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.