Agrobacterium tumefaciens-Mediated Nicotiana Plant Magnifection

The A. tumefaciens strains carrying the deconstructed virus vectors were incubated at 28 °C for 48 h in YEB/Rif/Cb or YEB/Rif/Km liquid medium (meat extract 5 g/L, yeast extract 1 g/L, peptone 5 g/L, sucrose 5 g/L, MgSO₄ 0.1 g/L, pH 7.0, rifampicin 50 mg/L and kanamycin 50 mg/L or carbenicillin 100 mg/L) until reaching an optical density (OD_600nm) of 1.5, once the optical density was obtained, a mixture of A. tumefaciens was prepared containing the vectors (pICH31070 with the scFv-anti-DEC205-OLLAS-VP6 gene, pICH20155, pICH15879, pICH14011 and pICH-GFP) necessary for the transformation of the plants. For the transformation of the plants with the gene, a mixture was prepared with equal volumes of the A. tumefaciens strains with the scFv-anti-DEC205-OLLAS-VP6 gene contained in pICH31070, pICH20155, or pICH15879 and pICH14011; for the expression of the positive control, the strains with the pICH20155 or pICH15879, pICH14011, and pICH-GFP vectors were mixed. Acetosyringone (200 mM) was added and the mixtures were incubated for two hours at room temperature. The mixtures were centrifuged at 5500 rpm for 15 min, the obtained pellets were resuspended in the infiltration buffer (10 mM 2-N-morpholino ethanesulfonic acid (MES) pH 5.5, 10 mM MgSO₄) to obtain a final dilution of 1:10 and have a DO_600nm of 0.15. The infection process of both Nicotiana benthamiana and Nicotiana sylvestris plants was carried out by wounding the underside of the leaf and the bacterial suspension was injected with the needleless syringe. Once infected, the plants were incubated at 25 °C with a photoperiod of 16 light hours and 8 dark hours for 10 to 12 days. Once the days of incubation were completed, the leaf tissue was harvested and macerated in mortar with liquid nitrogen and lyophilized for later use [10, 20, 25, 26].