The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection

Sean Chapman, Christine Faulkner, Eirini Kaiserli, Carlos Garcia-Mata, Eugene I Savenkov, Alison G Roberts, Karl J Oparka, John M Christie

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    164 Citations (Scopus)

    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 ( approximately 10 kDa) flavin-based alternative to GFP ( approximately 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.
    Original languageEnglish
    Pages (from-to)20038-20043
    Number of pages6
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume105
    Issue number50
    DOIs
    Publication statusPublished - 2008

    Keywords

    • Animals
    • Cryptochromes
    • Directed Molecular Evolution
    • Flavins
    • Flavoproteins
    • Fluorescence
    • Genes, Reporter
    • Genetic Engineering
    • Green Fluorescent Proteins
    • Luminescent Proteins
    • Microscopy, Confocal
    • Microscopy, Fluorescence
    • Oxygen
    • Photobleaching
    • Plant Viruses
    • Plants
    • Recombinant Fusion Proteins
    • Tobacco Mosaic Virus
    • Viral Proteins

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  • Cite this

    Chapman, S., Faulkner, C., Kaiserli, E., Garcia-Mata, C., Savenkov, E. I., Roberts, A. G., Oparka, K. J., & Christie, J. M. (2008). The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. Proceedings of the National Academy of Sciences of the United States of America, 105(50), 20038-20043. https://doi.org/10.1073/pnas.0807551105