Progress 07/30/01 to 07/15/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement (SCA) between the Agricultural Research Service (ARS) and University of Louisiana, Lafayette (ULL). Additional details of research can be found in the report for the inhouse research project, 6435-42000-019-00D, Identification and Enhancement of Seed-Based Biochemical Resistance in Crops to Aflatoxin Producing Pathogens. The project is aimed at enhancing resistance in cottonseed to infection by the fungus, Aspergillus flavus, that produces aflatoxin, a serious economic and food safety problem in U.S. agriculture. The report summarizes the continuation of the previous research project to identify promoters (triggers of gene expression) and structural genes that can enhance resistance to A. flavus in the cotton crop. The antifungal genes D4E1 and CPO-P have been placed under the control of the cottonseed storage protein (CSSP) promoter.
Constructs containing CSSP-D4E1 or CSSP- CPO-P have been transferred into tobacco and cotton to study the level of expression of these gene products in seed tissues and also to test the ability of seed present in these plants to resist invasion by A. flavus and other fungal pathogens. Studies are also continuing on the production and bioassay of one of the endogenous (internal) cotton defense proteins, an antifungal protein known as chitinase. It has been confirmed that this chitinase, if expressed at sufficient levels in plants, could be extremely effective against A. flavus based upon results of spore germination assays using purified plant extracts containing this protein. The gene has been expressed in a yeast system and is currently being purified in quantity to provide enough chitinase protein to perform additional efficacy assays against A. flavus and other plant pathogens. Using new equipment, we have modified our purification of cotton Class I chitinase. This new
purification scheme results in sufficient chitinase to enable preparation of a laboratory, antibody-based, assay of the chitinase to determine the concentrations necessary for efficacy against a variety of fungi, characterization of the biochemical properties of the enzyme, and studies of the other versions of the chitinase to determine their antifungal activities independently and in combinations. Purified cotton chitnase is currently being tested in vitro (laboratory) to determine its efficacy against A. flavus and other fungal pathogens. IMPACT: Characterization of antifungal proteins and their genes has led to new gene insertion strategies to improve resistance to aflatoxin contamination in cottonseed and other plants. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Chlan, Caryl A. 2005. Cotton Chitinases SAAS Bulletin of Biochemistry and
Biotechnology. 17:1-8.
Impacts
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement (SCA) between the Agricultural Research Service (ARS) and University of Louisiana, Lafayette (ULL). Additional details of research can be found in the report for the ARS parent research project, 6435-42000-016-00D, Field Identification of Pathogens Producing Aflatoxin Based on Detection of Gene Expression During Crop/Fungal Interactions. The project is aimed at enhancing resistance in cottonseed to infection by the fungus, Aspergillus flavus, that produces aflatoxin, a serious economic and food safety problem in U.S. agriculture. The report summarizes the continuation of the previous research project to identify promoters (triggers of gene expression) and structural genes that can enhance resistance to A. flavus in the cotton crop. The antifungal genes D4E1 and CPO-P have been placed under the control of the cottonseed storage protein (CSSP) promoter.
Constructs containing CSSP- D4E1 or CSSP-CPO-P have been transferred into tobacco and cotton to study the level of expression of these gene products in seed tissues and also to test the ability of seed present in these plants to resist invasion by A. flavus and other fungal pathogens. Studies are also continuing on the production and bioassay of one of the endogenous (internal) cotton defense proteins, an antifungal protein known as chitinase. It has been confirmed that this chitinase, if expressed at sufficient levels in plants, could be extremely effective against A. flavus based upon results of spore germination assays using purified plant extracts containing this protein. The gene has been expressed in a yeast system and is currently being purified in quantity to provide enough chitinase protein to perform additional efficacy assays against A. flavus and other plant pathogens. Using new equipment, we have modified our purification of cotton Class I chitinase. This new
purification scheme results in sufficient chitinase to enable preparation of a laboratory, antibody-based, assay of the chitinase to determine the concentrations necessary for efficacy against a variety of fungi, characterization of the biochemical properties of the enzyme, and studies of the other versions of the chitinase to determine their antifungal activities independently and in combinations. IMPACT: Characterization of antifungal proteins and their genes has led to new gene insertion strategies to improve resistance to aflatoxin contamination in cottonseed and other plants.
Impacts
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. Progress Report: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and University of Louisiana, Lafayette. Additional details of research can be found in the report for the parent CRIS 6435-42000-016-00D, "Field Identification of Pathogens Producing Aflatoxin Based on Detection of Gene Expression During Crop/Fungal Interactions." The project is aimed at enhancing resistance in cottonseed to infection by the fungus, Aspergillus flavus, that produces aflatoxin, a serious economic and food safety problem in U. S. agriculture. 1) The report summarizes the continuation of the previous research project to identify promoters (triggers of gene expression) and structural genes that can enhance resistance to A. flavus in the cotton crop. The antifungal genes D4E1 and CPO-P have been placed under the control of the cottonseed storage protein (CSSP)
promoter. Constructs containing CSSP-D4E1 or CSSP-CPO-P have been transferred into tobacco and cotton to study the level of expression of these gene products in seed tissues and also to test the ability of seed present in these plants to resist invasion by A. flavus and other fungal pathogens. 2) Studies are also continuing on the production and bioassay of one of the endogenous (internal) cotton defense proteins, a neutral chitinase. It has been confirmed that this chitinase, if expressed at sufficient levels in plants, could be extremely effective against A. flavus based upon results of spore germination assays using purified plant extracts containing this protein. The gene has been expressed in a yeast system and is currently being purified in quantity to provide enough chitinase protein to perform additional efficacy assays against A. flavus and other plant pathogens. We are also in the process of improving our purification scheme for cotton chitinases to individually isolate
and test other cotton chitianses that co-purify with the neutral chitinase. Recently we have expanded our efforts to study a chitinase gene from corn that may also be highly effective against A. flavus. Genes for chitinases with effective antifungal activities would be useful additions to the other antifungal genes being inserted into cotton for enhancing resistance in cottonseed to infection by A. flavus.
Impacts
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