Agroinfiltration of Nicotiana benthamiana Leaves

Expression plasmids were transformed by electroporation into A. tumefaciens strain GV3101 (harboring pSOUP) and plated on LB agar media with rifampicin (25 μg/mL), kanamycin (50 μg/mL), and tetracycline (10 μg/mL) selection. Starter cultures were prepared by inoculating a single colony in 2 mL of LB with rifampicin, kanamycin and tetracycline selection, and grown overnight at 28°C in a shaking incubator. Starter culture was used to inoculate the working culture (typically 20–50 mL), which was grown overnight under the same media, selection and incubation conditions. Working cultures were harvested by centrifugation at 24°C and 2,400 × g for 15 min. Cell pellets were resuspended in 10 mM MgCl₂ and the OD₆₀₀ adjusted to 0.5 prior to the addition of acetosyringone (200 μM). The resulting cell cultures were stored in the dark at room temperature for either 4 or 24 h prior to infiltration.

Nicotiana benthamiana plants were grown at room temperature and 16 h light per day, under LumiBar LED strip lighting (LumiGrow) intensity setting four. Seedlings were grown for approximately 3–4 weeks prior to agroinfiltration. Three expanded leaves per plant were infiltrated by applying pressure on the abaxial surface of the leaf with a disposable 5 mL syringe containing the Agrobacterium suspension. Each leaf was treated as a biological replicate. Agroinfiltrated plants were incubated for a specified period (typically 72 h) in a growth chamber set to 22°C with 16 h light per day. Agroinfiltrated leaves were harvested individually, snap-frozen in liquid nitrogen, powdered using a ball mill tissue-lyser (Retsch), and stored at -80°C prior to measurement in dual LUC assays.