Figure 1.

Effects of introduction of termination codons in ORF3 on the accumulation of TAS2-derived tasiRNAs in N. benthamiana. A, Structure of pri-TAS1a and pri-TAS2. The red bars and the red arrows show ORF3s and the miR173 cleavage sites, respectively. The numbers indicate the nucleotide positions on each transcript that correspond to the initiation codons, the miR173 cleavage sites, and the termination codons, respectively. The blue bars represent putative upstream ORFs. Downstream ORFs are omitted from this diagram. B, Structure of the plasmids used for transient expression analysis in N. benthamiana. Black and white arrows show the MIR and TAS2-related genes, respectively. The mutated nucleotides are shown by red characters. The termination codons generated in the mutated constructs are shown by boxed characters. C, Accumulation of TAS2-derived tasiRNAs in Agrobacterium-infiltrated N. benthamiana leaves. Cultures of Agrobacterium tumefaciens derived from two independent colonies for each construct were infiltrated into two independent plants, and leaves were separately harvested 48 h after infiltration. After RNA extraction, total RNA (10 μg) from each plant was applied to each lane of denaturing 15% polyacrylamide gel, separated by electrophoresis, and transferred to a nylon membrane. The RNA blot was hybridized with a ³²P-labeled probe complementary to TAS2 RNA. The probe was stripped, and the same blot was reprobed with probes complementary to miR171a and miR173, respectively. D, Effect of the position of termination codons on tasiRNA production from pri-TAS2. sRNA (10 μg) was used for the analysis. The experiment was carried out as described in C. The intensity of each band was quantified and the ratio of TAS2-3′D3(+) to miR173 was normalized with the leftmost lane.