Plant growth and agroinfiltration

Cassava plants were propagated vegetatively by planting properly lignified stem cuttings in soil. Cassava and Nicotiana benthamiana plants were grown in a greenhouse at 25 °C under a 16/8-h photoperiod. 3-week and 5-month-old cassava plants, and 2-week-old N.benthamiana seeding were used for agroinoculation. For agroinfiltration of recombinant CsCMV clones, the CsCMV-NC-based constructs were transformed into Agrobacterium tumefaciens GV3101 with pSoup helper plasmid, respectively. A single colony of A. tumefaciens strain GV3101 for each viral construct were gown overnight in Luria–Bertani medium containing rifampicin (25 mg/L) and kanamycin (50 mg/L) at 28 °C. Subsequently, overnight bacterial cultures were centrifuged at 2,500g for 10 min and were resuspended in agroinfiltration buffer (10 mM MgCl₂, 10 mM 2-(N-Morpholino) ethanesulfonic acid [pH 5.5], and 100 µM acetosyringone) for reaching an optical density of 0.8 at 600 nm (OD₆₀₀) [42]. The agrobacterium suspension was kept at room temperature for 3 h in the dark before agroinfiltration. For cassava, the back sides of four healthy and fully developed leave in the middle of each plant were selected for agroinfiltration using a 1-mL needleless syringe. Injections were performed at 8–10 spots on both sides of the main vein per leaf to enlarge the infiltrated leaf area. About 10 µL of agrobacterium suspension was used for each spot. N. benthamiana leaves were agroinfiltrated as described previously [43].