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TMV-Gate vectors: gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins.

Kagale S, Uzuhashi S, Wigness M, Bender T, Yang W, Borhan MH, Rozwadowski K - Sci Rep (2012)

Bottom Line: Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins.We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors.Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts.

View Article: PubMed Central - PubMed

Affiliation: Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan, Canada.

ABSTRACT
Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts.

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Related in: MedlinePlus

Nuclear localizsation of NLS-mCherry expressed using TMV-Gate vectors.(A) Schematic diagram showing constructs used for analysis of subcellular localisation of NLS-mCherry. mCherry carrying a nuclear localization signal (NLS) was introduced unmodified into pMW388. A NLS-mCherry variant without translational stop codon was linked in-frame with the coding sequence of YFP-3xFLAG or CFP-3xHA in pMW390 and pMW391, respectively. Annotated features as per Figure 1. (B) Epifluorescent microscopy images showing nuclear localisation of NLS-mCherry (upper panel), NLS-mCherry linked to YFP-3xFLAG (middle panel) or NLS-mCherry linked to CFP-3xHA in cells of N. benthamiana leaves infiltrated with A. tumefaciens carrying the constructs described in (A) and imaged 3 DPI. The nucleus was detected by staining cells with DAPI. Images were taken using YFP, CFP, mCherry or DAPI filter sets as indicated. Scale bar, 20 µM. DIC, differential interference contrast.
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f6: Nuclear localizsation of NLS-mCherry expressed using TMV-Gate vectors.(A) Schematic diagram showing constructs used for analysis of subcellular localisation of NLS-mCherry. mCherry carrying a nuclear localization signal (NLS) was introduced unmodified into pMW388. A NLS-mCherry variant without translational stop codon was linked in-frame with the coding sequence of YFP-3xFLAG or CFP-3xHA in pMW390 and pMW391, respectively. Annotated features as per Figure 1. (B) Epifluorescent microscopy images showing nuclear localisation of NLS-mCherry (upper panel), NLS-mCherry linked to YFP-3xFLAG (middle panel) or NLS-mCherry linked to CFP-3xHA in cells of N. benthamiana leaves infiltrated with A. tumefaciens carrying the constructs described in (A) and imaged 3 DPI. The nucleus was detected by staining cells with DAPI. Images were taken using YFP, CFP, mCherry or DAPI filter sets as indicated. Scale bar, 20 µM. DIC, differential interference contrast.

Mentions: In recent years, translational fusion of candidate proteins to a fluorescent protein combined with microscopy has become a powerful tool for defining protein function through determining its subcellular localisation. TMV-Gate vectors designed to express C-terminal YFP or CFP fusion proteins include an in-frame 3xFLAG or 3xHA epitope tag, respectively (Figure 1), and thus enable simultaneous analyses, including (i) in planta evaluation of subcellular localisation of fluorescent protein fusions and (ii) immunodetection, affinity purification and analysis of protein-protein interaction by virtue of the epitope tags attached at the C-terminal ends of the fusion proteins. To test the suitability of these vectors for studying subcellular localisation of proteins, the mCherry red fluorescent protein linked to a nuclear localisation signal derived from the SV40 large T-antigen (NLS-mCherry) was recombined with pMW388; additionally, a variant lacking the stop codon (NLS-mCherryΔSTOP) was recombined with the TMV-Gate vectors pMW390 or pMW391 to generate in-frame fusions with YFP-3xFLAG or CFP-3xHA, respectively (Figure 6A). The resultant constructs, designated as pMW412, pSK215 and pSK216, respectively (Table I), were agroinfiltrated into N. benthamiana leaves and subcellular localisation of the fusion proteins assessed by fluorescent microscopy. The subcellular fluorescence signal emitted by NLS-mCherry overlapped with the DAPI (4′,6-diamidino-2-phenylindole; a nuclear stain) signal (Figure 6B), demonstrating that NLS-mCherry localizes to the nucleus and can be utilized as a positive control in nuclear localisation experiments. As expected, both NLS-mCherry-YFP-3xFLAG and NLS-mCherry-CFP-3xHA fusion proteins localized to the nucleus, as deduced by comparing the fluorescence signal with the DAPI signal (Figure 6B). These results demonstrate pMW390 and pMW391 TMV-Gate vectors can be utilized for detection of subcellular localisation of proteins in planta.


TMV-Gate vectors: gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins.

Kagale S, Uzuhashi S, Wigness M, Bender T, Yang W, Borhan MH, Rozwadowski K - Sci Rep (2012)

Nuclear localizsation of NLS-mCherry expressed using TMV-Gate vectors.(A) Schematic diagram showing constructs used for analysis of subcellular localisation of NLS-mCherry. mCherry carrying a nuclear localization signal (NLS) was introduced unmodified into pMW388. A NLS-mCherry variant without translational stop codon was linked in-frame with the coding sequence of YFP-3xFLAG or CFP-3xHA in pMW390 and pMW391, respectively. Annotated features as per Figure 1. (B) Epifluorescent microscopy images showing nuclear localisation of NLS-mCherry (upper panel), NLS-mCherry linked to YFP-3xFLAG (middle panel) or NLS-mCherry linked to CFP-3xHA in cells of N. benthamiana leaves infiltrated with A. tumefaciens carrying the constructs described in (A) and imaged 3 DPI. The nucleus was detected by staining cells with DAPI. Images were taken using YFP, CFP, mCherry or DAPI filter sets as indicated. Scale bar, 20 µM. DIC, differential interference contrast.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3500846&req=5

f6: Nuclear localizsation of NLS-mCherry expressed using TMV-Gate vectors.(A) Schematic diagram showing constructs used for analysis of subcellular localisation of NLS-mCherry. mCherry carrying a nuclear localization signal (NLS) was introduced unmodified into pMW388. A NLS-mCherry variant without translational stop codon was linked in-frame with the coding sequence of YFP-3xFLAG or CFP-3xHA in pMW390 and pMW391, respectively. Annotated features as per Figure 1. (B) Epifluorescent microscopy images showing nuclear localisation of NLS-mCherry (upper panel), NLS-mCherry linked to YFP-3xFLAG (middle panel) or NLS-mCherry linked to CFP-3xHA in cells of N. benthamiana leaves infiltrated with A. tumefaciens carrying the constructs described in (A) and imaged 3 DPI. The nucleus was detected by staining cells with DAPI. Images were taken using YFP, CFP, mCherry or DAPI filter sets as indicated. Scale bar, 20 µM. DIC, differential interference contrast.
Mentions: In recent years, translational fusion of candidate proteins to a fluorescent protein combined with microscopy has become a powerful tool for defining protein function through determining its subcellular localisation. TMV-Gate vectors designed to express C-terminal YFP or CFP fusion proteins include an in-frame 3xFLAG or 3xHA epitope tag, respectively (Figure 1), and thus enable simultaneous analyses, including (i) in planta evaluation of subcellular localisation of fluorescent protein fusions and (ii) immunodetection, affinity purification and analysis of protein-protein interaction by virtue of the epitope tags attached at the C-terminal ends of the fusion proteins. To test the suitability of these vectors for studying subcellular localisation of proteins, the mCherry red fluorescent protein linked to a nuclear localisation signal derived from the SV40 large T-antigen (NLS-mCherry) was recombined with pMW388; additionally, a variant lacking the stop codon (NLS-mCherryΔSTOP) was recombined with the TMV-Gate vectors pMW390 or pMW391 to generate in-frame fusions with YFP-3xFLAG or CFP-3xHA, respectively (Figure 6A). The resultant constructs, designated as pMW412, pSK215 and pSK216, respectively (Table I), were agroinfiltrated into N. benthamiana leaves and subcellular localisation of the fusion proteins assessed by fluorescent microscopy. The subcellular fluorescence signal emitted by NLS-mCherry overlapped with the DAPI (4′,6-diamidino-2-phenylindole; a nuclear stain) signal (Figure 6B), demonstrating that NLS-mCherry localizes to the nucleus and can be utilized as a positive control in nuclear localisation experiments. As expected, both NLS-mCherry-YFP-3xFLAG and NLS-mCherry-CFP-3xHA fusion proteins localized to the nucleus, as deduced by comparing the fluorescence signal with the DAPI signal (Figure 6B). These results demonstrate pMW390 and pMW391 TMV-Gate vectors can be utilized for detection of subcellular localisation of proteins in planta.

Bottom Line: Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins.We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors.Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts.

View Article: PubMed Central - PubMed

Affiliation: Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan, Canada.

ABSTRACT
Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts.

Show MeSH
Related in: MedlinePlus