Subcellular localization of the endoplasmic reticulum-targeted green fluorescent protein-elastin-like polypeptide fusion protein (pPGEK) in Nicotiana benthamiana leaves. (A) Electron microscopy confirmed the location of numerous newly-formed endoplasmic reticulum- (ER-)derived protein bodies (PBs) (examples indicated by asterisks) in the cytoplasm of the leaf cells. (B-D) Progressively higher magnifications of the PBs seen in (A). (B) The novel PBs occupied the cytosolic space between the tonoplast (indicated by an arrowhead) and the plasma membrane (indicated by an arrow). (C) The PBs were clearly surrounded by a membrane that appears to no longer be contiguous with the ER. (D) The PB membrane was decorated with ribosomes (indicated with arrows), strongly suggesting that the PBs were originally derived from the rough ER. (E, F) Immunogold localization confirmed the presence of green fluorescent protein-elastin-like polypeptide (GFP-ELP) inside the novel cytoplasmic PBs in ultrathin sections of N. benthamiana leaves using anti-ELP (E) and anti-GFP (F) antibodies detected with goat anti-rabbit or anti-mouse IgG conjugated to 15 nm gold particles. No significant immunolabeling was observed in other subcellular compartments or wild-type plants. The different subcellular compartments were labeled: Cp, chloroplast; CW, cell wall; Mt, mitochondria; LB, lipid body; LV, lytic vacuole; *, induced protein body. Bar, 2 μm (A); 500 nm (B, C, E, F); 100 nm (D).