Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi

The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed vi a the chloroplast genome to obtain high levels of expression in transgenic tobacco (Nicotiana tabacum var. Petit Havana) plants. Polymerase chain reac tion products and Southern blots confirmed integration of MSI-99 into the c hloroplast genome and achievement of homoplasmy, whereas northern blots con firmed transcription. Contrary to previous predictions, accumulation of MSI -99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. This may be due to differences in the lipid comp osition of plastid membranes compared with the membranes of susceptible tar get microbes. In vitro assays with protein extracts from T-1 and T-2 plants confirmed that MSI-99 was expressed at high levels to provide 88% (T-1) an d 96% (T-2) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. When germinated in the absence of spectinomycin selec tion, leaf extracts from T-2 generation plants showed 96% inhibition of gro wth against P. syringae pv tabaci. In addition, leaf extracts from transgen ic plants (T-1) inhibited the growth of pregerminated spores of three funga l species, Aspergillus flavus, Fusarium moniliforme, and Verticillium dahli ae, by more than 95% compared with non-transformed control plant extracts. In planta assays with the bacterial pathogen P. syringae pv tabaci resulted in areas of necrosis around the point of inoculation in control leaves, wh ereas transformed leaves showed no signs of necrosis, demonstrating high-do se release of the peptide at the site of infection by chloroplast lysis. In planta assays with the fungal pathogen, Colletotrichum destructivum, showe d necrotic anthracnose lesions in non-transformed control leaves, whereas t ransformed leaves showed no lesions. Genetically engineering crop plants fo r disease resistance via the chloroplast genome instead of the nuclear geno me is desirable to achieve high levels of expression and to prevent pollen- mediated escape of transgenes.