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United States Department of Agriculture

Agricultural Research Service

Title: Control of Preharvest Aflatoxin Contamination in Cotton - Update on New Antifungal Pepetides and Chloroplast Expression

Authors
item Rajasekaran, Kanniah
item Cary, Jeffrey
item Ruhlman, T - UNIV OF NEW ORLEANS
item Cleveland, Thomas

Submitted to: Multicrop Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop-The Peanut Foundation
Publication Type: Abstract Only
Publication Acceptance Date: October 28, 2004
Publication Date: February 15, 2005
Citation: Rajasekaran, K., Cary, J.W., Ruhlman, T.A., Cleveland, T.E. 2005. Control of Preharvest Aflatoxin Contamination in Cotton - Update on New Antifungal Peptides and Chloroplast Expression. In: Proceedings of the 2004 Multicrop Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop-The Peanut Foundation, October 25-28, 2004, Sacramento, California. p. 36.

Technical Abstract: In order to combat the problem of preharvest aflatoxin contamination, we have made many advances using transgenic approaches in our lab. Most notably, we have demonstrated the antifungal activities of transgenic tobacco or cotton plants expressing the antifungal peptide D4E1 or the chloroperoxidase (cpo-p) that tolerate or resist invasion by many microbial pathogens, including Aspergillus flavus. We are currently multiplying seed of selected cotton transformants for field testing against soil-borne vascular pathogens and A. flavus. Meanwhile, we are also continually evaluating several new gene constructs for efficacy against A. flavus. Some of the approaches are listed below: 1) Following the promising results on A. flavus control that we demonstrated earlier with transgenic tobacco and cotton plants expressing the synthetic lytic peptide D4E1, we are currently addressing some of the problems inherent in the synthetic peptide technology: a) by making modified synthetic peptides that are more potent against A. flavus, yet not harmful to the plant; b) by making peptides that are large enough to be identified in planta and analyzed by immunoassays or western blots; c) targeting the expression of these peptides to plastids or peroxisomes to shield them from proteolytic degradation. 2) Antifungal activities of purothionin and hordothionin (provided by Dr. R. Skadsen, ARS, Madison, Wisconsin) were examined in our lab. These proteins, when assayed in vitro against germinating conidia of A. flavus, showed an inhibitory level of 10 uM. Cotton transformation experiments have been carried out with the horodthionin (Hth 1) construct driven by the cottonseed storage protein promoter (provided by Dr. Caryl Chlan, University of Louisiana, Lafayette, LA). Analyses of these plants are yet to be carried out for expression and phenotype in cottonseeds. 3) We have also engineered a synthetic cpo-p gene in an effort to optimize the codon usage of this bacterial gene for expression in plants. Analysis of crude extracts from transgenic tobacco leaves (nuclear expressing) indicate ample enzyme activity compared to the non-optimized version. We are in the process of making proper gene constructs with the optimized version for chloroplast transformation as well. 4) We have made new constructs with the antifungal gene, MOD 1, a synthetic gene that encodes the active form of RIP 1 from corn. Results from Dr. Weissinger's lab indicate that RIP 1 is not phytotoxic and is effective against A. flavus in transgenic peanuts. The efficacy of this gene will be tested in both tobacco and cotton in our lab. 5) In order to improve the efficiency of antifungal gene expression, we have been evaluating expression of transgenes in tobacco chloroplast transformants. Results are being presented in a separate poster (Ruhlman et al).

Last Modified: 9/22/2014