DCL1 interacts with PIF4.

<p><b>(A)</b> Yeast two-hybrid assays show the interactions between PIF4 and DCL1, or PIF4 and HYL1. Co-transformed yeast colonies were spotted on the selective SC medium minus Trp and Leu, then grown on SC medium minus His, Trp, and Leu supplemented with 5mM 3-amino-1, 2, 4-triazole (3-AT). <b>(B)</b> MBP pull-down assay shows the interaction between PIF4 and the double strand RNA binding domains (RBD) of DCL1. <i>In vitro</i> pull-down assay using <i>E</i>. <i>coli</i>–expressed MBP, MBP-DCL1-RBD, GST and GST-PIF4. GST- PIF4 was incubated with MBP-DCL1-RBD bound to amylose resin. The upper panel (input): an anti-GST antibody was used to detect GST or GST- PIF4. The bottom panel (pull-down): an anti-MBP antibody was used to detect MBP-DCL1-RBD. <b>(C)</b> BiFC assays show the interaction between PIF4 or the C-terminal fragment of PIF4 (PIF4-C) and DCL1or HYL1 in tobacco epidermal cells. No interactions were detected between PIF4 and YFP, PIF4 and DCL1-9, or the N-terminal fragment of PIF4 (PIF4-N) and DCL1. (Scale bar = 10 μm). <b>(D)</b> Co-IP assay shows the interaction between PIF4 and DCL1. <i>Arabidopsis</i> plants were transformed with <i>pPIF4</i>::<i>PIF4-HA</i> and <i>pDCL1</i>::<i>DCL1-YFP</i>. The extracts from 4-day-old seedlings were incubated with anti-HA–conjugated agarose. The pellet was analyzed by immunoblotting with anti-HA and anti-GFP antibodies. <b>(E)</b> Yeast two-hybrid assays show that the C-terminal fragments of PIF4 interact with DCL1-RBD. Diagrams of the PIF4 fragments (F1-F4) and domains within F1-F4 are shown at the bottom panel: F1, aa 1–255; F2, aa 123–431; F3, aa 211–431; F4, aa 255–431; F5, aa 256–374; APB, active phytochrome binding motif; bHLH domain, basic helix-loop-helix domain.