EXPRESSION OF GIANT SILKMOTH CECROPIN-B GENES IN TOBACCO

Cecropin B is a small antibacterial peptide from the giant silkmoth Hy alophora cecropia. To reveal the potential of this peptide for enginee ring bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secre tion. All constructs were cloned in a plant expression vector and intr oduced in tobacco via Agrobacterium tumefaciens. A cDNA-derived cecrop in B gene construct lacking the amino-terminal signal peptide was poor ly expressed in transgenic plants at the mRNA level, whereas plants ha rbouring a full-length cDNA-derived construct containing the insect si gnal peptide, showed increased cecropin B-mRNA levels. Highest express ion was found in plants harbouring a construct with a plant-gene-deriv ed 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 speci fic protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zw itter-ionic or non-ionic detergents, could prevent this degradation. N evertheless, transgenic tobacco plants were evaluated for resistance t o Pseudomonas solanacearum, the causal agent of bacterial wilt of many crops, and P. syringae pv. tabaci the causal agent of bacterial wildf ire, which are highly susceptible to cecropin B in vitro. No resistanc e was found. These experiments indicate that introduction and expressi on of cecropin B genes in tobacco does not result in detectable cecrop in B protein levels and resistance to bacterial infections, most likel y due to degradation of the protein by endogenous proteases.