Fig. 3: T101 of TAA1 is targeted by TMK4 kinase.

a Yeast two-hybrid assay of multiple protein kinases (bait) and TAA1 protein (prey). Three independent biological repeats. b In vitro pull-down assay showing GST-TMK4 kinase ___domain (KD) can be pulled down by TAA1-His proteins. His-Sumo proteins were used as a negative control. TMK4 KD was detected via western blot using an anti-GST antibody. TAA1-His and His-Sumo shown by Coomassie blue staining (CBB). Three independent repeats showed similar results. c TMK4 was coimmunoprecipitated with TAA1 in protoplasts. 35S-TMK4-HA and 35S-TAA1-Flag were transiently coexpressed in Arabidopsis protoplasts. Single transformation of 35S-TMK4-HA was used as control. Three independent repeats showed similar results. d In vitro kinase assay of TMK4 kinase ___domain (KD) on TAA1 protein. Three biological repeats. e Mass spectrometric analysis of the phosphor-peptide intensity in Col-0 and tmk4-1. The intensity indicates the phosphorylation level of T101 containing phosphor-peptide (93aa–116aa). Standardization and another two repeats were shown in Supplementary Fig. 10b, c. f Quantification of TAA1 enzymatic activity with or without TMK4KD phosphorylation. E. coli-purified His-sumo-TMK4KD and TAA1 proteins were mixed together to perform a phosphorylation reaction triggered by ATP before the in-gel transaminase catalytic reaction (Methods section). ATP+ indicated the phosphorylation reaction between TMK4KD and TAA1 with ATP, and ATP− indicated the abolished phosphorylation reaction without ATP. P values were shown as indicated (n represents five biological repeats); two-sided t-test. Centre line in box represents mean and whiskers show minimum to maximum.