Green fluorescence protein (GFP) reporter assay
The A. tumefaciens strain GV3101 containing one of the binary vector (35s:5NBts-GFP or 35s:5NBmts-GFP ,35s: miR398b, 35s:miR157 ) was cultured in LB medium overnight at 28°C. The A. tumefaciens samples with different vectors were collected and mixed at the indicated optical density (O.D.) using a DU800 spectrophotometer (Beckman Coulter). The A. tumefaciens harboring different vectors were infiltrated into tobacco leaves and the transfected cells were imaged by confocal laser scanning microscopy (Olympus FV1200) 60 h after inoculation. For western blot analysis, 100 mg of infiltrated leaf tissues was grounded into fine powder in liquid nitrogen and homogenized in 400 μl of tobacco protein extraction buffer (25 mM Tris-HCl, PH=7.5; 150 mM NaCl; 1 mM EDTA; 0.5% Triton X-100; 5% glycerol; 1% protein inhibitor; 2 mM phenylmethanesulfonyl fluoride). The same amount of extracted total protein was separated on a 15% SDS-PAGE gel, and transferred to a polyvinylidene fluoride membrane (Millipore). A 10,000-fold-diluted Rabbit Anti-GFP sera (Ab290, Abcam) and Secondary-Goat Anti-Rabbit IgG H&L (HRP) (Ab205718, Abcam) were used to detect GFP accumulation and total protein was stained with Coomassie brilliant blue (R250) for a loading control.