Abstract
Agrobacterium most likely can transform virtually all known plant species, and experimental protocols for Agrobacterium-mediated genetic transformation of yet more plant species, ecotypes, and cultivars are published almost on a daily basis. Interestingly, the Agrobacterium host range is not limited to the plant kingdom, and it has been shown to transform many species of fungi and even prokaryotes. The ability of Agrobacterium to genetically transform HeLa cells further widens the range of potential hosts of Agrobacterium to include humans and perhaps other animal species. Furthermore, because mammalian cells significantly differ from plant cells, they provide a useful experimental system for identification and functional characterization of plant-specific factors involved in the transformation process. Here, we present basic procedures for transfection and Agrobacterium-mediated genetic transformation of mammalian cells. We also demonstrate the use of mammalian cells for studies of the cellular components of the genetic transformation pathway.
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Gaudin, V., Vrain, T., and Jouanin, L. (1994). Bacterial genes modifying hormonal balances in plants. Plant Physiol. Biochem. 32, 11–29.
Bracha-Drori, K., Shichrur, K., Katz, A., et al. (2004). Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J. 40, 419–427.
Walter, M., Chaban, C., Schütze, K., et al. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438.
Bevan, M.W. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 1811–1821.
Draper, J., Scott, R., Armitage, P., and Walden, R. (eds.) (1988) Plant Genetic Transformation and Gene Expression, A Laboratory Manual. Blackwell Scientific Publications Ltd., London, UK.
Clough, S.J. and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.
Gelvin, S.B. (1998). The introduction and expression of transgenes in plants. Curr. Opin. Biotechnol. 9, 227–232.
Tzfira, T. and Citovsky, V. (2000). From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium-plant cell interaction. Mol. Plant Pathol. 1, 201–212.
Tzfira, T., Li, J., Lacroix, B., and Citovsky, V. (2004). Agrobacterium T-DNA integration: molecules and models. Trends Genet. 20, 375–383.
Tzfira, T. and Citovsky, V. (2002). Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol. 12, 121–129.
Gelvin, S.B. (2003). Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev. 67, 16–37.
Gelvin, S.B. (2000). Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 223–256.
Vergunst, A.C., Schrammeijer, B., den Dulk-Ras, A., de Vlaam, C.M.T., Regensburg-Tuink, T.J., and Hooykaas, P.J.J. (2000). VirB/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290, 979–982.
Christie, P.J. and Vogel, J.P. (2000). Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol. 8, 354–360.
Ding, Z., Atmakuri, K., and Christie, P.J. (2003). The outs and ins of bacterial type IV secretion substrates. Trends Microbiol. 11, 527–535.
Cascales, E. and Christie, P.J. (2004). Definition of a bacterial type IV secretion pathway for a DNA substrate. Science 304, 1170–1173.
Kelly, B.A. and Kado, C.I. (2002). Agrobacterium-mediated T-DNA transfer and integration into the chromosome of Streptomyces lividans. Mol. Plant Pathol. 3, 125–134.
Piers, K.L., Heath, J.D., Liang, X., Stephens, K.M., and Nester, E.W. (1996). Agrobacterium tumefaciens-mediated transformation of yeast. Proc. Natl. Acad. Sci. USA 93, 1613–1618.
Bundock, P., den Dulk-Ras, A., Beijersbergen, A., and Hooykaas, P.J.J. (1995). Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14, 3206–3214.
Bundock, P. and Hooykaas, P.J.J. (1996). Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc. Natl. Acad. Sci. USA 93, 15,272–15,275.
de Groot, M.J., Bundock, P., Hooykaas, P.J.J., and Beijersbergen, A.G. (1998). Agrobacterium tumefaciens-mediated transformation of filamentous fungi [published erratum appears in Nat. Biotechnol. 16, 1074 (1998)]. Nat. Biotechnol. 16, 839–842.
Michielse, C.B., Salim, K., Ragas, P., et al. (2004). Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer. Mol. Genet. Genomics 271, 499–510.
Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C., and Citovsky, V. (2001). Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl. Acad. Sci. USA 98, 1871–1876.
Pelczar, P., Kalck, V., Gomez, D., and Hohn, B. (2004). Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells. EMBO Rep. 5, 632–637.
Tzfira, T., Frankmen, L., Vaidya, M., and Citovsky, V. (2003). Site-specific integration of Agrobacterium T-DNA via double-stranded intermediates. Plant Physiol. 133, 1011–1023.
Chilton, M.-D.M. and Que, Q. (2003). Targeted integration of T-DNA into the tobacco genome at double-strand breaks: new insights on the mechanism of T-DNA integration. Plant Physiol. 133, 956–965.
van Attikum, H. and Hooykaas, P.J.J. (2003). Genetic requirements for the targeted integration of Agrobacterium T-DNA in Saccharomyces cerevisiae. Nucleic Acids Res. 31, 826–832.
Tzfira, T., Vaidya, M., and Citovsky, V. (2001). VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity. EMBO J. 20, 3596–3607.
Tzfira, T., Vaidya, M., and Citovsky, V. (2002). Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis VIP1 gene. Proc. Natl. Acad. Sci. USA 99, 10,435–10,440.
Citovsky, V., Kapelnikov, A., Oliel, S., et al. (2004). Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro. J. Biol. Chem. 279, 29,528–29,533.
Britt, A.B. and May, G.D. (2003). Re-engineering plant gene targeting. Trends Plant Sci. 8, 90–95.
Pelczar, P., Kalck, V., and Kovalchuk, I. (2003). Different genome maintenance strategies in human and tobacco cells. J. Mol. Biol. 331, 771–779.
Felgner, P.L., Gadek, T.R., Holm, M., et al. (1987). Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84, 7413–7417.
Brewer, C.B. and Roth, M.G. (1991). A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin. J. Cell Biol. 114, 413–421.
Tzfira, T., Jensen, C.S., Wangxia, W., Zuker, A., Altman, A., and Vainstein, A. (1997). Transgenic Populus: a step-by-step protocol for its Agrobacterium-mediated transformation. Plant Mol. Biol. Rep. 15, 219–235.
Ausubel, F.M., Brent, R., Kingston, R.E., et al. (1987). Current Protocols in Molecular Biology, Greene Publishing-Wiley Interscience, New York.
Hajdukiewicz, P., Svab, Z., and Maliga, P. (1994). The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994.
Binns, A.N. and Thomashow, M.F. (1988). Cell biology of Agrobacterium infection and transformation of plants. Annu. Rev. Microbiol. 42, 575–606.
Zambryski, P.C. (1989). Agrobacterium-plant cell DNA transfer. In: Mobile DNA (Berg, D.E. and Howe, M.M. eds.), American Society for Microbiology, Washington, DC, pp. 309–333.
Tinland, B. (1996). The integration of T-DNA into plant genomes. Trends Plant Sci. 1, 178–184.
Gielen, J., Terryn, N., Villarroel, R., and Van Montagu, M. (1999). Complete nucleotide sequence of the T-DNA region of the plant tumour-inducing Agrobacterium tumefaciens Ti plasmid pTiC58. J. Exp. Bot. 50, 1421–1422.
Ballas, N. and Citovsky, V. (1997). Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein. Proc. Natl. Acad. Sci. USA 94, 10,723–10,728.
Tzfira, T. and Citovsky, V. (2001). Comparison between nuclear import of nopaline-and octopine-specific VirE2 protein of Agrobacterium in plant and animal cells. Mol. Plant Pathol. 2, 171–176.
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Tzfira, T., Kunik, T., Gafni, Y., Citovsky, V. (2006). Mammalian Cells. In: Wang, K. (eds) Agrobacterium Protocols Volume 2. Methods in Molecular Biology, vol 344. Humana Press. https://doi.org/10.1385/1-59745-131-2:435
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DOI: https://doi.org/10.1385/1-59745-131-2:435
Publisher Name: Humana Press
Print ISBN: 978-1-58829-843-0
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