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. 2014 Jan 8;9(1):e84891. doi: 10.1371/journal.pone.0084891

Figure 3. TIFY8 is a nuclear transcriptional repressor.

Figure 3

A–C, TIFY8 localizes to the nucleus. Confocal root tip imaging of 4-day-old Arabidopsis seedlings overexpressing the TIFY8-GFP fusion protein (A), free GFP (B) or SV40-NLS fused GFP (C), respectively. Propidium iodide staining was performed prior to imaging to enhance the visualization of the cells. D, NINJA, but not TIFY8, interacts directly with TPL in Y2H assays. Co-transformation of the PJ69-4A yeast strain with TIFY8 or NINJA and the N-terminal fragment of TPL (TPL-N) in pGADT7 or pGBKT7 vectors, respectively. Transformed yeast were spotted on control medium lacking Leu and Trp (-2) or selective medium additionally lacking His (-3). E, TIFY8 recruits TPL through interaction with NINJA in Y3H assays. Co-transformation of the PJ69-4A yeast strain with TIFY8 and TPL-N in Gateway-compatible pGADT7 and pGBKT7 vectors, respectively, together with NLS-3xFLAG-6xHis tagged NINJA in the pMG426 vector. Transformed yeast were spotted on control medium lacking Leu, Trp and Ura (-3) or selective medium additionally lacking His (-4). A negative control is provided by substitution of NINJA by the empty pMG426 vector. F, TIFY8 acts as a transcriptional repressor in transient expression assays. Transactivation activity in tobacco protoplasts transfected with a pUAS–fLUC reporter construct, effector constructs fused to GAL4DBD, and a 35S:rLUC normalization construct. Error bars represent ±SE of eight biological replicates. Asterisks represent significant differences (***, p<0.001, one-way ANOVA, Tukey HSD's Post Hoc test).