Figure 4.

Validation of the predicted vd-sRNA:target mRNA complex formation by an artificial microRNA. (A) Duplexes predicted to be formed by complexes of amiRNAs and GFP reporter constructs containing the Chloride Channel CLC-b-like and the RPS3a-like mRNA target sequences. (B) N. benthamiana leaves were agro-infiltrated with (1) empty pBIN61 vector (EV) plus GFP:CLCb; (2) amiR:(+)191 plus GFP:CLCb; (3) EV plus GFP:RPS3a; and, (4) amiR:(−)69 plus GFP:RPS3a. At 3-dpi, the leaves were photographed under UV illumination. N. benthamiana leaves were agroinfiltrated in the same combinations as in (B). (C) At 3-dpi, total RNA extracts were subjected to RNA gel blot analyses with either GFP (top panel) or 7SL (lower panel) radiolabeled probes. Full size gel blots are presented in Fig. S2. The signals in (C) were quantified and expressed as a ratio of the GFP to the 7SL signals. For each set of experiments, the ratio of GFP to 7SL obtained with EV plus GFP:XX (control) was set at a value of 1. The additional bars indicate the relative GFP/7SL ratio for each amiRNA (as indicated) expressed with its respective GFP:XX. (D) At 3 dpi, total protein extracts were subjected to immunoblotting with anti- GFP (top panel) and anti-PEPC (lower panel) antibodies. Full size immunoblots are presented in Fig. S3. The immunoblot signals from (D) were quantified and expressed as a ratio of the GFP to the PEPC signals. For each set of experiments, the ratio of GFP to PEPC obtained with EV plus GFP:C11-vdXX (control) was set at a value of 1. The additional bars indicate the relative GFP/PEPC ratio for each amiRNA (as indicated) expressed with its respective GFP target. Each experiment was performed at least three times. Error bars indicate SD. The asterisks indicate statistically significant for paired t-test (P <  0.05).