24 well tissue culture plates

PEG-Ca mediated transformation followed a previously published protocol [17] (link) with some modifications. The protoplasts of Arabidopsis were suspended in 100 µl of MMG solution at a density of 10⁷ (and the maize protoplasts at 5×10⁶) cells/ml in a 2 ml round-bottom centrifuge tube containing 15 µg of DNA and mixed well. An equal volume of 40% PEG solution (40% PEG4000, 0.2 M mannitol and 100 mM CaCl₂⋅2H₂O) was added to the protoplast-DNA mix drop-wise with gentle shaking for 25 min. The mixture was diluted with 440 µl of cell culture solution which, depending on the species, was: (Arabidopsis: 154 mM NaCl, 125 mM CaCl₂⋅2H₂O, 5 mM KCl, 2 mM MES pH5.7) or (maize: 0.6 M mannitol, 4 mM KCl, 4 mM MES, pH 5.7). The protoplasts were then centrifuged at 112×g, the supernatant removed, the protoplasts re-suspended in 1 ml cell culture solution, and plated in the wells of 24-well tissue culture plates (NEST Biotechnology, China). The protoplasts were incubated at 23°C (Arabidopsis) or 25°C (maize) for 18 h prior to harvest for luciferase activity determination.

Viral culture of SARS-CoV-2 was performed in biosafety level-3 (BSL-3) facility. SARS-CoV-2 was isolated from a COVID-19 patient in our locality. SARS-CoV-2 was propagated in Vero cells in minimum essential medium (MEM) (Gibco, Waltham, MA, USA) supplemented with 1% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). Vero cells were seeded onto 24-well tissue culture plates (NEST, Wuxi, China), with 1 mL of MEM with 10% FBS, at 1 × 10⁶ cells/mL in 24 wells plate and incubated at 37 °C in a 5% carbon dioxide (CO₂) incubator for 24 h until >90% confluence (~0.2 × 10⁶ cells). Vero cells were washed once with phosphate-buffered saline (PBS) (Oxoid, Waltham, MA, USA) and inoculated with three multiplicity of infection (MOI) of SARS-CoV-2 in serum-free MEM, and then incubated at 37 °C. After 1 h of infection, unbound virus was removed by washing with 1 mL of PBS twice. The infected cells were maintained in MEM with 1% FBS until virus-induced cytopathic effect (CPE) was observed after two days. The supernatant was collected and was spun down at 2000× g to remove cell debris for subsequent RNA extraction.