Immunomodulation of plant function by in vitro selected single-chain Fv intrabodies.

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.

[1]  A. Hiatt,et al.  Production of antibodies in transgenic plants , 1989, Nature.

[2]  I. Tomlinson,et al.  Antibody arrays for high-throughput screening of antibody–antigen interactions , 2000, Nature Biotechnology.

[3]  P. D. Nagy,et al.  Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance , 2004, Nature Biotechnology.

[4]  R. Kontermann Intrabodies as therapeutic agents. , 2004, Methods.

[5]  Marc Van Montagu,et al.  Efficient octopine Ti plasmid-derived vectors for Agrobacterium- mediated gene transfer to plants , 1985, Nucleic Acids Res..

[6]  A. Cattaneo,et al.  Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack , 1993, Nature.

[7]  E. Weiler,et al.  Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. , 1995, The Plant journal : for cell and molecular biology.

[8]  K. D. Hardman,et al.  Single-chain antigen-binding proteins. , 1988, Science.

[9]  J. Scheller,et al.  Purification of Spider Silk-elastin from Transgenic Plants and Application for Human Chondrocyte Proliferation , 2004, Transgenic Research.

[10]  P. Hirsch,et al.  A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid , 1983, Nature.

[11]  R. Fischer,et al.  Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen , 2004, Nature Biotechnology.

[12]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[13]  Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly , 2001 .

[14]  C. Milstein,et al.  Continuous cultures of fused cells secreting antibody of predefined specificity , 1975, Nature.

[15]  W. Crosby,et al.  Modulation of flavonoid metabolism in Arabidopsis using a phage-derived antibody , 2004, Molecular Breeding.

[16]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[17]  A. Schots,et al.  Phage display-selected single-chain antibodies confer high levels of resistance against Tomato spotted wilt virus. , 2005, The Journal of general virology.

[18]  K. Wittrup,et al.  Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display. , 2004, Nucleic acids research.

[19]  Y. Shoyama,et al.  Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants , 2003, Plant Cell Reports.

[20]  U. Conrad,et al.  Single-Chain Fv Antibodies Expressed in Plants , 1998 .

[21]  G. Whitelam,et al.  Synthesis of a Functional Anti–Phytochrome Single–Chain Fv Protein in Transgenic Tobacco , 1992, Bio/Technology.

[22]  T. Rabbitts,et al.  Selection of antibodies for intracellular function using a two-hybrid in vivo system. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[23]  A. Bradbury Cloning Hybridoma cDNA by RACE , 2001 .

[24]  Annemarie Honegger,et al.  Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework* , 2002, The Journal of Biological Chemistry.

[25]  U. Conrad,et al.  Immunomodulation of ABA function affects early events in somatic embryo development , 2001, Plant Cell Reports.

[26]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[27]  I. Yamaguchi,et al.  Anti‐herbicide single‐chain antibody expression confers herbicide tolerance in transgenic plants , 2003, FEBS letters.

[28]  Rainer Fischer,et al.  Plant-based production of biopharmaceuticals. , 2004, Current opinion in plant biology.

[29]  S. Chen,et al.  Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Construction of scFv from Hybridoma by Two-Step Cloning , 2001 .

[31]  S. He,et al.  Expression of a Functional Antizearalenone Single-Chain Fv Antibody in Transgenic ArabidopsisPlants , 2000, Applied and Environmental Microbiology.

[32]  J. Fry,et al.  A simple and general method for transferring genes into plants. , 1985, Science.

[33]  W. Hartung,et al.  Prevention of stomatal closure by immunomodulation of endogenous abscisic acid and its reversion by abscisic acid treatment: physiological behaviour and morphological features of tobacco stomata , 2002, Planta.

[34]  D. Heineke,et al.  Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in transgenic potato plants , 2001, Planta.

[35]  R. Franconi,et al.  Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library , 2005, Plant Molecular Biology.

[36]  T. Clackson,et al.  Making antibody fragments using phage display libraries , 1991, Nature.

[37]  U. Conrad,et al.  Immunomodulation of phytohormones and functional proteins in plant cells. , 2001, Trends in plant science.

[38]  S. Jobling,et al.  Immunomodulation of enzyme function in plants by single-domain antibody fragments , 2003, Nature Biotechnology.

[39]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[40]  A. Depicker,et al.  The plantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance , 2000, Plant Molecular Biology.

[41]  U. Conrad,et al.  Immunomodulation of function of small heat shock proteins prevents their assembly into heat stress granules and results in cell death at sublethal temperatures. , 2004, The Plant journal : for cell and molecular biology.

[42]  G. Winter,et al.  Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. , 1996, Journal of molecular biology.

[43]  Michael D. McLean,et al.  Immunomodulation confers herbicide resistance in plants. , 2004, Plant biotechnology journal.

[44]  M. Neuberger,et al.  Expression and targeting of intracellular antibodies in mammalian cells. , 1990, The EMBO journal.

[45]  S. Schillberg,et al.  Immunomodulation of polyamine biosynthesis in tobacco plants has a significant impact on polyamine levels and generates a dwarf phenotype. , 2005, Plant Biotechnology Journal.

[46]  Lucy J. Holt,et al.  By-passing selection: direct screening for antibody-antigen interactions using protein arrays. , 2000, Nucleic acids research.

[47]  M. Stocks Intracellular antibodies: a revolution waiting to happen? , 2006, Current opinion in molecular therapeutics.

[48]  Rainer Fischer,et al.  Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies , 2000, Plant Molecular Biology.

[49]  J. Scheller,et al.  Considerations on antibody-phage display methodology. , 2005, Combinatorial chemistry & high throughput screening.

[50]  U. Conrad,et al.  Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity , 1998, Plant Molecular Biology.

[51]  G. P. Smith,et al.  Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. , 1985, Science.

[52]  U. Conrad,et al.  Seed‐specific immunomodulation of abscisic acid activity induces a developmental switch , 1997, The EMBO journal.

[53]  S. Schillberg,et al.  Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells , 1999 .

[54]  H. Katinger,et al.  Efficient transformation of Agrobacterium spp. by electroporation. , 1989, Nucleic acids research.

[55]  M. Nakajima,et al.  Expression of a functional single-chain antibody against GA24/19 in transgenic tobacco. , 1999, Bioscience, biotechnology, and biochemistry.

[56]  G. Winter,et al.  Phage antibodies: filamentous phage displaying antibody variable domains , 1990, Nature.

[57]  S. Schillberg,et al.  Antibody-Based Resistance to Plant Pathogens , 2004, Transgenic Research.