Fig. 4

DGK5α has compromised diacylglycerol kinase activity and cannot be phosphorylated by BIK1. A DGK5α exhibits a compromised diacylglycerol kinase activity. The recombinant GST-MBP, HIS-DGK5β, or HIS-DGK5α proteins were incubated with [¹⁴C]-DOG in a reaction buffer containing ATP for 30 min. Chloroform-soluble products were separated by the thin-layer chromatography (TLC) plate, and PA was detected by autoradiography (Autorad., top). The amount of PA levels is quantified based on the band intensities using ImageJ software. The PA level produced by DGK5β (second lane) was set as 1, and relative PA levels were labeled on the bottom of the autoradiography panel. The protein loading is shown by CBB staining on the bottom. B The cpl3-3 mutant displays the elevated PA production. Cell lysates from WT and cpl3-3 seedlings with/without 100 nM flg22 treatment for 10 min were incubated with [¹⁴C]-DOG in a reaction buffer for 60 min. Total lipids were separated by the TLC plate placed in an acidic solvent system, and PA was detected by autoradiography (top panel). The protein loading is shown by CBB staining on the bottom. The band intensities corresponding to PA were quantified using the ImageJ software. The PA level without flg22 treatment in WT was set as 1, and relative PA levels were labeled on the bottom of the autoradiography panel. C BIK1 interacts with DGK5β, but not DGK5α, in an in vitro pull-down assay. Recombinant GST-BIK1 proteins and GST-MBP proteins (control) were immobilized on glutathione sepharose beads and incubated with HIS-DGK5β or HIS-DGK5α proteins for the pull-down assay. Eluted proteins were subjected to immunoblotting with an α-HIS or α-GST antibody (PD: GST; top two panels), and proteins before the pull-down assay are shown as input (middle two panels). The total proteins are stained by CBB (bottom panel). D BIK1 phosphorylates DGK5β, but not DGK5α in vitro. The in vitro kinase assay was performed using purified GST-BIK1 or GST-BIK1^KM as a kinase and GST, GST-BAK1^KD, GST-DGK5β, or GST-DGK5α as the substrates using [γ-³²P]-ATP. BIK1^KM is a BIK1 kinase-dead mutant, and BAK1^KD is the BAK1 truncation with the kinase domain alone, which does not carry auto-phosphorylation activity. Phosphorylation was analyzed by autoradiography (Autorad.) (top), and protein loading is shown by CBB staining (bottom). E DGK5β, but not DGK5α, shows the upper shifted band upon flg22 treatment in the presence of BIK1. DGK5β-HA or DGK5α-HA was co-expressed with BIK1-FLAG in protoplasts from WT plants, followed by 100 nM flg22 treatment for 10 min. Total proteins were separated with Mn.²⁺-Phos-tag (top panel) or regular SDS-PAGE (middle two panels), followed by immunoblotting with an α-HA or α-FLAG antibody. Rubisco stained by CBB from the regular SDS-PAGE serves as a loading control (bottom panel)