Transient Gene Expression in Plant Cells Mediated by Agrobacterium tumefaciens: Application for the Analysis of Virulence Loci

Yoshioka, Yasushi; Takahashi, Yūzō; Matsuoka, Ken; Nakamura, Kenzo; Koizumi, Jun; Kojima, Mineo; Machida, Yasunori

doi:10.1093/oxfordjournals.pcp.a029013

Cited by 25 publications

(10 citation statements)

References 52 publications

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“…Stable transformation was performed as described by Yoshioka et al (1996) with some modi®cations. Brie¯y, the plasmids pMAT037, pMAT037/HA-pAPX, and pMAT037/CAT-pAPX were transferred independently into Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method of HoÈ fgen and Willmitzer (1988).…”

Section: Cell Culture and Transformationmentioning

confidence: 99%

Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain

Mullen

Lisenbee

Flynn

et al. 2001

Planta

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Peroxisomal ascorbate peroxidase (APX) (EC 1.11.1.11) was shown recently to sort through a subdomain of the ER (peroxisomal endoplasmic reticulum; pER), and in certain cases, alter the distribution and/or morphology of peroxisomes and pER when overexpressed transiently in Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells. Our goal was to gain insight into the dynamics of peroxisomal membrane protein sorting by characterizing the structure and formation of reorganized peroxisomes and pER. Specifically, we test directly the hypothesis that the observed phenomenon is due to the oligomerization of cytosol-facing, membrane-bound polypeptides. a process referred to as membrane "zippering". Results from differential detergent permeabilization experiments confirmed that peroxisomal APX is a C-terminal "tail-anchored" (Cmatrix-Ncytosol) membrane protein with a majority of the polypeptide facing the cytosol. Transient expression of several APX chimeras whose passenger polypeptides can form dimers or trimers resulted in the progressive formation of "globular" peroxisomes and circular pER membranes. Stable expression of the trimer-capable fusion protein yielded suspension cultures that reproducibly maintained a high degree of peroxisomal globules but relatively few detectable pER membranes. Electron micrographs revealed that the globules consisted of numerous individual peroxisomes, seemingly in direct contact with other peroxisomes and/or mitochondria. These peroxisomal clusters or aggregates were not observed in cells transiently expressing monomeric versions of APX. These findings indicate that the progressive, independent "zippering" of peroxisomes and pER is due to the post-sorting oligomerization of monomeric, cytosol-facing polypeptides that are integrally inserted into the membranes of "like" organelles. The dynamics of this process are discussed, especially with respect to the involvement of the microtubule cytoskeleton.

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Section: Cell Culture and Transformationmentioning

confidence: 99%

Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain

Mullen

Lisenbee

Flynn

et al. 2001

Planta

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“…Recent studies suggested the activity of VirD2 NLSs to be universal because the recombinant protein accumulated in the nuclei of microinjected Xenopus oocytes and Drosophila embryos (14). Although activity of both VirD2 NLSs was documented in the studies listed above, only the C-terminal NLS has been found to be involved in tumorigenicity of Agrobacterium (15)(16)(17)(18).…”

mentioning

confidence: 99%

Interaction of the DNA modifying proteins VirD1 and VirD2 of Agrobacterium tumefaciens : Analysis by subcellular localization in mammalian cells

Relić

Andjelković

Rossi

et al. 1998

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

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Interaction between Agrobacterium tumefaciens and plants provides a unique example of interkingdom gene transfer. Agrobacterium, a plant pathogen, is capable to stably transform the plant cell with a segment of its own DNA called T-DNA (transferred DNA). This process depends, among others, on the specialized bacterial virulence proteins VirD1 and VirD2 that excise the T-DNA from its adjacent sequences. Subsequent to transfer to the plant cell, the virulence protein VirD2, through its nuclear localization signal (NLS), is believed to guide the T-DNA to the nucleus. The T-DNA then is integrated into the plant genome. Although both of these proteins are essential for bacterial virulence, physical interaction of them has not been analyzed so far. We studied associations between these proteins by expressing them in mammalian cells and by testing for intracellular localization and colocalization. When expressed in human cells [HeLa, human embryo kidney (HEK) 293], the VirD2 protein homogeneously distributed over the nucleoplasm. The presence of any of two NLSs, on the N and C termini of VirD2, was sufficient for its efficient nuclear localization whereas deletion of both NLSs rendered the protein cytoplasmic. However, this double NLS mutant was translocated to the nucleus in the presence of wild-type VirD2 protein, implying VirD2-VirD2 interaction. The VirD1 protein, by itself localized in the cytoplasm, moved to the nucleus when coexpressed with the VirD2 protein, suggesting VirD1-VirD2 interaction. This interaction was confirmed by coimmunoprecipitation tests. Of interest, both proteins coimported to the nucleus showed a similar, peculiar sublocalization. The data are discussed in terms of functions of the VirD proteins. In addition, coimport of proteins into nuclei is suggested as a useful system in studying individual protein-protein interactions.

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“…1995). An acvB Tn5 insertion was shown to be avirulent (Wirawan et al, 1996, Yoshioka et al, 1996 suggesting that this protein, possibly in association with the T -strand, is involved in some essential step of the transport of the T-DNA. The VirE2 protein is the most abundant protein produced in A. tumefaciens after induction of the virulence genes by plant phenolic compounds (Stachel,I 986a).…”

Section: Ll T-dna Processingmentioning

confidence: 99%

The Rhizobiaceae

1998

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Transient Gene Expression in Plant Cells Mediated by Agrobacterium tumefaciens: Application for the Analysis of Virulence Loci

Cited by 25 publications

References 52 publications

Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain

Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain

Interaction of the DNA modifying proteins VirD1 and VirD2 of Agrobacterium tumefaciens : Analysis by subcellular localization in mammalian cells

The Rhizobiaceae

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