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

Plant virus-based nanoparticles can be produced in plants on a large scale and are easily modified to introduce new functions, making them suitable for applications such as vaccination and drug delivery, tissue engineering, and in vivo imaging. The latter is often achieved using green fluorescent protein and its derivatives, but the monovalent fluorescent protein iLOV is smaller and more robust. Here, we fused the iLOV polypeptide to the N-terminus of the Potato virus X (PVX) coat protein, directly or via the Foot-and-mouth disease virus 2A sequence, for expression in Nicotiana benthamiana. Direct fusion of the iLOV polypeptide did not prevent the assembly or systemic spread of the virus and we verified the presence of fusion proteins and iLOV hybrid virus particles in leaf extracts. Compared to wild-type PVX virions, the PVX particles displaying the iLOV peptide showed an atypical, intertwined morphology. Our results confirm that a direct fusion of the iLOV fluorescent protein to filamentous PVX nanoparticles offers a promising tool for imaging applications.