Abstract

Fluorescent proteins (FPs) based on green fluorescent protein (GFP) are widely used throughout cell biology to study protein dynamics, and have extensive use as reporters of virus infection and spread. However, FP-tagging of viruses is limited by the constraints of viral genome size resulting in FP loss through recombination events. To overcome this, we have engineered a smaller (approximate to 10 kDa) flavin-based alternative to GFP (approximate to 25 kDa) derived from the light, oxygen or voltage-sensing (LOV) domain of the plant blue light receptor, phototropin. Molecular evolution and Tobacco mosaic virus (TMV)based expression screening produced LOV variants with improved fluorescence and photostability in planta. One variant in particular, designated iLOV, possessed photophysical properties that made it ideally suited as a reporter of subcellular protein localization in both plant and mammalian cells. Moreover, iLOV fluorescence was found to recover spontaneously after photobleaching and displayed an intrinsic photochemistry conferring advantages over GFP-based FPs. When expressed either as a cytosolic protein or as a viral protein fusion, iLOV functioned as a superior reporter to GFP for monitoring local and systemic infections of plant RNA viruses. iLOV, therefore, offers greater utility in FP-tagging of viral gene products and represents a viable alternative where functional protein expression is limited by steric constraints or genome size.

Published in

Proceedings of the National Academy of Sciences
2008, Volume: 105, number: 50, pages: 20038-20043

SLU Authors

• Savenkov, Eugene

  • Department of Plant Biology, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Food Science
Forest Science
Renewable Bioenergy Research


Publication identifier

DOI: https://doi.org/10.1073/pnas.0807551105

Permanent link to this page (URI)

https://res.slu.se/id/publ/20416