Luciferase reporter assay
One hundred microliters of the Agrobacterium strain GV3101
containing the pGreenII 0800-target site-LUC plasmid were
combined with 900 μl of the Agrobacterium strain GV3101
containing either the 35s:pri miR398b or35s:primiR157plasmid. The mixture was incubated (28°C, 200 rpm, shaker) to a final
concentration (OD600 = 1.0), and resuspended in infiltration buffer (10
mM MES, pH 5.6, 10 mM MgCl2 and 150 μM acetosyringone) as described
previously (Hellens et al., 2005). The same volume of mixed suspensions
were injected into leaves of Nicotiana benthamiana by
agro-infiltration. After 48 h, LUC luminescence was examined using a
cryogenically cooled CCD camera (Lumazome PyLoN 2048B) as described
previously (Chen et al., 2008). LUC and RLUC activities were measured as
counts per second using a multimode plate reader
(PerkinElmer)
using the Promega kit (N1630).