Fig. 1

MPK3 phosphorylates HD2B in vitro and interacts with HD2B. a In vitro phosphorylation assays of MBP (left), GST (middle), and HD2B-GST (right) by MPK3 (3), MPK4 (4), and MPK6 (6). MPK3, 4, and 6 were immunoprecipitated from flagellin-treated root cell suspensions. For each assay, Coomassie staining of the gel (CBB) is shown on the left as a protein loading control and the autoradiography (Autorad) is shown on the right. MPK3, 4, and 6 were able to phosphorylate MBP, but not GST. HD2B phosphorylation was observed with all three MPKs, but MPK3 showed the highest activity. Arrows indicate full-length forms of all substrate proteins. IgG indicates immunoglobulin G used for immunoprecipitation of the MAPKs. Asterisks indicate an unknown phosphoprotein in the MPK4 lanes, or MPK6 autophosphorylation in the MPK6 lanes. b In vitro phosphorylation assays of WT HD2B (WT) and the mutant HD2B protein forms HD2B-T249E and HD2B-T249E/S266D. Coomassie staining of the gel (CBB) is shown as a protein loading control. c Cell fractionation assay of MPK3 localization. Proteins from total, cytoplasmic, and nuclear cell extracts were subjected to SDS-PAGE and analyzed by immunoblotting with anti-MPK3 antibody. Anti-H3 and anti-PEPC antibodies were used as controls for nuclear and cytoplasmic proteins, respectively. d Immunostaining of MPK3 and HD2B performed on plants expressing a GFP-HD2B fusion. Antibodies used to reveal the presence of the proteins are noted above the panels. DAPI staining was used to label the nuclei. MPK3 could be detected both in the cytoplasm and in the nucleus while GFP-HD2B was only observed in the nucleus. e BiFC analysis of HD2B interaction with MPK3 in epidermal cells of Agrobacterium-infiltrated Nicotiana benthamiana. Empty vectors were used as controls. Fluorescence indicates that yellow fluorescent protein C terminal fragment (YFPc)-HD2B interacts with YFPn-MPK3. YFPc-HD2B does not interact with empty YFPn vector and YFPn-MPK3 does not interact with empty YFPc vector Overlay indicates merging the yellow fluorescent protein (YFP) and light transmission images