Figure 1

Progression and persistence of VIGS in excised leaf disks . Three-week-old N. benthamiana plants were inoculated with Agrobacterium carrying TRV::NbPDS and TRV::NbChlH constructs and grown in the greenhouse along with TRV::GFP (vector control) and wild-type non-inoculated plants. A) Leaf disks were collected from upper non-inoculated newly developed leaves at 8 dpi and incubated on MS medium. Since silencing of NbPDS and NbChlH genes in leaves is expected to produce discoloration, change in phenotype (from dark green to white or yellowing) was monitored and photographs were taken at two-day intervals. B) Leaf disks were collected from upper non-inoculated newly developed leaves from silenced plants at 20 dpi and incubated on MS medium and CIM. Persistence of silencing phenotype (white or yellow) was monitored for 20 more days, and photographs were taken at 20 and 40 dpi. The upper panel shows the respective phenotype and the lower panel shows RT-PCR that was performed using first-strand cDNA as a template, synthesized from total RNA (2 μg) primed by oligo-(dT)₁₅, with 30 PCR cycles. Plates containing leaf disks were maintained under day/night period of 16 h/8 h at light intensity of 100 μmol m^-2 s^-1 in both cases. NbPDS, N. benthamiana phytoene desaturase; NbChlH, N. benthamiana Mg-chelatase H subunit; GFP, green fluorescent protein; dpi, days post-inoculation; MS, Murashige and Skoog medium; CIM, callus induction medium; TRV2-CP, Tobacco rattle virus RNA2 coat protein; EF1α, Elongation factor-1 alpha as loading control.