FIGURE 4.

Validating the action of a slmiRNA on FRG3 by transient co-expression in Nicotiana benthamiana leaves. (A) Quantification of FRG3 transcript levels in total RNA isolated from N. benthamiana leaves using qRT-PCR. qRT-PCR was performed as described in the Section “Materials and Methods” using FRG3 primers. Values were normalized to N. benthamiana actin. ^∗∗∗ indicate significant differences when compared to the corresponding control plants in the same treatments at p < 0.001. (B) Determination of FRG3 protein levels. Crude total protein (extracts isolated from N. benthamiana leaves infiltrated with different constructs were separated on SDS-PAGE gels and then used to prepare Western blots. Blots were reacted with a FLAG antiserum. A duplicate gel stained with Coomassie was used as loading control. Similar results were observed for three biological replicates. (C) Identification of the slmiRNA cleavage site on the target gene mRNA using 5′RACE. Total RNA samples were obtained as described above and subjected to 5′ RACE as described in the Section “Materials and Methods.” The arrows denote the detected cleavage sites, while the ratios indicate the fraction of events detected (out of 20 clones analyzed).)