Split-ubiquitin-based membrane yeast two-hybrid (MYTH) system

Protein interactions were tested using the split-ubiquitin-based MYTH system (MoBiTec), with introduced Gateway cloning sequences (Strzalka et al., 2015). Bait (pDHB1Gateway) and prey (pPR3-NGateway) vectors containing full-length phototropins or their N- or C-terminal domains (according to Aihara et al., 2008) were prepared as described for BiFC vectors, using the primers given in Supplementary Table S2. Yeast transformation and handling were described elsewhere (Strzalka et al., 2015). For scoring interactions, transformed yeast plated on agar plates were kept in 30 °C either in darkness or under blue light (~20 μmol m⁻² s⁻¹, 470nm) for 3 d. Each experiment was repeated at least three times.