Fig. 5.

In vitro phosphorylation of GBNV N protein. a Phosphorylation of the N protein (5 μg) was carried out by mixing γ³²P ATP (1 μCi) and the soluble fraction of tobacco plant sap in 25 mM HEPES buffer, pH 7.2, containing 2 mM MnCl₂, incubation for 30 min at 30°C, and analysis by SDS-PAGE. The left panel shows an autoradiogram, and the right panel shows the same gel stained with Coomassie blue. Lane 1, N protein with plant sap; lane 2, N protein alone without plant sap (negative control); lane 3, plant sap alone without N protein (negative control), lane 4: molecular mass markers; lanes 5 and 6, reaction in the presence of 2 mM EGTA and 2 mM EDTA, respectively. b Effect of metal ions on phosphorylation of the N protein. The phosphorylation reaction was carried out without any metal ions (lane 1) or with varying concentrations (0.5, 1 and 2 mM) of MnCl₂ (lanes 2, 3, and 4, respectively). Similarly, the reaction was carried out separately with 0.5, 1 and 2 mM MgCl₂ (lanes 5-7) and CaCl₂ (lanes 8-10). Lane 11, molecular mass markers. The left panel shows an autoradiogram, and the right panel shows the same gel stained with Coomassie blue. c The phosphorylation reaction was carried out with 1 μCi of γ ³²P GTP as the phosphoryl group donor instead of labeled ATP. Lane 1, plant sap alone; lane 2, N protein alone; lane 3, reaction carried out in the absence of any metal ions; lanes 4, 5 and 6, reaction carried out in the presence of 2 mM MnCl₂, 2 mM MgCl₂ and 2 mM CaCl₂, respectively; lane 7, molecular mass markers. d Phosphorylation of the N protein (5 μg) was carried out with 200 ng of purified recombinant tobacco CK2 instead of plant sap as described in the legend to Fig. 5 a. Lane 1, CK2 alone without N protein; lane 2, N protein alone without CK2; lane 3, N protein with CK2; lane 4, molecular mass markers; lane 5, BSA (Calbiochem) with CK2 (negative control); lane 6, histone H1 (Sigma) with CK2 (positive control); lane 7, histone H1 (Sigma) without CK2. e Phosphorylation reaction carried out with 100 ng of purified recombinant chickpea CDPK instead of plant sap. Lane 1, CDPK alone without N protein (negative control); lane 2, N protein alone without CDPK; lane 3, N protein with CDPK; lane 4, molecular mass markers; lane 5, histone H1 (Sigma) with CDPK (positive control); lane 6, BSA (Calbiochem) with CDPK (negative control)