Measurement of luciferase activity in tobacco

Leaves at six days post-infiltration were used for the luciferase analysis. Luminescence of six leaf samples from three plants was analyzed for each analysis with a luminometer, TriStar LB941 (Berthold Technologies). The detection unit of Tristar LB941 is a low-noise photomultiplier, which can detect from 340 to 650 nm. No emission filter set was used, because the autofluorescence from living rice cells are negligible.

For measurement of Nluc activity in leaf tissue lysate, the Nano-Glo® Luciferase Assay System (Promega) was used according to the manufacturer’s instructions. A piece of leaf disc 5 mm×5 mm square (ca. 2.5 mg) was cut out from the infiltrated region and ground in 100 µl of Passive lysis buffer (Promega) with a pestle. To avoid overloading, the lysate was further diluted 10,000-fold with Passive Lysis buffer. Luminescence was measured with TriStar LB941 immediately after mixing 5 µl of the diluted lysate with 5 µl of Nano-Glo Luciferase Assay Reagent (Promega) on a 96-well titer plate.

For measurement of Fluc activity in leaf tissue lysate, the Dual-Luciferase® Reporter Assay System (Promega) was used according to the manufacturer’s instructions. After the cell lysate was prepared in the same way as above, it was further diluted 100-fold to avoid overloading. Luminescence was measured immediately after mixing 2 µl of the diluted lysate with 10 µl of LARII reagent in the kit on a 96-well titer plate.

For measurement of Nluc activity in live cells, the Nano-Glo® Luciferase Assay System and the Nano-Glo® Live Cell Assay System (Promega) were used according to the manufacturer’s instructions. 30 µl of Nano-Glo Luciferase Assay Reagent or Nano-Glo® Live Cell Reagent was infiltrated into the Agro-infiltrated leaf and a piece of leaf disc 5 mm×5 mm square was removed. After 3 min incubation, the leaf disc was placed into a 96-well plate and luminescence was measured. For measurement of Fluc activity in live cells, 30 µl of 20 mM D-luciferin solution was infiltrated and luminescence was analyzed in the same way as the Nluc analysis in live cells.