4.5. Subcellular Localization and BiFC

To observe interactions between proteins using bimolecular fluorescence complementation (BiFC), we introduced gene fragments into pENTR-D-TOPO vectors (Invitrogen, Carlsbad, CA, USA) and then transferred them to their destination vectors by LR recombination (Promega, Madison, WI, USA) [25]. pGEM-gw-VC vector was used for fusion of Venus Carboxyl-terminus (VC) with OsDREB1A, OsDREB1B, and OsDREB1C, pGEM-gw-VN vector used for fusion of Venus Amino-terminus (VN) with OsPP2C06, OsPP2C09, osPP2c30, and OsPYL/RCAR3. Generated plasmids were introduced into rice protoplasts as indicated pairs using the PEG-mediated method [32]. The ER-mCherry reporter was used as internal control. Fluorescence signals were captured using a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). The combination of excitation wavelength/detection range of emission for Venus signals was 488 nm (solid state laser)/ with a bandpass of 505–561. Signal intensities on captured images were analyzed using Leica Application SuiteX provided by the manufacturer (Leica Microsystems, Wetzlar, Germany) with default settings (threshold 30%, background 20%).