Fig. 3

SAPK10 physically interacts with OsSLAC1 and phosphorylates the N-terminal region of OsSLAC1 in vitro. a Yeast two hybridization assays. SAPK10 was used as bait (BD-SAPK), and the N-terminal region and C-terminal regions of OsSLAC1 were used as prey (AD-N-OsSLAC2 and AD-C-OsSLAC1). b Subcellular localization of SAPK10-GFP in rice protoplasts. c BiFC analyses of VN-SAPK10 and OsSLAC1-VC using rice protoplasts. ER-mCherry was used as an internal control. VN-Hd3A was used as a negative control. Scale bars are 10 μm. d The relative BiFC signal intensities depict the average ± SD of over 20 cells emitting the fluorescence signals. The value was calculated as Venus signal/mCherry signal. e Co-IP analysis using GFP-N-terminal SLAC1 and SAPK10-Flag coexpressed in rice protoplasts. These experiments were repeated three times and a representative result was presented. f In vitro kinase assay for His-SAPK10 activity with GST-N-OsSLAC1. The ³²P radioactivity was detected using a phosphor image analyzer (upper panel) and polyacrylamide gel was stained using Coomassie brilliant blue R250 (lower panel). GST-N-terminal region OREB1 (GST-N-OREB1) was used as a control