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

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.

SUBMITTER: Chapman S

PROVIDER: S-EPMC2604982 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe mBFP is an improved variant of NADPH-dependent blue fluorescent protein that was originally identified from the non-bioluminescent pathogenic bacteria Vibrio vulnificus CKM-1. To explore the application of mBFP in plants, the mBFP gene expression was driven by one of the three promoters, namely, leaf-specific (RbcS), hypoxia-inducible (Adh) or auxin-inducible (DR5) promoters, in different plant tissues such as leaves, roots and flowers under diverse treatments. In addition, the expressed mBFP protein was targeted to five subcellular compartments such as cytosol, endoplasmic reticulum, apoplast, chloroplast and mitochondria, respectively, in plant cells.ResultsWhen the mBFP was transiently expressed in the tobacco leaves and floral tissues of moth orchid, the cytosol and apoplast exhibited brighter blue fluorescence than other compartments. The recombinant mBFP-mS1C fusion protein exhibited enhanced fluorescence intensity that was correlated with more abundant RNA transcripts (1.8 fold) as compared with a control. In the root tips of horizontally grown transgenic Arabidopsis, mBFP could be induced as a reporter under hypoxia condition. Furthermore, the mBFP was localized to the expected subcellular compartments, except that dual targeting was found when the mBFP was fused with the mitochondria-targeting signal peptide. Additionally, the brightness of mBFP blue fluorescence was correlated with NADPH concentration.ConclusionThe NADPH-dependent blue fluorescent protein could serve as a useful reporter in plants under aerobic or hypoxic condition. However, to avoid masking the mitochondrial targeting signal, fusing mBFP as a fusion tag in the C-terminal will be better when the mBFP is applied in mitochondria trafficking study. Furthermore, mBFP might have the potential to be further adopted as a NADPH biosensor in plant cells. Future codon optimization of mBFP for plants could significantly enhance its brightness and expand its potential applications.

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

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