Pgbkt7 vector

The nuclear indicator construct NLS-RFP, the reporter construct Gal4:GUS and the effector constructs GD (Gal4 DNA Binding Domain), CAT, GL3, GD-GL3, GD-GL1, GD-TTG1 used for protoplasts transfection have been described previously [44 (link), 49 (link), 50 (link)].
To make the HA (Human influenza hemagglutinin)-tagged OsGL3B construct, and GFP (Green fluorescent protein) tagged and/or GD-tagged OsGL1s, OsGL3B and OsTTG1A constructs for protoplast transfection, the full-length ORF (open-reading frame) of the corresponding genes were amplified by RT-PCR using RNA isolated from rice seedlings, or synthesized (for OsGL3B, OsGL1C, OsGL1D and OsGL1E) by Sangon Biotech Co., Ltd, and cloned in-frame with an N-terminal HA, GFP or GD tag and under the control of the double 35S promoter into pUC19 vector [50 (link), 51 (link)].
To make 35S:OsTTG1A construct for plant transformation, the HA tagged OsTTG1A construct in pUC19 was digested with NdeI and AflII and subcloned into the binary vector pPZP211 vector [52 (link)].
To generate bait and prey constructs for yeast-two-hybrid assays, OsGL3B was cloned into pGBKT7 vector (Oebiotech), and GL1 and TTG1 and their homolog genes in rice were cloned into pGADT7 vector (Oebiotech).

A tomato cDNA library cloned into the pGADT7 vector (Shanghai OE Biotech Co., Ltd., Shanghai, China) was used in protein–protein interaction screens. The ToCV-p27 gene was amplified directly from the ToCV-BJ isolate using the primer pair BD-p27-F/BD-p27-R (Table S1). The recombinant plasmid pGBKT7-p27 was cloned into the PGBKT7 vector (Shanghai OE Biotech Co., Ltd., Shanghai, China) by double digestion. PGBKT7-p27 was transferred to the Y2H Gold strain, and then it was coated on a SD/-Trp medium plate and cultured in a 30 °C incubator for observation under 5 d. Single colonies were selected and added to 50 mL of SD/-Trp medium for shock culture for 20 h until the OD600 were 0.8 and then fused with 1 mL of the tomato library for 24 h. Microscopical observation observed the formation of zygotes in the shape of a clover, and the resuspended cells were centrifuged at low speed and added to YPDA. The bacterial solution (300 μL) was diluted to 3 mL with 0.9% NaCl containing Kan (50 μg/mL) and spread in SD/-Trp-Leu + X-α-Gal + 125 ng/mL of AbA medium plates. These plates were coated and cultured upside down in a 30 °C incubator for approximately five days to observe colony growth in the solid plates. Then, blue-white spot screening was performed on the SD/-Ade-His-Leu-Trp + X-α-Gal + 125 ng/mL AbA plate.