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Expression of giant silkmoth cecropin B genes in tobacco

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Abstract

Cecropin B is a small antibacterial peptide from the giant silkmothHyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco viaAgrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance toPseudomonas solanacearum, the causal agent of bacterial wilt of many crops, andP. syringae pv.tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin Bin vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.

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References

  • Boman, H. G., Boman, I. A., Andreu, D., Li Z. Q., Merrifield, R. B., Schlenstedt, G. and Zimmermann, R. (1989) Chemical synthesis and enzymic processing of precursor forms of cecropins A and B.J. Biol. Chem. 264, 5852–60.

    Google Scholar 

  • Boman, H. G. and Hultmark, D. (1987) Cell-free immunity in insects.Ann. Rev. Microbiol. 41, 103–26.

    Google Scholar 

  • Bradford, M. M. (1976) A rapid and sensitive method for the quantization of microgram quantitaties of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–54.

    Google Scholar 

  • Christensen, B., Fink, J., Merrifield, R. B. and Mauzerall, D. (1988) Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes.Proc. Natl Acad. Sci. USA 85, 5072–6.

    Google Scholar 

  • Deen, C., Claassen, E., Gerritse, K., Zegers, N. D. and Boersma, W. J. A. (1990) A novel carbodiimide coupling method for synthetic peptides: enhanced anti-peptide antibody responses.J. Immunol. Meth. 129, 119–25.

    Google Scholar 

  • Denecke, J., Botterman, J. and Deblaere, R. (1990) Protein secretion in plant cells can occur via a default pathway.Pl. Cell 2, 51–9.

    Google Scholar 

  • De Vries, S., Hoge, H. and Bisseling, T. (1991) Isolation of total and polysomal RNA from plant tissues. In Gelvin, B., Schilperoort, R. A. and Verma, D. P. S. eds,Plant Molecular Biology Manual, pp. B6/1–13. Dordrecht: Kluwer Academic Publishers.

    Google Scholar 

  • De Wit, P. J. G. M. and Spikman, G. (1982) Evidence for the occurrence of race and cultivar-specific elicitors of necrosis in intercellular fluids of compatible interactions ofCladosporium fulvum and tomato.Physiol. Pl. Pathol. 21, 1–11.

    Google Scholar 

  • Engst, K. and Shaw, P. D. (1992) Identification of alysA-like gene required for tabtoxin biosynthesis and pathogenicity inPseudomonas syringae pv.tabaci strain PTBR2.024.Mol. Pl. Microbe Inter. 5, 322–9.

    Google Scholar 

  • Florack, D. E. A., Dirkse, W. G., Visser, B., Heidekamp, F. and Stiekema, W. J. (1994) Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting.Pl. Mol. Biol. 24, 83–96.

    Google Scholar 

  • Goto, M. (1992)Fundamentals of Bacterial Plant Pathology. San Diego, USA: Academic Press, Inc.

    Google Scholar 

  • Hightower, R., Baden, C., Penzes, E. and Dunsmuir, P. (1994) The expression of cecropin peptide in transgenic tobacco does not confer resistance toPseudomonas syringae pv.tabaci.Pl. Cell Rep. 13, 295–9.

    Google Scholar 

  • Hoekema, A., Kastelein, R., Vasser, M. and De Boer, H. A. (1987) Codon replacement in thePGK1 gene ofSaccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.Mol. Cell. Biol. 7, 2914–24.

    Google Scholar 

  • Hofsten, P. van, Faye, I., Kockum, K., Lee, J. Y., Xanthopoulos, K. G., Boman, I. A., Boman, H. G., Engström, Å., Andreu D. and Merrifield R. B. (1985) Molecular cloning, cDNA sequencing, and chemical synthesis of cecropin B fromHyalophora cecropia.Proc. Natl Acad. Sci. USA 82, 2240–3.

    Google Scholar 

  • Holak, T. A., Engström, Å., Kraulis, P. J., Lindeberg, G., Bennich, H., Jones, T. A., Gronenborn, A. M. and Clore G. M. (1988) The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study.Biochem. 27, 7620–9.

    Google Scholar 

  • Hultmark, D., Engström, A., Bennich, H., Kapur, R. and Boman, H. G. (1982) Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from cecropia pupae.Eur. J. Biochem. 127, 207–17.

    Google Scholar 

  • Jaynes, J. M., Xanthopoulos, K. G., Destéfano-Beltrán, L. and Dodds, J. H. (1987) Increasing bacterial disease resistance utilizing antibacterial genes from insects.BioEssays 6, 263–70.

    Google Scholar 

  • Jaynes, J. M., Nagpala, P., Destéfano-Beltrán, L., Huang, J. H., Kim, J. H., Denny, T. and Cetiner, S. (1993) Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused byPseudomonas solanacearum.Pl. Sci. 89, 43–53.

    Google Scholar 

  • Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W. (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J. 6, 3901–7.

    Google Scholar 

  • Kay, R., Chan, A., Daley, M. and McPherson, J. (1985) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes.Science 236, 1299–302.

    Google Scholar 

  • Klement, Z. (1963) Rapid detection of pathogenicity of phytopathogenic pseudomonads.Nature 199, 299–300.

    Google Scholar 

  • Koncz, C. and Schell, J. (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type ofAgrobacterium binary vector.Mol. Gen. Genet. 204, 383–96.

    Google Scholar 

  • Kunkel, T. A. (1985) Rapid and efficient site-directed mutagenesis without phenotypic selection.Proc. Natl Acad. Sci. USA 82, 488–92.

    Google Scholar 

  • Larebeke, N. van, Engler, G., Holster, M., Elsacker, S. van den, Zaenen, I., Schilperoort, R. A. and Schell, J. (1977) Large plasmid inAgrobacterium tumefaciens essential for crown gall inducing activity.Nature 252, 169–70.

    Google Scholar 

  • Lidholm, D. A., Gudmundsson, G. H., Xanthopoulos, K. G. and Boman, H. G. (1987) Insect immunity: cDNA clones coding for the precursor forms of cecropins A and D, antibacterial proteins fromHyalophora cecropid.FEBS Lett. 226, 8–12.

    Google Scholar 

  • Lütcke, H. A., Chow, K. C., Mickel, F. S., Moss, K. A., Kern, H. F. and Scheele, G. A. (1987) Selection of AUG initiation codons differs in plants and animals.EMBO J. 6, 43–8.

    Google Scholar 

  • Martin, C. and French, E. R. (1985) Bacterial wilt of potato:Pseudomonas solanacearum. Technical information bulletin 13 16 pp. Lima, Peru: International Potato Center.

    Google Scholar 

  • McGuire, R. G., Jones, J. B. and Sasser, M. (1986) Tween media for semiselective isolation ofXanthomonas campestris pv.vesicatoria from soil and plant material.Pl. Dis. 70, 887–91.

    Google Scholar 

  • Murray, E. E., Rocheleau, T., Eberle, M., Stock, C., Sekar, V., and Adang, M. (1991) Analysis of unstable RNA transcripts of insecticidal crystal protein genes ofBacillus thuringiensis in transgenic plants and electroporated protoplasts.Pl. Mol. Biol. 16, 1035–50.

    Google Scholar 

  • Nordeen, R. O., Sinden, S. L., Jaynes, J. M. and Owens, L. D. (1992) Activity of cecropin SB37 against protoplasts from several plant species and their bacterial pathogens.Pl. Sci. 82, 101–7.

    Google Scholar 

  • Perlak, F. J., Fuchs, R. L., Dean, D. A., McPherson, S. L. and Fischhoff, D. A. (1991) Modifications of the coding sequence enchances plant expression of insect control protein genes.Proc. Natl. Acad. Sci. USA 88, 3324–8.

    Google Scholar 

  • Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989)Molecular Cloning: a Laboratory Manual, Cold Spring Harbor, USA: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  • Sanger, F., Nicklen, S. and Coulson, A. R. (1977) DNA sequencing with the chain-terminating inhibitors.Proc. Natl Acad. Sci. USA 74, 5463–7.

    Google Scholar 

  • Shaner, G. and Finney, R. E. (1977) The effect of nitrogen fertilization on the expression of slow-mildewing resistance in Knox wheat.Phytopathol 67, 1051–6.

    Google Scholar 

  • Steiner, H. (1982). Secondary structure of the cecropins: antibacterial peptides from the mothHyalophora cecropia.FEBS Lett. 137, 283–7.

    Google Scholar 

  • Stewart, W. (1971) Isolation and proof of structure of wildfire toxin.Nature 229, 174–8.

    Google Scholar 

  • Winstead, N. N. and Kelman, A. (1952) Inoculation techniques for evaluating resistance toPseudomonas solanacearum.Phytopathol 42, 628–34.

    Google Scholar 

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Florack, D., Allefs, S., Bollen, R. et al. Expression of giant silkmoth cecropin B genes in tobacco. Transgenic Research 4, 132–141 (1995). https://doi.org/10.1007/BF01969415

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