Fig. 1.
Interaction between AtCSP3 and candidate proteins (#1–38) revealed by the yeast two-hybrid assay. Positive interactors were selected by histidine auxotrophy (growth on His- plate) and galactose-induced β-galactosidase activity. Arabidopsis thailiana (Col-0) plants were grown at 22 °C under 16-h light/8-h dark conditions. Cold-acclimated (5 days at 4 °C) three-week-old seedlings were harvested for cDNA library preparation. The CloneMiner cDNA Library Construction Kit (Invitrogen) was utilized to construct a cDNA library based on the entry vector pDONR222. The library was subsequently transferred into pJG4-5 (Kaminaka et al. 2006) using LR Clonase (Invitrogen). For bait construction, a PCR-amplified AtCSP3 cDNA was cloned into pENTR/D-TOPO (Invitrogen) and then transferred into the destination vector, pEG202, using LR clonase II (Invitrogen). The Arabidopsis cDNA library (1 × 105 independent clones) in pJG4-5 was transformed into Saccharomyces cerevisiae EGY48 cells (Kaminaka et al. 2006) containing pEG202-AtCSP3 (bait) and pJK103 (reporter). As a positive control (C), interaction between AtLSD1 proteins was utilized (Kaminaka et al. 2006). Identification of candidate proteins is listed in Table 1