Figure 4.

Phytochromes Inhibit the Interaction of COP1 with SPA Proteins.

(A) Yeast three-hybrid analysis of the effects of phyA and phyB on the COP1-SPA1 interaction. COP1 and SPA1 were expressed as a standard yeast two-hybrid protein-protein interaction pair (BD-COP1 and AD-SPA1). Phytochromes were coexpressed as additional proteins, with C-terminal nuclear localization signals (phyA/B-NLS). Yeast cells were grown on chromophore-supplemented plates for 72 h under either constant darkness (D), R, or FR light, and the interaction of COP1 and SPA1 detected using ONPG. Values are the average of nine assays; error bars display 1 sd.

(B) Yeast three-hybrid analysis, including a LUC fusion of COP1 (BD-LUC-COP1) and AD-SPA1, performed as in (A) under constant R light. Left, X-Gal filter lift assays; right, in vivo luciferase activity. Plates were sprayed with 5 mM luciferin and imaged using a CCD camera. Arbitrary light signal intensity is indicated in the adjacent scale.

(C) Yeast three-hybrid analysis of the effects of phyA on the COP1-SPA2/4 interaction. Performed as for (A) in either darkness (Pr) or constant R light (Pfr), and interaction was detected by X-Gal filter lift assay.

Immunoblot analysis of BD-LUC-COP1, AD-SPA1, and phyA-NLS protein levels is shown in Supplemental Figure 6.