The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection.
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.
Chapman
Sean
S
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.
Faulkner
Christine
C
Kaiserli
Eirini
E
Garcia-Mata
Carlos
C
Savenkov
Eugene I
EI
Roberts
Alison G
AG
Oparka
Karl J
KJ
Christie
John M
JM
D000818:Animals; D056931:Cryptochromes; D019020:Directed Molecular Evolution; D005415:Flavins / Q000737:chemistry; D005420:Flavoproteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D005453:Fluorescence; D017930:Genes, Reporter; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000378:metabolism; D038761:Photobleaching; D010942:Plant Viruses / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010944:Plants / Q000821:virology*; D011993:Recombinant Fusion Proteins; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects
D056931:Cryptochromes; D005415:Flavins; D005420:Flavoproteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D010100:Oxygen