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United States Department of Agriculture

Agricultural Research Service

Title: Expressing Antifungal Genes in the Chloroplast Genome

Authors
item Ruhlman, Tracey - UNO, NOLA
item Rajasekaran, Kanniah
item Cary, Jeffrey

Submitted to: Multicrop Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop-The Peanut Foundation
Publication Type: Abstract Only
Publication Acceptance Date: September 1, 2004
Publication Date: October 25, 2004
Citation: Ruhlman, T., Rajasekaran, K., Cary, J.W. 2004. Expressing antifungal genes in the chloroplast genome [abstract]. Multicrop Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop-The Peanut Foundation. October 25-28, 2004, Sacramento, California. p. 41.

Technical Abstract: Phytopathogens and mycotoxin producing saprophytes can significantly impact the value of commodity crops and cause concern for food and feed safety. Introduction of microbial resistance may be achieved through genetic manipulation of susceptible crop species. Genetic transformation of plastid genomes via particle bombardment offers many benefits over nuclear transformation. For example, proteins can accumulate to high levels (up to 50 percent of total soluble protein) due to increased expression; proteins of interest are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic proteases; maternal inheritance of oganellar genomes allows for control of transgene escape through pollen and single copy insertion through homologous recombination eliminates complications such as transgene silencing. Our research aims to confer resistance to microbial pathogens and mycotoxin producers by stable integration and expression of novel antifungal genes such as chloroperoxidase (cpo-p) or D4E1 in the plastid genome. Tobacco transformants were identified by repeated rounds of selection on regeneration media with toxic levels of spectinomycin, resistance to which was conferred by the aminoglycoside-3'-adenyltransferase (aadA) gene in the chloroplast transformation cassette. PCR analysis with primers specific for our transgene has confirmed integration in the plastid genome. Western analysis confirms the presence of the gene product in higher abundance in chloroplast transformants over nuclear. Preliminary gene expression analyses have indicated that antimicrobial peptides/proteins produced within chloroplasts have higher levels of antifungal proteins due to orgnanelle proliferation and presumably protection of proteins within the chloroplast envelope. Following cell and organelle lysis that occurs as a result of microbial invasion, antifungal proteins will be released at high concentration at the site of infection, thus inhibiting pathogen advance and spread of disease and/or production of mycotoxins.

Last Modified: 11/22/2014
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