Expression of a Chimeric Magainin Gene in Potato Confers Improved Resistance to the Phytopathogen Erwinia carotovora

Abstract

Magainin peptides originally identified from Xenopus laevis have cytotoxic effects against a range of prokary- otic organisms without harmful effects on higher eukaryotes. The mechanism of cytotoxicity of the peptides is by disrup- tion of membranes, which causes osmolysis. Magainin peptides are known to inhibit the in vitro growth of many phytopa- thogens including Erwinia carotovora, the causative agent of soft rot disease in potato. A synthetic gene was constructed based on the antimicrobial peptide magainin II. The coding sequence for the modified magainin II, magaininD, was con- structed using potato codon preference. Modifications included a point mutation previously shown to reduce proteolytic cleavage, and three substitutions known to increase activity of the peptide against prokaryotic organisms. Agrobacterium- mediated transformation was used to generate transgenic potato plants. Each line was tested for expression of the trans- gene at RNA and protein levels using RT-PCR and western analyses. Lines were identified that expressed both RNA and protein of the magaininD transgene. Field trials with the transgenic magaininD potato plants were conducted over three seasons, and harvested tubers were evaluated in bioassays for resistance to Erwinia carotovora ssp. atroseptica. Results from bioassays identified potato lines with significantly improved resistance to soft rot compared with control lines. The same lines were determined to have higher levels of expression of the transgene-derived peptide. The result demonstrated that the design, construction and transformation of a synthetic antimicrobial magainin gene was a successful strategy in introducing a novel form of disease resistance in potato plants.