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. 2016 Aug 16;12(8):e1006252. doi: 10.1371/journal.pgen.1006252

Fig 5. PI4Kγ5 interacts with a membrane-bound TF ANAC078 in planta and phosphorylates ANAC078 in vitro.

Fig 5

A. ANAC078 interacts with PI4Kγ5 in yeast cells. Yeast cells co-transformed with pGBKT7-ANAC078 and pGADT7-PI4Kγ5 were diluted 10, 102, 103 and 104 times and grown on synthetic dropout (SD) medium lacking Leu, Trp, His, and Ade [SD(-Leu-Trp-His-Ade)], supplemented with X-α-gal. Growth of cells was observed after transformation for 2 days and yeast cells co-transformed with pGBKT7-ANAC078 and empty pGADT7 were used as negative control. B. Firefly luciferase complementation assay confirmed the ANAC078-PI4Kγ5 interaction in planta. C. Transient expression analysis showed that GFP-ANAC078 co-localizes with RFP-PI4Kγ5 in Arabidopsis protoplasts. Bar = 50 μm. D. PI4Kγ5 phosphorylates ANAC078 and ANAC078(-TM) in vitro. Recombinantly expressed His-PI4Kγ5, His-ANAC078, His-ANAC078(-C) (deletion of C-terminus), and His-ANAC078(-TM) (deletion of N-terminal transmembrane region) were used for kinase assay. Anti-His and anti-pThr antibodies were used to detect the protein input (upper panel) and phosphorylated proteins (bottom panel), respectively. E. Western blot analysis revealed that ANAC078(-C) amount was significantly reduced in pi4kγ5–1. The 7th and 8th rosette leaves (~100 mg) of WT or pi4kγ5–1 plants expressing cMyc-ANAC078 (cMyc-ANAC078 in WT; or cMyc-ANAC078 in pi4kγ5–1) were used for protein extraction and western blot analysis (upper panel). A mouse cMyc antibody was used to detect the cMyc-ANAC078 or cMyc-ANAC078(-C) proteins. CBB staining indicated the equal protein loading (bottom panel). F. Expression of ANAC078(-C) driven by PI4Kγ5 promoter rescued the leaf serration of pi4kγ5–1, while full ANAC078 could not. The 7th rosette leaf was shown. Bars = 1 cm. G. Fluorescence observation revealed the fluorescence of GFP-ANAC078 in the nucleus and plasma membrane of WT protoplasts, while only in plasma membrane but not nucleus of pi4kγ5–1 protoplasts. Serration of the 7th and 8th rosette leaves of 28-day-old WT or pi4kγ5–1 plants were used for protoplast preparation. Bar = 20 μm.