Dual-LUC reporter (DLR) assay

The interaction of GbMYC2 with GbCCD7 and GbCCD8b promoters was examined using a dual-LUC reporter (DLR) assay. The reporter plasmid pGreenII 0800-LUC (Hellens et al. 2005) contained the LUC gene, controlled by GbCCD7 and GbCCD8b promoters with G-box binding elements upstream. The Renilla LUC-encoding gene driven by the 35S promoter was used as an internal control. To construct the effector plasmid, the CDS of GbMYC2 was introduced into the overexpression vector pGreenII 62-SK to generate p35S-GbMYC2. Subsequently, A. tumefaciens strain GV3101 cultures were transformed with the pCCD-LUC reporter or p35S-GbMYC2 effector plasmid, or with the empty vector used as a control. Equal amounts of A. tumefaciens strain GV3101 cultures containing pCCD-LUC and p35S-GbMYC2 constructs were mixed and co-infiltrated into N. benthamiana leaves. After 12 h in the dark, plants were returned to normal growth conditions for 24 h, after which leaves were uniformly sprayed with 0.2 mg mL⁻¹ LUC substrate. The fluorescence signal was detected after 8 min in darkness, and the LUC assay was conducted using the DLR assay system and a GloMax 20-20 luminometer (Promega, Madison, WI, USA).