Glomax 96 microplate luminometer system

Plasmids for secretory proteins were constructed by cloning full-length cDNAs of MoSPAB1 and MoSPAB1^ΔSP into the pCAMBIA1300 vector under the control of the 35 S promoter. The 1689-bp Bsr-d1 promoter was cloned into the pGreenII-0800 vector, forming a reporter construct. Renilla luciferase (REN) driven by the CaMV 35 S promoter was used as the internal control. Secretory protein constructs and reporters were used to co-transform tobacco leaf epidermal cells by Agrobacterium-mediated infiltration as described previously^{41 (link)}.
For rice protoplasts, the plasmids for secretory proteins were constructed by cloning full-length cDNAs of MYBS1, MoSPAB1^ΔSP, CfSPAB1^ΔSP, and CsSPAB1^ΔSP individually into the pGreenII 62-SK vector under the control of the 35 S promoter. This method was also described previously^{42 (link)}. Diluted D-Luciferin potassium salt (1 mM) (Coolaber, China) was smeared on the tobacco leaf surface, and a live imaging instrument (Bio-Rad ChemiDoc XRS) was used to detect fluorescence. LUC and REN activities were detected using the Dual-Lumi II Luciferase Assay Kit (Beyotime, China) in the GLOMAX96 Microplate Luminometer system (Promega), according to the manufacturer’s manual. Preparation and subsequent transfection of rice protoplasts were performed as described previously^{38 (link)}. Relative LUC/REN ratios were calculated.