A versatile vector system for multiple gene expression in plants

Chung, Sang-Min; Frankman, Ellen L.; Tzfira, Tzvi

doi:10.1016/j.tplants.2005.06.001

Cited by 117 publications

(125 citation statements)

References 8 publications

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“…Generation of Arabidopsis transgenic plants expressing VBF in the antisense orientation was described previously (25). In parallel experiments, transgenic plants expressing VBF in the sense orientation were produced by cloning the VBF coding sequence into the EcoRI-NdeI sites of pBluescript II (Stratagene), removing it as an EcoRI-SalI fragment and inserting it into the EcoRI-SalI sites of pSAT4-35SP-MCS-35ST (44). The subsequent experimental steps were as described for production of the antisense plants (25).…”

Section: Discussionmentioning

confidence: 99%

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF ^VBF ubiquitin–ligase complex pathway

Zaltsman

Gafni

Citovsky

2012

Proc. Natl. Acad. Sci. U.S.A.

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One the most intriguing, yet least studied, aspects of the bacterium–host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium . During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA—as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins—into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity.

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Section: Discussionmentioning

confidence: 99%

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF ^VBF ubiquitin–ligase complex pathway

Zaltsman

Gafni

Citovsky

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…8A for combinations) were constructed using the pSAT and pRCS2 vectors available from Chung et al (22). Specifically, the ss-RFP-HDEL ORF (encoding the red fluorescent protein (RFP) fused to the N-terminal Arabidopsis chitinase signal sequence and the C-terminal HDEL ER retrieval signal (23)) was amplified from pRTL2-ss-RFP-HDEL using the sticky-end PCR technique (24).…”

Section: Methodsmentioning

confidence: 99%

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

O'Quin

Bourassa

Zhang

et al. 2010

Journal of Biological Chemistry

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Changes in ambient temperature represent a major physiological challenge to membranes of poikilothermic organisms. In plants, the endoplasmic reticulum (ER)-localized omega-3 fatty-acid desaturases (Fad3) increase the production of polyunsaturated fatty acids at cooler temperatures, but the FAD3 genes themselves are typically not up-regulated during this adaptive response. Here, we expressed two closely related plant FAD3 genes in yeast cells and found that their enzymes produced significantly different amounts of omega-3 fatty acids and that these differences correlated to differences in rates of protein turnover. Domain-swapping and mutagenesis experiments revealed that each protein contained a degradation signal in its N terminus and that the charge density of a PEST-like sequence within this region was largely responsible for the differences in rates of protein turnover. The half-life of each Fad3 protein was increased at cooler temperatures, and protein degradation required specific components of the ER-associated degradation pathway including the Cdc48 adaptor proteins Doa1, Shp1, and Ufd2. Expression of the Fad3 proteins in tobacco cells incubated with the proteasomal inhibitor MG132 further confirmed that they were degraded via the proteasomal pathway in plants. Collectively, these findings indicate that Fad3 protein abundance is regulated by a combination of cis-acting degradation signals and the ubiquitin-proteasome pathway and that modulation of Fad3 protein amounts in response to temperature may represent one mechanism of homeoviscous adaptation in plants.

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“…The pSAT family of plasmids is composed of plasmids that have been designed to facilitate (1) the overexpression of target genes under the control of various promoters and terminators (Chung et al, 2005;Tzfira et al, 2005); (2) the analysis of protein-protein interactions by using the bimolecular fluorescence complementation and multicolor bimolecular fluorescence complementation assays (Citovsky et al, 2006;Lee et al, 2008); (3) the fusion of target genes to various autofluorescence proteins ; (4) RNA interferencemediated down-regulation ; (5) the expression of ZFNs (Tovkach et al, 2009; (6) Gateway-mediated gene cloning Chakrabarty et al, 2007); and (7) the expression of epitope-tagged proteins (T. Tzfira, unpublished data). Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs.…”

Section: Discussionmentioning

confidence: 99%

“…We followed the basic design of the pAUX (Goderis et al, 2002) and its successor pSAT (Chung et al, 2005;Tzfira et al, 2005; family of plasmids to facilitate the assembly of multiple genes into a single binary plasmid. In the pSAT system, functional plant expression cassettes are individually cloned into different pSAT plasmids (e.g.…”

Section: Design Of the Binary Vector And Novel Psatsmentioning

confidence: 99%

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors

Zeevi¹,

Liang²,

Arieli³

et al. 2011

Plant Physiol.

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Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems. A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells. The majority of these systems, however, are limited to the cloning and transfer of just a single gene of interest. Thus, plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants. Here, we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc finger nucleases (ZFNs) and homing endonucleases. Our system is composed of a modified binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors. By combining the use of designed ZFNs and commercial restriction enzymes, multiple plant expression cassettes were sequentially cloned into the acceptor binary vector. Using this system, we produced binary vectors that carried up to nine genes. Arabidopsis (Arabidopsis thaliana) protoplasts and plants were transiently and stably transformed, respectively, by several multigene constructs, and the expression of the transformed genes was monitored across several generations. Because ZFNs can potentially be engineered to digest a wide variety of target sequences, our system allows overcoming the problem of the very limited number of commercial homing endonucleases. Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs, and since our cloning system is based on ZFN and homing endonucleases, it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids.

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A versatile vector system for multiple gene expression in plants

Cited by 117 publications

References 8 publications

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF ^VBF ubiquitin–ligase complex pathway

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF ^VBF ubiquitin–ligase complex pathway

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors

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A versatile vector system for multiple gene expression in plants

Cited by 117 publications

References 8 publications

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF VBF ubiquitin–ligase complex pathway

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF VBF ubiquitin–ligase complex pathway

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors

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Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF ^VBF ubiquitin–ligase complex pathway

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF ^VBF ubiquitin–ligase complex pathway