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Recombinant Proteins From Plants pp 289–312Cite as

Immunomodulation of Plant Function by In Vitro Selected Single-Chain Fv Intrabodies

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 483))

Summary

In this chapter, we discuss and compare the different concepts and examples as well as present the basic protocols for applying intrabody-based approaches in plants for the investigation of cell functions and plant cell–pathogen interactions. The immunomodulation strategy, a molecular technique that allows to interfere with cellular metabolism, signal transduction pathways, or pathogen infectivity, is based on the ectopic expression of genes encoding specific recombinant antibodies. This needs basic prerequisites to be successfully applied as resources and techniques to isolate specific recombinant antibodies with sufficient binding parameters to bind and to block even low-concentrated targets or to compete successfully with substrates and ligands. Also techniques and constructs to efficiently transform plants and to target recombinant antibodies to selected compartments are important requirements. Basic protocols for all these techniques are provided.

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References

  1. Biocca, S., Neuberger, M. S. and Cattaneo,A. (1990) Expression and targeting of intracellular antibodies in mammalian cells. EMBO J. 9, 101–108.

    CAS  PubMed  Google Scholar 

  2. Marasco, W. A., Haseltine, W. A. and Chen, S. Y. (1993) Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl. Acad. Sci. USA 90, 7889–7893.

    Article  CAS  PubMed  Google Scholar 

  3. Kontermann, R. E. (2004) Intrabodies as therpeutic agents. Methods 34, 163–170.

    Article  CAS  PubMed  Google Scholar 

  4. Stocks, M. (2006) Intracellular antibodies: A revolution waiting to happen ? Curr. Opin. Mol. Ther. 8, 17–23.

    CAS  PubMed  Google Scholar 

  5. De Jaeger, G., De Wilde, C., Eeckhout, D., Fiers, E. and Depicker, A. (2000) Theplantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance. Plant Mol. Biol. 43, 419–428.

    Article  PubMed  Google Scholar 

  6. Conrad, U. and Manteuffel, R. (2001) Immunomodulation of phytohormones and functional proteins in plant cells. Trends Plant Sci. 6, 399–402.

    Article  CAS  PubMed  Google Scholar 

  7. Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S. and Whitlow, M. (1988) Single-chain antigen-binding proteins. Science 242, 423–426.

    Article  CAS  PubMed  Google Scholar 

  8. Dübel, S. and Kontermann, R. E. (2001) Recombinant antibodies, in:Antibody Engineering(Kontermann, R. and Dübel, S., eds.), Springer-Verlag, Berlin Heidelberg, pp. 3–18.

    Google Scholar 

  9. Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.

    Article  CAS  PubMed  Google Scholar 

  10. Burmester, J. and Plückthun, A. (2001) Construction of scFv fragments from hybri-doma or spleen cells by PCR assembly, inAntibody Engineering(Kontermann, R. and Dübel, S., eds.), Springer-Verlag, Berlin Heidelberg, pp. 19–40.

    Google Scholar 

  11. Breitling, F., Moosmayer, D., Brocks, B. and Dübel, S. (2001) Construction of scFv from hybridoma by two-step cloning of the VH and VL domains, inAntibody Engineering(Kontermann, R. and Dübel, S., eds.), Springer-Verlag, Berlin Heidelberg, pp. 41–55.

    Google Scholar 

  12. Bradbury, A. (2001) Cloning hybridoma cDNA by RACE, inAntibody Engineering(Kontermann, R. and Dübel, S., eds.), Springer-Verlag, Berlin Heidelberg, pp. 56–64.

    Google Scholar 

  13. Smith, G. P. (1985) Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317.

    Article  CAS  PubMed  Google Scholar 

  14. McCafferty, J., Griffiths, A. D., Winter, G. and Chiswell, D. J. (1990) Phage antibodies: Filamentous phage displaying antibody variable domains. Nature 348, 552–554.

    Article  CAS  PubMed  Google Scholar 

  15. Clackson, T., Hoogenboom, H. R., Griffiths, A. D. and Winter, G. (1991) Making antibody fragments using phage display libraries. Nature 352, 624–628.

    Article  CAS  PubMed  Google Scholar 

  16. Conrad, U. and Scheller, J. (2005) Considerations on antibody-phage display methodology. Comb. Chem. High Throughput Screen. 8, 117–126.

    Article  CAS  PubMed  Google Scholar 

  17. Low, N. M., Holliger, P. H. and Winter, G. (1996) Mimicking somatic hypermutation: Affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J. Mol. Biol. 260, 359–368.

    Article  CAS  PubMed  Google Scholar 

  18. Swers, J. S., Kellogg, B. A. and Wittrup, K. D. (2004) Shuffled antibody libraries created byin vivohomologous recombination and yeast surface display. Nucleic Acids Res. 32, e36.

    Article  PubMed  Google Scholar 

  19. Visintin, M., Tse, E., Axelson, H., Rabbitts, T. H. and Cattaneo, A. (1999) Selection of antibodies for intracellular function using a two-hybridin vivosystem. Proc. Natl. Acad. Sci. USA 96, 11723–11728.

    Article  CAS  PubMed  Google Scholar 

  20. der Maur, A. A., Zahnd, C., Fisher, F., Spinelli, S., Honegger, A., Cambillau, C.,Escher, D., Pluckthun, A. and Barberis, A. (2002) Direct in vivo screening of intra-body libraries constructed on a highly stable single-chain framework. J. Biol. Chem. 277, 45075–45085.

    Article  Google Scholar 

  21. Hiatt, A., Cafferkey, R. and Bowdish, K. (1989) Production of antibodies in trans-genic plants. Nature 342, 76–78.

    Article  CAS  PubMed  Google Scholar 

  22. Fischer, R., Stöger, E., Schillberg, S., Chris-tou, P. and Twyman, R. (2004) Plant-based production of biopharmaceuticals. Curr. Opin. Plant Biol. 7, 152–158.

    Article  CAS  PubMed  Google Scholar 

  23. Stöger, E., Vaquero, C., Torres, E., Sack,M., Nicholson, L., Drossard, J., Williams, S., Keen, D., Perrin, Y., Christou, P. and Fischer, R. (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol. Biol. 42, 583–590.

    Article  PubMed  Google Scholar 

  24. Owen, M., Gandecha, A., Cockburn, B. and Whitelam, G. (1992) Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco. Biotechnology (N.Y.) 10, 790–794.

    Google Scholar 

  25. Conrad, U. and Fiedler, U. (1998) Compartment-specific accumulation of recom-binant immunoglobulins in plant cells: An essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol. Biol. 38, 101–109.

    Article  CAS  PubMed  Google Scholar 

  26. Spiegel, H., Schillberg, S., Sack, M., Holzem, A., Nähring, J., Monecke, M., Liao, Y.-C. and Fischer, R. (1999) Accumulation of Antibody fusion proteins in the cytoplasm and ER of Plants. Plant Sci. 149, 63–71.

    Article  CAS  Google Scholar 

  27. Jobling, S. A., Jarman, C., Teh, M.-M., Holmberg, N., Blake, C. and Verhoeyen, M. E. (2003) Immunomodulation of enzyme function in plants by single-domain antibody fragments. Nat. Biotechnol. 21, 77–80.

    Article  CAS  PubMed  Google Scholar 

  28. Santos, M. O., Crosby, W. L. and Winkel, B. S. J. (2004) Modulation of flavonoid metabolism in Arabidopsis using a phage-derived antibody. Mol. Breed. 13, 333–343.

    Article  CAS  Google Scholar 

  29. Nölke, G., Schneider, B., Fischer, R. and Schillberg, S. (2005) Immunomodulation of polyamine biosynthesis in tobacco plants has a significant impact on polyamine levels and generates a dwarf phenotype. Plant Bio-technol. J. 3, 237–247.

    Article  Google Scholar 

  30. Miroshnichenko, S., Tripp, J., zur Nieden, U., Neumann, D., Conrad, U. and Man-teuffel, R. (2005) Immunomodulation of function of small heat shock proteins prevents their assembly into heat stress granules and results in cell death at sublethal temera-tures. Plant J. 41, 269–281.

    Article  CAS  PubMed  Google Scholar 

  31. Artsaenko, O., Peisker, M., zur Nieden, U., Fiedler, U., Weiler, E. W., Müntz, K. and Conrad, U. (1995) Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. Plant J. 8, 745–750.

    Article  CAS  PubMed  Google Scholar 

  32. Phillips, J., Artsaenko, O., Fiedler, U., Horstmann, C., Mock, H.-P., Müntz, K. and Conrad, U. (1997) Seed-specific immunomodulation of abscisic acid activity induces a developmental switch. EMBO J. 16, 4489–4496.

    Article  CAS  PubMed  Google Scholar 

  33. Senger, S., Mock, H.-P., Conrad, U. and Manteuffel, R. (2001) Immunomodula-tion of ABA function affects early events in somatic embryo development. Plant Cell Rep. 20, 112–120.

    Article  CAS  Google Scholar 

  34. Wigger, J., Phillips, J., Peisker, M., Hartung,W., zur Nieden, U., Artsaenko, O., Fiedler, U. and Conrad, U. (2002) Prevention of stomatal closure by immunomodulation of endogenous abscisic acid and its reversion by abscisic acid treatment: Physiological behaviour and morphological fratures of tobacco stomata. Planta 215, 413–423.

    Article  CAS  PubMed  Google Scholar 

  35. Strauss, M., Kauder, F., Peisker, M., Son-newald, U., Conrad, U. and Heineke, D. (2001) Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in trans-genic potato plants. Planta 213, 361–369.

    Article  CAS  PubMed  Google Scholar 

  36. Shimada, N., Suzuki, Y., Nakajima, M., Conrad, U., Murofushi, N. and Yamagushi, I. (1999) Expression of a functional single-chain antibody against GA 24/19 in trans-genic tobacco. Biosci. Biotechnol. Biochem. 63, 779–783.

    Article  CAS  PubMed  Google Scholar 

  37. Putalun, W., Taura, F., Qing, W., Matsushita, H., Tanaka, H. and Shoyama, Y. (2003) Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasod-ine glycoside in plants. Plant Cell Rep. 22, 344–349.

    Article  CAS  PubMed  Google Scholar 

  38. Eto, J., Suzuki, Y., Ohkawa, H. and Yamaguchi, I. (2003) Anti-herbicide single-chain antibody expression confers herbicide tolerance in transgenic plants. FEBS Lett. 550, 179–184.

    Article  CAS  PubMed  Google Scholar 

  39. Almquist, K. C., Niu, Y., Mclean, M. D., Mena, F. L., Yau, K. Y. F., Brown, K., Bran-dle, J. E. and Hall, J. C. (2004) Immu-nomodulation confers herbicide resistance in plants. Plant Biotechnol. J. 2, 189–197.

    Article  CAS  PubMed  Google Scholar 

  40. Leps, B. (2003) Anti-Quinmerac Einket-tenantikörperexpression in transgenen Tabakpflanzen.Thesis, University of Halle-Wittenberg, Germany.

    Google Scholar 

  41. Leps, M. (2002) Expression von Einketten-antikörpern gegen das Fungizid Kresoxim-methyl in transgenen Tabakpflanzen. Thesis, University of Halle-Wittenberg, Germany.

    Google Scholar 

  42. Yuan, Q., Hu, W., Pestka, J. J., He, S. Y. and Hart, L. P. (2000) Expression of a functional antizearalenone singel-chain Fv antibody in transgenic Arabidopsis plants. Appl. Environ. Microbiol. 66, 3499–3505.

    CAS  Google Scholar 

  43. Tavladoraki, P., Benvenuto, E., Trinca, S., De Martinis, D., Cattaneo, A. and Galeffi, P. (1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366, 469–472.

    Article  CAS  PubMed  Google Scholar 

  44. Schillberg, S., Zimmermann, S., Zhang, M.-Y. and Fischer, R. (2001) Antibody-based resistance to plant pathogens. Transgenic Res. 10, 1–12.

    Article  CAS  PubMed  Google Scholar 

  45. Peschen, D., Li, H.-P., Fischer, R., Kreu-zaler, F. and Liao, Y.-C. (2004) Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen. Nat. Biotechnol. 22, 732–738.

    Article  CAS  PubMed  Google Scholar 

  46. Boonrod, K., Galetzka, D., Nagy, P. D., Conrad, U. and Krczal, G. (2004) Single-chain antibodies against a plant viral RNA-dependent RNA plymerase confer virus resistance. Nat. Biotechnol. 22, 856–862.

    Article  CAS  PubMed  Google Scholar 

  47. Villani, M. E., Roggero, P., Bitti, O., Ben-venuto, E. and Franconi, R. (2005) Immu-nomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library. Plant Mol. Biol. 58, 305–316.

    Article  CAS  PubMed  Google Scholar 

  48. Prins, M., Lohuis, D., Schots, A. and Gold-bach, R. (2005) Phage display-selected single-chain antibodies confer hifg levels of resistance against Tomato spotted wilt virus. J. Gen. Virol. 86, 2107–2113.

    Article  CAS  PubMed  Google Scholar 

  49. de Wildt, R. M., Mundy, C. R., Gorick,B. D. and Tomlinson, I. M. (2000) Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat. Bio-technol. 18, 989–994.

    Article  Google Scholar 

  50. Scheller, J., Henggeler, D., Viviani, A. and Conrad, U. (2004) Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation. Transgenic Res. 13, 51–57.

    Article  CAS  PubMed  Google Scholar 

  51. Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.

    Article  CAS  Google Scholar 

  52. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  CAS  PubMed  Google Scholar 

  53. Conrad, U., Fiedler, U., Artsaenko, O. and Phillips, J. (1998) Single-chain Fv antibodies expressed in plants, in Methods in Biotechnology, Vol. 3: Recombinant proteins from plants (Cunningham, C. and Porter, A. J. R., eds.), Humana, Totowa, NJ, pp. 103–127.

    Chapter  Google Scholar 

  54. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  55. Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M. and Leemans, J. (1985) Efficient octopine Ti plasmid-derived vectors for Agrobac-terium-mediated gene transfer to plants. Nucleic Acids Res. 13, 4777–4788.

    Article  CAS  PubMed  Google Scholar 

  56. Hoekema, A., Hirsch, P. R., Hooykaas, P. J. J. and Schilperoort, R. A. (1983) A binary plant vector strategy based on sepa-ration of vir - and T-region of the Agrobac-terium tumefaciens Ti-plasmid. Nature 303, 179–180.

    Article  CAS  Google Scholar 

  57. Mattanovich, D., Rüker, F., da Câmara Machado, A., Laimer, M., Regner, F., Stein-kellner, H., Himmler, G. and Katinger, H. (1989) Efficient transformation of Agro-bacterium spp. by electroporation. Nucleic Acids Res. 16, 6747.

    Article  Google Scholar 

  58. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Anal. Bio-chem. 72, 248–254.

    CAS  Google Scholar 

  59. Holt, L. J., Büssow, K., Walter, G. and Tom-linson, I. M. (2000) By-passing selection: Direct screening for antibody-antigen interactions using protein arrays. Nucleic Acids Res. 28, e72.

    Article  CAS  PubMed  Google Scholar 

  60. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G. and Fraley, R. T. (1985) A simple and general method for transferring genes into plants. Science 227, 1229–1231.

    Article  CAS  Google Scholar 

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Acknowledgments

The authors thank Doreen Floss for critically reading the manuscript, Silke Krause for helpful comments on phage display technology, and Birgit Schäfer for recording the photographs in Fig. 17.5.

Author information

Authors and Affiliations

  1. Dept. Cell Biology and Plant Physiology, University of Regensburg, Regensburg, Germany

    Manfred Gahrtz

  2. Molecular Genetics/Phytoantibodies, Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

    Udo Conrad

Authors
  1. Manfred Gahrtz
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  2. Udo Conrad
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Editors and Affiliations

  1. CNRS, Université de Rouen, Mont Saint Aignan, France

    Loïc Faye  & Véronique Gomord  & 

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Gahrtz, M., Conrad, U. (2009). Immunomodulation of Plant Function by In Vitro Selected Single-Chain Fv Intrabodies. In: Faye, L., Gomord, V. (eds) Recombinant Proteins From Plants. Methods in Molecular Biology™, vol 483. Humana Press. https://doi.org/10.1007/978-1-59745-407-0_17

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  • DOI: https://doi.org/10.1007/978-1-59745-407-0_17

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-978-9

  • Online ISBN: 978-1-59745-407-0

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