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Developing an Extraction Protocol to Identify a Molecule Responsible for Powdery Mildew Disease Resistance
Powdery white mildew is a fungal disease that infects crops, decreasing quality of growth and yield. Interestingly, in A. thaliana there is a gene mutation which appears to enable the resistance to powdery mildew disease and acts through a single molecule. Using different separation techniques, we are in the process of isolating and identifying the targeted analyte. Plant extracts containing the metabolite are first processed through C18 solid phase extraction (SPE) followed by weak anion exchange (WAX) SPE. The interaction with the WAX cartridge suggests that the compound carries a negative charge. The extraction is further fractionated using hydrophilic interaction liquid chromatography (HILIC) by separating into two fractions based on hydrophilicty. Each fraction is analyzed by nuclear magnetic resonance (NMR) followed by matrix assisted laser desorption/ionization (MALDI) in an effort to identify the molecule. The subsequent fractions are then subjected to a size exclusion method collected into 12 distinct fractions. Based on our bioassay, the target molecule eluted last, suggesting that the molecule is a small compound. Currently, we are exploring various diffusion experiments using diffusion ordered spectroscopy (DOSY) to determine the molecular weights present in the final mixture Ultimately, we will continue to purify the mixture until a single compound is isolated and we will then use single crystal X-ray diffraction for identification.
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