Fungal infection is a serious problem in both the production and storage of food. Not only can fungi cause severe losses in food production but can also produce potent carcinogens in stored grain. Plants produce small proteins called defensins that are involved in defense against fungal pathogens. Defensins are cysteine-rich proteins that inhibit the growth of a broad range of fungi at low concentrations. The long-range goal of this project is to understand the mode of action of these natural antifungal agents and to eventually apply that knowledge to protect both crops and consumers. Plant defensins are highly varied in their amino acid sequences, but they share common structural elements. Two defensins, MsDef1 and MsDef2, have been isolated from alfalfa. While their amino acid sequences are 65% identical, they have markedly different antifungal activity in both spectrum and morphological effect on fungi. Recent work by the PI has shown for the first time that one of these defensins, MsDef1, blocks a specific calcium channel in mammalian cells, whereas MsDef2 does not. In contrast, a structurally related highly potent antifungal defensin isolated from radish does not block this calcium channel. It is proposed that MsDef1 inhibits fungal growth through disruption of a calcium gradient in the fungal hyphae and that plant defensins have different modes of action. Work from the PI's lab also suggests that fungal and mammalian calcium channels share more functional similarity than previously thought. This project will directly test these hypotheses by taking advantage of the electrophysiological, molecular, and genetic tools available in the model fungus Neurospora crassa. The three-dimensional structures of the alfalfa defensins will be determined and these structures will be used to ascertain the structural motif required for calcium channel recognition and the active sites of these peptides will be determined by mutational analysis. In addition, the ability of these peptides to block a specific calcium channel in fungal cells will be determined to further examine the evolutionary connection between the fungal and animal calcium channels. This research will not only aid in our understanding of how defensins arrest the growth of fungi but will also pave the way for the development of disease resistant transgenic crops.
