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United States Department of Agriculture

Agricultural Research Service

Research Project: IDENTIFICATION AND ENHANCEMENT OF SEED-BASED BIOCHEMICAL RESISTANCE IN CROPS TO AFLATOXIN PRODUCING PATHOGENS

Location: Food and Feed Safety Research

Title: Expression of chloroperoxidase from Pseudomonas pyrrocinia in tobacco plastids for fungal resistance

Authors
item Ruhlman, Tracey - UNIV OF CENTRAL FLORIDA
item Rajasekaran, Kanniah
item Cary, Jeffrey

Submitted to: Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 19, 2014
Publication Date: N/A

Interpretive Summary: Development of transgenic crops resistant to preharvest aflatoxin contamination can be achieved by transforming the nucleus or chloroplasts of the target plants. Important benefits of chloroplast transformation include higher levels of gene expression and prevention of transgene escape through pollen. A higher level of transgene expression is essential to control Aspergillus fungus which causes the aflatoxin contamination. We have demonstrated in this report significant control of Aspergillus and other fungal pathogens through chloroplast-transformed tobacco plants expressing an antifungal chloroperoxidase gene of bacterial source. Transgenic approaches such as plastid biotechnology are maturing beyond the introductory model systems into crop species and hold the promise to deliver efficacy in the field. By applying this expertise through creative collaborative projects aimed at controlling infestation and subsequent contamination of commodities we should be able to provide a higher level of safety to consumers of agricultural products in the future.

Technical Abstract: While genetic improvement of susceptible crop species may enhance resistance to microbial pathogens and facilitate reduced pesticide load, the possibility for transmission of novel genes to wild relatives has hampered acceptance of genetically modified (GM) crops in some markets. Chloroplast transformation presents an attractive alternative to nuclear transformation and offers the potential to ameliorate environmental concerns. Most agronomically important species exhibit maternal inheritance of organellar genomes eliminating the threat of transgene escape through pollen and gene silencing is absent. The chloroperoxidase (cpo-p) gene from Pseudomonas pyrocinnia was transformed into the chloroplast genome (plastome) of Nicotiana tabacum. Transformants were identified by selective regeneration on media with 500 mg L-1 spectinomycin, resistance to which was conferred by the aminoglycoside-3’-adenyltransferase (aadA) gene in the plasmid vector. Southern analysis has confirmed integration in the plastome. Western analysis confirms the presence of the CPO-P in higher abundance in transplastomic plants than in cpo-p nuclear transformants. Northern analysis of primary transformants for cpo-p showed 15-fold higher transcript abundance than nuclear transformants; yet this extent of enhancement was not observed in western blot, enzyme or bioassay, indicating a bottleneck at the post-transcriptional level. In an effort to enhance the rate of translation a new construct was developed replacing the single ribosome binding site between aadA and cpo-p with the tobacco psbA 5’ UTR.

Last Modified: 9/21/2014
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