Location: Food and Feed Safety Research
Project Number: 6054-42000-025-03-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 30, 2013
End Date: Jul 29, 2018
There are four objectives that will be addressed in this work: 1) identify if the antifungal peptide, D4E1, is being produced by cells of transgenic plants (either nuclear or plastid transformed tobacco, corn or cotton plants) expressing the D4E1 gene and in what quantities and where is the peptide localizing within the cell; 2) identify cotton cultivars or progenitors with enhanced natural resistance to Aspergillus (A.) flavus infection; 3) determine the mechanism of action of the D4E1 antifungal peptide against A. flavus and other select plant pathogens such as Fusarium verticillioides or Verticillium dahliae; and 4) develop ribonucleic acid interference (RNAi) vectors for transformation into cotton or corn to target A. flavus genes involved in growth and/or aflatoxin production.
Production and localization of the D4E1 peptide will be made possible by transforming tobacco plants with a hemagglutinin (HA)-tagged version of the D4E1 gene. A specific antibody for the HA-tag will be used to immunolocalize and quantify the HA-tagged peptide by Transmission Electron Microscopy. Seed collected from a number of different cotton cultivars (20-40) will be tested for levels of resistance to infection by Aspergillus (A.) flavus using a kernel screening assay (KSA) technique. Cultivars demonstrating increased resistance will have their seed bulked and a second round of KSAs performed to identify the most resistant cultivar(s). This cultivar(s) will then be analyzed by proteomic and genomic techniques to identify proteins/genes involved in increased resistance to A. flavus infection and/or aflatoxin production. To determine the mechanism of action of D4E1 on A. flavus a series of assays using the dye SYTOX Green will be performed. SYTOX Green cannot penetrate intact cells, and its uptake is a measure of membrane integrity. If D4E1 is lytic in nature then SYTOX Green will be able to penetrate the treated fungal hyphae causing the fluorescence emission of SYTOX to increase by more than 500-fold. We have developed a SYTOX-microtiter plate assay to monitor fungal membrane integrity following exposure to D4E1. In this assay, A. flavus spores are incubated with varying concentrations of D4E1 in the presence of SYTOX Green and the emission values quantified. Ribonucleic acid interference (RNAi) has been shown to be a powerful tool to study downregulation of expression of target genes. Genes known to be critical to A. flavus growth and/or aflatoxin production will be the basis for development of RNAi vectors for transformation into corn or cotton. Transformed plants will be analyzed for the presence of siRNAs produced by the plants dicer machinery. Plants producing the siRNAs will be inoculated with A. flavus and the level of fungal infection and aflatoxin production will be determined by standard molecular and biochemical techniques. Plants demonstrating increased resistance to A. flavus infection will be further studied under greenhouse and field conditions for resistance to fungal infection during abiotic stress conditions such as drought.