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Plant Physiol, November 2001, Vol. 127, pp. 852-862
Expression of an Antimicrobial Peptide via the Chloroplast Genome
to Control Phytopathogenic Bacteria and Fungi
Gerald
DeGray,
Kanniah
Rajasekaran,
Franzine
Smith,
John
Sanford, and
Henry
Daniell*
Department of Molecular Biology and Microbiology and
Center for Discovery of Drugs and Diagnostics, 12722 Research Parkway,
University of Central Florida, Orlando, Florida 32826-3227 (G.D.,
H.D.); United States Department of Agriculture, Agricultural Research
Service, Southern Regional Research Center, New Orleans, Louisiana
70124-4305 (K.R.); and Sanford Scientific Inc., 877 Marshall Road,
Waterloo, New York 13165 (F.S., J.S.)
The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of
expression in transgenic tobacco (Nicotiana tabacum var.
Petit Havana) plants. Polymerase chain reaction products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and
achievement of homoplasmy, whereas northern blots confirmed 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
composition of plastid membranes compared with the membranes of
susceptible target microbes. In vitro assays with protein extracts from
T1 and T2 plants confirmed that MSI-99 was expressed at high levels to provide 88% (T1) and 96%
(T2) inhibition of growth against Pseudomonas
syringae pv tabaci, a major plant pathogen. When
germinated in the absence of spectinomycin selection, leaf extracts
from T2 generation plants showed 96% inhibition of growth
against P. syringae pv tabaci. In
addition, leaf extracts from transgenic plants (T1)
inhibited the growth of pregerminated spores of three fungal species,
Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae, 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, whereas transformed leaves showed no signs of necrosis,
demonstrating high-dose release of the peptide at the site of infection
by chloroplast lysis. In planta assays with the fungal pathogen,
Colletotrichum destructivum, showed necrotic anthracnose
lesions in non-transformed control leaves, whereas transformed leaves
showed no lesions. Genetically engineering crop plants for disease
resistance via the chloroplast genome instead of the nuclear genome is
desirable to achieve high levels of expression and to prevent
pollen-mediated escape of transgenes.
*
Corresponding author; e-mail daniell{at}mail.ucf.edu; fax
407-384-2062.
© 2001 American Society of Plant Physiologists
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