Transient Expression in Nicotiana benthamiana

Plasmids were introduced into Agrobacterium tumefaciens AGL1 (Lazo et al., 1991) by electroporation. Electroporated cells were recovered in yeast extract peptone medium for 2 h at 28°C and selected on yeast extract peptone agar medium containing 25 μg mL⁻¹ rifampicin, 50 μg mL⁻¹ carbenicillin, and 50 μg mL⁻¹ kanamycin. Transformants were grown overnight at 28°C and 225 rpm. Agrobacteria cells were harvested and resuspended in MilliQ water to a final OD₆₀₀ of 1. Equal volumes of agrobacteria suspensions were mixed and introduced into N. benthamiana leaves (approximately 4 weeks old) using a blunt syringe. An expression construct carrying the open reading frame of p19 (Hearne et al., 1990) was always coinfiltrated to prevent posttranscriptional gene silencing (Voinnet et al., 1999; Lakatos et al., 2004). Two plants were infiltrated per combination, and infiltrations were repeated to confirm results. After infiltration, the plants were kept in the greenhouse (21°C day/19°C night) until leaf material was harvested.