Structure-Activity Relationships and Modes of Action of Plant Antifungal Defensins

真菌感染是食品生产和储存中的一个严重问题。真菌不仅会导致粮食生产的严重损失，而且还会在储存的谷物中产生强致癌物。植物产生一种叫做防御素的小蛋白质，这种蛋白质参与防御真菌病原体。防御素是富含半胱氨酸的蛋白质，在低浓度下抑制广泛的真菌生长。该项目的长期目标是了解这些天然抗真菌剂的作用模式，并最终将这些知识应用于保护作物和消费者。植物防御素的氨基酸序列差异很大，但它们具有共同的结构元件。从苜蓿中分离到两种防御素MsDef 1和MsDef 2。虽然它们的氨基酸序列有65%的相同性，但它们对真菌的抗菌活性在谱和形态上都有明显的差异。PI最近的工作首次表明，这些防御素之一MsDef1阻断哺乳动物细胞中的特定钙通道，而MsDef2则没有。相反，从萝卜中分离出的一种结构相关的高效抗真菌防御素并不阻断这种钙通道。 有人提出，MsDef 1抑制真菌生长，通过破坏真菌菌丝中的钙梯度，植物防御素具有不同的作用模式。PI实验室的工作还表明，真菌和哺乳动物的钙通道比以前认为的具有更多的功能相似性。该项目将利用模式真菌粗糙脉孢菌中可用的电生理、分子和遗传工具直接测试这些假设。苜蓿防御素的三维结构将被确定，这些结构将用于确定钙通道识别所需的结构基序，这些肽的活性位点将通过突变分析来确定。此外，这些肽阻断真菌细胞中特定钙通道的能力将被确定，以进一步研究真菌和动物钙通道之间的进化联系。这项研究不仅有助于我们了解防御素如何阻止真菌生长，还将为抗病转基因作物的开发铺平道路。

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.