Figure 9. Gene silencing of NbGAPDH-A inhibits the localization of RCNMV MP to the cortical VRCs.

(A) Representative CLSM images of ALSV- or ALSV/gsGAP-infected N. benthamiana mesophyll (upper panels) and epidermal cells (lower panels) infiltrated with Agrobacterium cultures that contained pBICR1/MsG2fsMP, which expressed recombinant RCNMV RNAs encoding MP-GFP (Figure S1K). The images were obtained at 38 h after infiltration. Scale bar = 30 µm. The images include the merged DIC and GFP channels, and they represent confocal projections of 20 optical sections at 1 µm intervals, ranging from the surface to the middle of the cells. (B) The leaf samples in (A) were subjected to epifluorescence microscopy and the percentage of fluorescent cells containing cortical punctates was determined. The data shown are the totals from three replicate assays. (C) Protoplasts prepared from ALSV- or ALSV/gsGAP-infected N. benthamiana plants were inoculated with a mixture of in vitro transcripts of pUCR1-MsG and pRNA2fsMP (Figure S1L) [10]. Representative CLSM images of the protoplasts at 12 hpi. Scale bar = 30 µm. The images include the merged DIC and GFP channels (panels 1 and 2), or GFP channel (panel 3). They represent confocal projections of 15 optical sections at 2 µm intervals, ranging from the surface to the center of the cells. (D) The protoplast samples in (C) were subjected to Western blotting. Proteins extracted from 2×10⁴ protoplasts were loaded in each lane. MP-GFP was detected using a rabbit polyclonal antibody against GFP. The numbers below the image represent the relative accumulation levels (means ± SE) of the proteins obtained using the Image Gauge program (Fuji Film), which were calculated from two independent experiments. Asterisk indicates a not significant (P>0.05; Student's t-test) difference compared with the accumulation of proteins in the protoplasts from ALSV-infected N. benthamiana. RbcL is a Coomassie brilliant blue-stained gel image, which shows the large subunit of Rubisco proteins.