Multifunctional roles of γb in accumulation of BSMV RNAs.
Panel A: RNA silencing suppressor activities of different γb mutants. Circles in the photos indicate regions of N. benthamiana leaves infiltrated with A. tumefaciens harboring plasmids expressing γb derivatives and GFP. Regions infiltrated with wild-type γb or the empty pGD vector (EV) served as positive or negative controls. Leaves were observed at 3 dpi under UV illumination. Panels B and C: Northern blot analyses of BSMV RNAs isolated from infiltrated tissue. N. benthamiana leaf tissues were agroinfiltrated with pCaBS-α and/or various pCaBS-γ derivatives shown in S2C Fig. Combinations of vector constructs used for Northern blot assays are indicated at the top of each lane. At 3 dpi, total RNA was isolated from the inoculated leaves and subjected to Northern blot analysis. Note: The left and middle series (I and II) of images in Panels B and C depict plus-strand RNAs and images on the right side (III) show minus-strand RNAs. BSMV plus-strand (+) RNAs were detected with the 3′-UTR probe, and plus-strand (+) RNAα was visualized with the plus-strand RNAα specific probe. Minus-strand (-) RNAα was detected with the minus-strand RNAα probe. Bands corresponding to RNAα, RNAγ and subgenomic (sg) RNAγ are indicated along the right sides of the northern blots. Note: Due to reasons that have yet to be identified, RNAγγbATGm expressing tissues consistently accumulated lower levels of subgenomic RNA (γsg) when co-inoculated with RNAα than tissue RNAα + RNAγΔγb. Methylene blue stained ribosomal RNAs were used to show equal loading. Intensities of the RNAα-specific bands were measured, normalized and analyzed statistically using procedures described in the Materials and Methods. Quantifications of RNAα accumulation from different lanes in panels B and C are shown in the bar graphs. The graphic data represent the means of three independent experiments. Error bars indicate standard error (n = 3). In each bar chart, different letters above bars denote statistically significant differences according to the Duncan's multiple range test (P < 0.05). In Panel C, lane 6 shows analysis of BSMV RNA accumulation in leaves expressing the γb1-85 protein in which the αa-interacting regions had been deleted, and lane 7 shows RNA accumulation in tissue expressing γb1-85m protein in which the RNA binding activities have been eliminated.