Hyperexpression of an endoplasmic reticulum-targeted elastin-like polypeptide fusion induces the formation of protein bodies in leaves. (A) Accumulation of endoplasmic reticulum- (ER-)targeted green fluorescent protein (GFP), with or without an elastin-like polypeptide (ELP) tag, when transiently co-expressed with the p19 suppressor of gene silencing in the leaves (n = 8) of Nicotiana benthamiana plants. ***, significant difference (P < 0.001). (B) Confocal image of the ER-targeted GFP control protein (pPGK) demonstrating the open polygonal network consistent with ER-localization. (C-H) In the presence of an ELP fusion tag, the ER-targeted GFP (pPGEK) was detected in brightly fluorescing spherical-shaped particles distributed throughout the cells of the leaf. (C) The novel PBs were closely associated with the ER tubules as small punctuate structures early on in the PB-formation process. (D) With time, the PB-like organelles continued to grow and appeared to be released from the ER into the cytoplasm, where they remained. (E-H) The PBs obtained various sizes and tended to cluster together within the cell, although the distribution pattern was quite variable. The majority of PBs had an observable diameter of between 0.5 and 1.0 μm, but larger PBs were seen at lower frequencies with some approaching diameters of 8.0 μm. (I) Deglycosylation of an ER-targeted GFP-ELP fusion engineered to contain an N-glycosylation motif (GELVSNGTVT). Total protein extracts (3 μg/lane) from agro-infiltrated plant tissue expressing pPNGEK were incubated for 24 h in the presence (+) or absence (-) of peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) and then subjected to sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotted with an anti-GFP antibody. Bar, 10 μm (B-F); 5 μm (G, H).