title: Expression of giant silkmoth cecropin B genes in tobacco.
abstract: Cecropin B is a small antibacterial peptide from the giant silkmoth Hyalophora 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 via Agrobacterium 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 to Pseudomonas solanacearum, the causal agent of bacterial wilt of many crops, and P. syringae pv. tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin B in 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.
authors.affiliation: Department of Molecular Biology, DLO Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen, Netherlands.
authors.forename: D
authors.initials: D
authors.lastname: Florack
authors.forename: S
authors.initials: S
authors.lastname: Allefs
authors.forename: R
authors.initials: R
authors.lastname: Bollen
authors.forename: D
authors.initials: D
authors.lastname: Bosch
authors.forename: B
authors.initials: B
authors.lastname: Visser
authors.forename: W
authors.initials: W
authors.lastname: Stiekema
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007301:Insect Hormones / Q000096:biosynthesis / Q000235:genetics*; D019476:Insect Proteins; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D014170:Transformation, Genetic
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D007301:Insect Hormones; D019476:Insect Proteins; C031162:cecropin B protein, Insecta