Research Article

Systemic Infection of Nicotiana benthamiana with Potato virus X Nanoparticles Presenting a Fluorescent iLOV Polypeptide Fused Directly to the Coat Protein

Figure 3

Analyses of PVX-infected N. benthamiana leaves expressing iLOV. (a–d) Leaf extracts (20 μl) were separated by SDS-PAGE and proteins were (a) stained with Coomassie Brilliant Blue, (b, c) transferred onto a nitrocellulose membrane for western blotting, or (d) detected under UV light. M: P7712 Prestained Protein Standard (NEB), 1: negative control, plant extract from noninfected N. benthamiana leaves, 2: plant extract from pPVX-iLOV-CP infected leaves, 3: plant extract from pPVX-iLOV-2A-CP infected leaves, 4: positive control, PVX particles, and 5: positive control, plant extract of iLOV expressed by TMV-derived vector under control of a subgenomic promoter-like sequence [57]. Antibody detection: (b) anti-PVX and and (c) anti-iLOV and . iLOV-CP and iLOV-2A-CP fusion proteins are highlighted with asterisks, and the free iLOV polypeptide is highlighted with an arrow. (e–h) Electron micrographs of leaf extracts expressing recombinant PVX particles displaying iLOV. (e) iLOV-CP, (f) iLOV-2A-CP, and (g) PVX particles captured with MAC58, detected with anti-PVX, and labeled with GAR 15 nm gold conjugate. Bar = (e) 200 nm, (f, g) 500 nm, and (h) 1 μm.