A LexA-based yeast two-hybrid system for studying light-switchable interactions of phytochromes with their interacting partners

Abstract

Phytochromes are a family of photoreceptors in plants that perceive the red (R) and far-red (FR) components of their light environment. Phytochromes exist in vivo in two forms, the inactive Pr form and the active Pfr form, that are interconvertible by treatments with R or FR light. It is believed that phytochromes transduce light signals by interacting with their signaling partners. A GAL4-based light-switchable yeast two-hybrid (Y2H) system was developed two decades ago and has been successfully employed in many studies to determine phytochrome interactions with their signaling components. However, several pairs of interactions between phytochromes and their interactors, such as the phyA-COP1 and phyA-TZP interactions, were demonstrated by other assay systems but were not detected by this GAL4 Y2H system. Here, we report a modified LexA Y2H system, in which the LexA DNA-binding domain is fused to the C-terminus of a phytochrome protein. The conformational changes of phytochromes in response to R and FR light are achieved in yeast cells by exogenously supplying phycocyanobilin (PCB) extracted from Spirulina. The well-defined interaction pairs, including phyA-FHY1 and phyB-PIFs, are well reproducible in this system. Moreover, we show that our system is successful in detecting the phyA-COP1 and phyA-TZP interactions. Together, our study provides an alternative Y2H system that is highly sensitive and reproducible for detecting light-switchable interactions of phytochromes with their interacting partners.