Artificial microRNA (amiR)-mediated silencing of DsRED expression in M. truncatula roots. A) Stable dsRED-expressing M. truncatula plants (35Spro::dsRED) were used for A. rhizogenes mediated root transformation. After transformation with the amiR-dsRed construct (right), parts of the roots lost dsRED fluorescence due to amiR-mediated gene silencing. Scale bar represents 5 mm. B) Relative levels of mature amiR-dsred determined by stem loop qRT-PCR and relative transcript levels of dsred measured by qRT-PCR as determined by qRT-PCR. Values shown are mean values of 3–4 biological replicates +/− standard deviation. Asterisks indicate statistical differences (P<0.05). C) Western blot analysis confirmed reduced accumulation of dsRED proteins in roots after transformation with the amiR-dsRed construct (a). Shown are three replicates of amiR-silenced root samples and two vector controls (v). Reduced accumulation of dsRED protein after amiR-dsred infiltration were visualized using an anti-dsRED antibody. Equal sample loading is shown by anti-RUBISCO activase.