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