Biochemical Studies of a Novel Fungal Protein Toxic to Plants

本研究旨在阐明小麦核腔菌（Pyrenophora tritici-repentis）毒性背后的基本生化机制。已经表明，蛋白质PtrToxA是第一个发现的蛋白质类寄主选择性毒素，是某些小麦品种上的疾病症状的原因。PtrToxA在功能上是一种显著的蛋白质，因为它似乎能够进入植物细胞，进入叶绿体，并导致细胞死亡。人们对这些事件是如何发生的知之甚少，但考虑到最近在蛋白质表征和结晶方面的进展，该系统现在有望取得重大突破。这项为期三年的合作研究计划将通过结构研究支持的生化方法研究PtrToxA的作用机制。具体目标是（1）进行参与PtrToxA毒性的植物伴侣蛋白的生物化学研究，（2）解决天然PtrToxA和选定突变体的三维结构，以及（3）设计，创建和表征PtrToxA的定点突变体，探测生物化学作用机制。从历史上看，毒素一直是阐明基本生化机制的非常有价值的工具，同样，这项工作有望为植物疾病提供见解，帮助建立毒素诱导细胞死亡的宝贵模型系统，并揭示植物中新的生化机制。此外，由于PtrToxA序列似乎与任何已知蛋白质无关，因此该结构将有助于绘制所有已知蛋白质折叠的努力。从实践的角度来看，该项目有可能导致有效的工具，以打击晒黑点。由于该项目的跨学科性质，参与该项目的人员，从本科生到主要研究人员，将接受异常广泛的培训，并将努力确保所有参与者都接触到生物化学，细胞生物学，和生物物理方面。

This project aims to elucidate the basic biochemical mechanisms behind the toxicity of Pyrenophora tritici-repentis, a fungus that causes tan spot disease of wheat. It has been shown that a protein, PtrToxA, which is the first discovered proteinaceous host-selective toxin, is responsible for disease symptoms on certain cultivars of wheat. Functionally PtrToxA is a remarkable protein, as it appears to be able to enter plant cells, enter the chloroplast, and cause cell death. Little is known about how these events occur, but given recent progress in characterizing and crystallizing the protein, the system is now poised for a major breakthrough. This collaborative three-year research program will study the PtrToxA mechanism of action through biochemical approaches supported by structural studies. Specific aims are to (1) conduct biochemical investigations of plant partner proteins involved in PtrToxA toxicity, (2) solve the three-dimensional structures of native PtrToxA and selected mutants, and (3) design, create, and characterize site-directed mutants of PtrToxA that probe biochemical mechanisms of action. Historically, toxins have been incredibly valuable tools for elucidating basic biochemical mechanisms, and similarly, this work is expected to provide insights into plant disease, to help establish an invaluable model system for toxin-induced cell death, and to uncover novel biochemical mechanisms in plants. Also, because the PtrToxA sequence appears unrelated to any known protein, the structure will contribute to efforts to map all known protein folds. From a practical perspective, this project has the potential to lead to effective tools to combat tan spot.Broader impact: The personnel involved in the project, from undergraduates through the principle investigators, will receive unusually broad training due to the interdisciplinary nature of the project, and efforts will be made to be sure all participants are exposed to both the biochemical, cell biological, and biophysical aspects of the project.