Figure 5.

The re-initiation supporting protein (RISP)–transactivator viroplasmin (TAV) complex mediates contacts between 60S ribosomal protein L24 and eIF3. (A) Mock-inoculated (M) and cauliflower mosaic virus (CaMV)-infected (I) turnip plants were used for co-immunoprecipitation with anti-P0–P1–P2 antibodies. The panels show immunoblotting of RISP, TAV, S6 or L13 present in input, normal human serum (HS) and the entire immunoprecipitate (IP) using appropriate rabbit polyclonal antibodies. (B–D) Immunofluorescence assay showing colocalization of endogenous 60S and TAV (B) 60S and RISP in systemically CaMV-infected epidermal cells of leaves of B. rapa plants at 15 days post-inoculation (dpi; C) and 60S and RISP in mock-infected epidermal cells (D). Double-immunolabelling was carried out using anti-TAV and anti-P0–P1–P2 antibodies (anti-60S) (B) or anti-60S and anti-RISP antibodies (C, D). The nucleus was stained with DAPI (blue). The lower panels in (B) and (C) represent higher magnification images of the insets in the upper panels. In the merge, DAPI fluorescence is blue, TAV is red, 60S is green and RISP is red. Round-shaped structures are indicated by arrows. Scale bars, 5 μm. (E) GST pull-down assays. GST–L24 attached to glutathione Sepharose 4B beads (lane 7) was mixed either with RISP (lane 8), or RISP and eIF3 (lanes 9 and 10), or RISP and TAV (lanes 11–12), or RISP, TAV and eIF3 (lanes 13–14); purified TAV, RISP and eIF3 are shown in lanes 1, 2 and 3, respectively. Unbound (U) and bound (B) fractions were analyzed by SDS–PAGE followed by Coomassie blue staining.