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The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2008 Dec 16; Vol. 105 (50), pp. 20038-43. Date of Electronic Publication: 2008 Dec 05. - Publication Year :
- 2008
-
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.
- Subjects :
- Animals
Cryptochromes
Directed Molecular Evolution
Flavins chemistry
Flavoproteins genetics
Flavoproteins metabolism
Flavoproteins radiation effects
Fluorescence
Genes, Reporter
Genetic Engineering
Green Fluorescent Proteins analysis
Green Fluorescent Proteins genetics
Green Fluorescent Proteins metabolism
Green Fluorescent Proteins radiation effects
Luminescent Proteins genetics
Luminescent Proteins metabolism
Luminescent Proteins radiation effects
Microscopy, Confocal
Microscopy, Fluorescence
Oxygen metabolism
Photobleaching
Plant Viruses genetics
Plant Viruses metabolism
Recombinant Fusion Proteins
Tobacco Mosaic Virus genetics
Tobacco Mosaic Virus physiology
Viral Proteins genetics
Viral Proteins metabolism
Viral Proteins radiation effects
Flavoproteins analysis
Luminescent Proteins analysis
Plant Viruses physiology
Plants virology
Viral Proteins analysis
Subjects
Details
- Language :
- English
- ISSN :
- 1091-6490
- Volume :
- 105
- Issue :
- 50
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 19060199
- Full Text :
- https://doi.org/10.1073/pnas.0807551105