS3.2-REM1.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP.

K. Box plot representing the mean peak value of ND diameter extracted from the Gaussian fit of Figure 4J. No significant difference was revealed using a Mann-Whitney test.

L. Boxplot of the proportion of Col-0/ProUbi10:mEOS3.2-REM1.2 detections found in ND five days after infiltration with free GFP (“mock”) or PlAMV-GFP. No significant difference was revealed using a Mann-Whitney test.

Group 1 REMs and CPK3 are in the same functional pathway and regulate each other PM diffusion upon PlAMV infection

A. Box plots of the mean area of PlAMV-GFP infection foci in cpk3-2, rem1.2 rem1.3 rem1.4 triple mutant and rem1.2 rem1.3 rem1.4/cpk3 #1 and #2 quadruple mutants. Three independent biological repeats were performed, with at least 23 foci per experiment and per genotype. Significant differences were revealed using a One-Way ANOVA followed by a Tukey’s multiple comparison test. Letters are used to discriminate between statistically different conditions (p<0.0001).

B. Representative images of PlAMV-GFP infection foci at 5 dpi in the different mutant backgrounds. Scale bar = 500µm.

C. Representative trajectories of cpk3-2/ProUbi10:mEOS3.2-REM1.2 five days after infiltration with either free GFP (“mock”) or PlAMV-GFP. Scale bar = 2µm.

D. Distribution of the diffusion coefficient (D), represented as log(D) for cpk3-2/ProUbi10:mEOS3.2-REM1.2 five days after infiltration with either free GFP (“mock”) or PlAMV-GFP. Data were acquired from at least 11 cells over the course of three independent experiments.

E. Box plot representing the mean peak value extracted from the Gaussian fit of the distribution of the diffusion coefficient (D) represented in Figure 5D. No significant difference was revealed using a Mann-Whitney test.

F. Mean square displacement (MSD) over time of cpk3-2/ProUbi10:mEOS3.2-REM1.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP.

G. Representative trajectories of rem1.2 rem1.3 rem1.4/ProUbi10:CPK3-mEOS3.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP. Scale bar = 2µm.

H. Distribution of the diffusion coefficient (D), represented as log(D) for rem1.2 rem1.3 rem1.4/ProUbi10:CPK3-mEOS3.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP. Data were acquired from at least 10 cells over the course of three independent experiments.

I. Box plot representing the mean peak value extracted from the Gaussian fit of the distribution of the diffusion coefficient (D) represented in Figure 5H. No significant difference was revealed using a Mann-Whitney test.

J. Mean square displacement (MSD) over time of rem1.2 rem1.3 rem1.4/ProUbi10:CPK3-mEOS3.2 five days after infiltration with free GFP (“mock”) or PlAMV-GFP.

Proposed “kiss-and-run” model describing REM1.2 and CPK3 interdependence

Upper panel: in healthy conditions, REM1.2 displays a smaller mobility at the PM than CPK3. Random interactions between the two proteins can occur.

Middle panel: upon PlAMV infection, CPK3 is activated. CPK3 activation and confinement are related but whether activation of the protein occurs before or after its confinement is not deciphered yet. Activation results in phosphorylation (+P) of local substrates, including REM.

Lower panel: REM phosphorylation induces its diffusion at the PM. The period during which CPK3 remains confined is not determined yet.