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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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Protein-protein interaction of AtDMC1G138D expressed using TMV-Gate vectors.(A) Yeast two-hybrid analysis demonstrating self-interaction of AtDMC1G138D. (B) Confirmation of AtDMC1G138D self-interaction by pull-down experiment. The FLAG-tagged AtDMC1G138D and 3xHA-tagged AtDMC1G138D immobilized on anti-FLAG and anti-HA agarose beads, respectively, were incubated with total protein extract of N. benthamiana leaf discs transiently expressing c-myc-tagged AtDMC1G138D or c-myc-tagged mGFP5 (negative control) and 3xFLAG-tagged AtDMC1G138D or 3xFLAG-tagged GusPlus (negative control), respectively; corresponding TMV-Gate vectors and recombinant plasmids are listed in Table I. After washing the affinity matrices, proteins were eluted using SDS-PAGE sample buffer, resolved by SDS-PAGE and visualized by immunoblotting with antibodies as indicated. WB, western blot.
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f5: Protein-protein interaction of AtDMC1G138D expressed using TMV-Gate vectors.(A) Yeast two-hybrid analysis demonstrating self-interaction of AtDMC1G138D. (B) Confirmation of AtDMC1G138D self-interaction by pull-down experiment. The FLAG-tagged AtDMC1G138D and 3xHA-tagged AtDMC1G138D immobilized on anti-FLAG and anti-HA agarose beads, respectively, were incubated with total protein extract of N. benthamiana leaf discs transiently expressing c-myc-tagged AtDMC1G138D or c-myc-tagged mGFP5 (negative control) and 3xFLAG-tagged AtDMC1G138D or 3xFLAG-tagged GusPlus (negative control), respectively; corresponding TMV-Gate vectors and recombinant plasmids are listed in Table I. After washing the affinity matrices, proteins were eluted using SDS-PAGE sample buffer, resolved by SDS-PAGE and visualized by immunoblotting with antibodies as indicated. WB, western blot.

Mentions: Protein-protein interactions play a central role in the maintenance and control of cellular and organismal processes. The study of protein-protein interactions has been greatly enhanced by epitope-tagging technology facilitating ‘pull-down’ experiments that can be used to detect and validate protein interactions. In order to establish the utility of epitope-tagging TMV-Gate vectors for analysing protein-protein interactions we employed AtDMC1. The yeast and human DMC1 orthologues have been shown to physically interact homotypically by yeast two-hybrid (Y2H) analysis and, in the case of human DMC1, by immunoprecipitation34. DMC1 DNA strand exchange activity in vitro is dependent upon ATP binding and hydrolysis mediated by an ATPase motif highly conserved between prokaryote and eukaryote RecA-like proteins35, including AtDMC1. Notably, mutating the ATP-binding motif in yeast DMC1 (yDMC1G126D) results in defective meiotic homologous recombination; however, homotypic interactions of yDMC1G126D have not been demonstrated because of instability of the Y2H plasmid expressing this protein34. In characterising AtDMC1 we created a variant (AtDMC1G138D) analogous to yDMC1G126D and sought to assess if it could interact homotypically. Self-interaction of AtDMC1G138D was initially analysed by Y2H assay wherein interaction of the bait and prey fusion proteins results in expression of a HIS3 reporter gene enabling growth of his3 auxotrophic yeast cells on a selective medium lacking histidine. When the AtDMC1G138D bait was expressed with the non-interacting empty prey vector no growth of yeast cells was detected in histidine-deficient plates containing 15 mM 3-amino-1,2,4-triazole (3-AT) which was added to suppress autoactivation of the reporter gene by the AtDMC1G138D bait. In contrast, co-expression of AtDMC1G138D as bait and prey enabled growth of yeast cells, indicating that AtDMC1G138D can interact homotypically (Figure 5A) and, as proposed for yDMC1G126D34, given the substitution of glycine by the relatively bulky and negatively charged aspartate likely does so independently of ATP binding.


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)

Protein-protein interaction of AtDMC1G138D expressed using TMV-Gate vectors.(A) Yeast two-hybrid analysis demonstrating self-interaction of AtDMC1G138D. (B) Confirmation of AtDMC1G138D self-interaction by pull-down experiment. The FLAG-tagged AtDMC1G138D and 3xHA-tagged AtDMC1G138D immobilized on anti-FLAG and anti-HA agarose beads, respectively, were incubated with total protein extract of N. benthamiana leaf discs transiently expressing c-myc-tagged AtDMC1G138D or c-myc-tagged mGFP5 (negative control) and 3xFLAG-tagged AtDMC1G138D or 3xFLAG-tagged GusPlus (negative control), respectively; corresponding TMV-Gate vectors and recombinant plasmids are listed in Table I. After washing the affinity matrices, proteins were eluted using SDS-PAGE sample buffer, resolved by SDS-PAGE and visualized by immunoblotting with antibodies as indicated. WB, western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Protein-protein interaction of AtDMC1G138D expressed using TMV-Gate vectors.(A) Yeast two-hybrid analysis demonstrating self-interaction of AtDMC1G138D. (B) Confirmation of AtDMC1G138D self-interaction by pull-down experiment. The FLAG-tagged AtDMC1G138D and 3xHA-tagged AtDMC1G138D immobilized on anti-FLAG and anti-HA agarose beads, respectively, were incubated with total protein extract of N. benthamiana leaf discs transiently expressing c-myc-tagged AtDMC1G138D or c-myc-tagged mGFP5 (negative control) and 3xFLAG-tagged AtDMC1G138D or 3xFLAG-tagged GusPlus (negative control), respectively; corresponding TMV-Gate vectors and recombinant plasmids are listed in Table I. After washing the affinity matrices, proteins were eluted using SDS-PAGE sample buffer, resolved by SDS-PAGE and visualized by immunoblotting with antibodies as indicated. WB, western blot.
Mentions: Protein-protein interactions play a central role in the maintenance and control of cellular and organismal processes. The study of protein-protein interactions has been greatly enhanced by epitope-tagging technology facilitating ‘pull-down’ experiments that can be used to detect and validate protein interactions. In order to establish the utility of epitope-tagging TMV-Gate vectors for analysing protein-protein interactions we employed AtDMC1. The yeast and human DMC1 orthologues have been shown to physically interact homotypically by yeast two-hybrid (Y2H) analysis and, in the case of human DMC1, by immunoprecipitation34. DMC1 DNA strand exchange activity in vitro is dependent upon ATP binding and hydrolysis mediated by an ATPase motif highly conserved between prokaryote and eukaryote RecA-like proteins35, including AtDMC1. Notably, mutating the ATP-binding motif in yeast DMC1 (yDMC1G126D) results in defective meiotic homologous recombination; however, homotypic interactions of yDMC1G126D have not been demonstrated because of instability of the Y2H plasmid expressing this protein34. In characterising AtDMC1 we created a variant (AtDMC1G138D) analogous to yDMC1G126D and sought to assess if it could interact homotypically. Self-interaction of AtDMC1G138D was initially analysed by Y2H assay wherein interaction of the bait and prey fusion proteins results in expression of a HIS3 reporter gene enabling growth of his3 auxotrophic yeast cells on a selective medium lacking histidine. When the AtDMC1G138D bait was expressed with the non-interacting empty prey vector no growth of yeast cells was detected in histidine-deficient plates containing 15 mM 3-amino-1,2,4-triazole (3-AT) which was added to suppress autoactivation of the reporter gene by the AtDMC1G138D bait. In contrast, co-expression of AtDMC1G138D as bait and prey enabled growth of yeast cells, indicating that AtDMC1G138D can interact homotypically (Figure 5A) and, as proposed for yDMC1G126D34, given the substitution of glycine by the relatively bulky and negatively charged aspartate likely does so independently of ATP binding.

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