Using GFP to study virus invasion and spread in plant tissues

Oparka, Karl J.; Roberts, Alison G.; Cruz, Simon Santa; Boevink, Petra C.; Prior, D. A. M.; Smallcombe, Anna

doi:10.1038/41145

Cited by 53 publications

(32 citation statements)

References 3 publications

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“…Plant virus-based vectors have been used extensively to express free GFP to localize viruses to specific cells and tissues (Oparka et al, 1997) and to express GFP fusion proteins to determine the subcellular localization of both plant and viral proteins (Oparka et al, 1997;Boevink et al, 1998;Gillespie et al, 2002). We wished to determine whether such vectors could be used in a high-throughput manner, in preference to stable transformation, to determine the subcellular localization of proteins expressed from random cDNA-gfp fusions.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Viral Expression of cDNA–Green Fluorescent Protein Fusions Reveals Novel Subcellular Addresses and Identifies Unique Proteins That Interact with Plasmodesmata

Escobar¹,

Haupt²,

Thow³

et al. 2003

Plant Cell

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196

102

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A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana . Libraries of random, partial cDNAs fused to the 5 Ј or 3 Ј end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus . Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5 Ј end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of Ͼ 20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles.

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Section: Introductionmentioning

confidence: 99%

High-Throughput Viral Expression of cDNA–Green Fluorescent Protein Fusions Reveals Novel Subcellular Addresses and Identifies Unique Proteins That Interact with Plasmodesmata

Escobar¹,

Haupt²,

Thow³

et al. 2003

Plant Cell

Self Cite

196

102

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“…Researchers have been fascinated by the use of GFP genes as markers in many hosts (Oparka et al, 1997;Harper et al, 1999;Harper and Stewart, 2000;Hudson et al, 2001). Various transgenic GFP genes have been developed (Halfhill et al, 2003), and one of them actually was used as a marker gene to detect GM plants in field experiments .…”

Section: Discussionmentioning

confidence: 99%

Development of Visible Markers for Transgenic Plants and their Availability for Environmental Risk Assessment

Tamaoki

Imai²,

Takahashi³

et al. 2006

Zeitschrift Für Naturforschung C

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Monitoring of transgenic plants in the field is important, but risk assessment has entailed laborious use of invisible marker genes. Here, we assessed three easily visible marker transgenes Ð green fluorescent protein (GFP), R, and Nicotiana tabacum homeobox (NTH) 15 genes Ð for their potential use as marker genes for monitoring genetically modified plants. Transgenic Arabidopsis thaliana plants for each of these genes were visibly distinguished from wild-type plants. We determined the germination rate, 3-week fresh weight, time to first flowering, and seed weight of the transgenic plants to evaluate whether the expression of these marker genes affected the growth of the host. Introduction of GFP gene had no effect on the evaluated parameters, and we then used the GFP gene as a marker to assess the outcrossing frequency between transgenic and two Arabidopsis species. Our results showed that the hybridization frequency between transgenic plants and Arabidopsis thaliana was 0.24%, and between transformants and Arabidopsis lyrata it was 2.6% under experimental condition. Out-crossing frequency was decreased by extending the distance between two kinds of plants. Thus, the GFP gene is a useful marker for assessing the whereabouts of transgenes/transformants in the field. We also demonstrated that the GFP gene is possibly applicable as a selection marker in the process of generation of transgenic plants.

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“…Green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an invaluable tool for monitoring gene expression and fusion protein localization in vivo or in situ, as well as for determination of cellular morphological changes (Chalfie et al, 1994;Hong et al, 1999b;Maniak et al, 1995;Oparka et al, 1997). A variant type of GFP (EGFP), a marker for visualization of apoptotic cell morphological changes (Hong et al, 1999b), was used in the present study to trace VP3 localization and monitor interaction with other proteins.…”

Section: Vp3 Localizationmentioning

confidence: 99%

Identification of Aquatic Birnavirus VP3 Death Domain and Its Dynamic Interaction Profiles in Early and Middle Replication Stages in Fish Cells

Hong¹,

Wu²

2012

Viral Genomes - Molecular Structure, Diversity, Gene Expression Mechanisms and Host-Virus Interactions

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Using GFP to study virus invasion and spread in plant tissues

Cited by 53 publications

References 3 publications

High-Throughput Viral Expression of cDNA–Green Fluorescent Protein Fusions Reveals Novel Subcellular Addresses and Identifies Unique Proteins That Interact with Plasmodesmata

High-Throughput Viral Expression of cDNA–Green Fluorescent Protein Fusions Reveals Novel Subcellular Addresses and Identifies Unique Proteins That Interact with Plasmodesmata

Development of Visible Markers for Transgenic Plants and their Availability for Environmental Risk Assessment

Identification of Aquatic Birnavirus VP3 Death Domain and Its Dynamic Interaction Profiles in Early and Middle Replication Stages in Fish Cells

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