Figure 7. Detection of HA-epitope-tagged RCY1 protein and RCY1 transcript in N. benthamiana leaves transiently expressingRCY1-HA constructs in which the RCY1 introns were replaced with COR15a or PRF3 introns.

RCY1 transcripts in N. benthamiana leaves agro-infiltrated with P_35S-gRCY1-HA, P_35S-gRCY1.ΔII-HA, P_35S-cRCY1.Ic-HA, P_35S-cRCY1.Ip-HA, or P_35S-cRCY1-HA were detected by northern hybridization. pRI201-AN (Vector) was used as an empty-vector control for agro-infiltration. As an internal control for RNA sample quantities, 18S rRNA is shown (A). Relative amounts of RCY1 transcripts in each line were measured by quantitative RT-PCR (B). HA-epitope-tagged RCY1 protein (α-HA) in N. benthamiana leaves transiently expressing P_35S-gRCY1-HA,P_35S-gRCY1.ΔII-HA, P_35S-cRCY1.Ic-HA, P_35S-cRCY1.Ip-HA, or P_35S-cRCY1-HA was immunologically detected using anti-HA monoclonal antibody. As an internal control for protein sample quantities, the large subunit of RuBisCO was visualized by staining with CBB (C). RCY1-HA protein amounts in each line were quantified by band intensity using Quantity One software (D). For all experiments, four independent plants transiently expressing each vector construct were analyzed. The averages of relative amounts of RCY1 transcript and protein ±SE are shown in B and D, respectively. In A and C, representative photographs are shown. The size of each band and the position of 18S rRNA were shown at right side of the panels. Data were subjected to analysis of variance and treatment means were compared by Tukey's test. Different letters indicate a statistically significant difference in the relative amount of RCY1 transcript (n  =  4, P<0.05).