|Rajasekaran, Kanniah - Rajah|
Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/27/2001
Publication Date: 11/1/2001
Citation: DEGRAY, G., RAJASEKARAN, K., SMITH, F., SANFORD, J., DANIELL, H. EXPRESSION OF AN ANTIMICROBIAL PEPTIDE VIA THE CHLOROPLAST GENOME TO CONTROL PHYTOPATHOGENIC BACTERIA AND FUNGI. PLANT PHYSIOLOGY. 2001. V. 127. P. 852-862. Interpretive Summary: Current protocols of genetic transformation of crop plants rely on introduction of new genes into the plant nucleus. This approach has raised the following two concerns: a) low expression of the introduced gene due to limited number of its copies (1-10) that land in the plant nucleus and b) possible escape of introduced genes to other related plants through pollen dispersal. This manuscript describes a novel procedure for transforming chloroplasts, the abundant green pigment bodies in plant cells that are responsible for photosynthesis. Chloroplast genes, either transformed or non-transformed, do not lend themselves for escape through pollen and the introduced genes in chloroplasts are expressed at a higher level, simply due to the fact that there are about 10,000 chloroplasts per cell in a mature leaf compared to one nucleus per cell. Scientists have demonstrated for the first time, in this manuscript, higher expression level of a disease resistance gene (MSI-99) in transgenic tobacco through transformation of chloroplasts. This gene produces a defense molecule that is similar to a natural one secreted from the skin of African clawed frog. Transgenic plants and their progenies have also been shown to resist or withstand several bacterial and fungal plant pathogens including mycotoxin-producing fungi. The principal users of information will be scientists involved in related research and those in the biotechnology industry.
Technical Abstract: The anti-microbial peptide MSI-99, an analogue of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco plants. PCR products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, while northern blots confirmed transcription. Accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. In vitro assays with protein extracts from T1 and T2 plants confirmed that MSI-99 was expressed at high levels to provide 88 percent and 96 percent inhibition of growth against Pseudomonas syringae pv. tabaci, a major plant pathogen. In addition, leaf extracts from transgenic plants (T1) inhibited the growth of pre-germinated spores of three fungal species, Aspergillus flavus, Fusarium moniliforme and Verticillium dahliae by more than 95 percent compared to non-transformed control plant extracts. In planta assays with the bacterial pathogen, Pseudomonas syringae pv. tabaci and with the fungal pathogen, Colletotrichum destructivum resulted in areas of necrosis around the point of inoculation in control leaves, while transformed leaves showed no signs of necrosis, demonstrating high dose release of the peptide at the site of infection by chloroplast lysis. 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.