Recombinant cystatins in plants

长期以来，蛋白酶抑制剂一直被认为是开发抗虫作物的潜在生物杀虫剂。近年来，研究还描述了这些蛋白质对植物自身生理的间接影响，可能有助于引入具有病原、耐寒性或抗旱性等新的农艺性状。利用功能蛋白质组学和蛋白质工程的最新进展，本项目旨在进一步了解重组蛋白酶抑制剂在植物中的作用，并扩大其在干旱条件下保护植物的作用。植物胱抑素蛋白家族的蛋白酶抑制剂将被用作该项目的模型，因为最近的一些报告表明，它们对胁迫条件下的植物产生了积极影响。与植物蛋白水解酶具有不同亲和力的候选半胱氨酸蛋白酶将首先从一系列半胱氨酸蛋白酶变异体中分离出来，该变异体是根据在本发现计划的最后一个资金周期中开发的一种新的蛋白质设计方法设计的。然后，选定的变异体将用于表达研究，以测试根据每个变异体的目标蛋白酶在植物中触发可变效应的可行性。最后，最有希望的变异体将被用于实现马铃薯的抗旱性，被选为具有重大农艺意义的干旱敏感作物模式。该项目的第一个成果将是在当前气候变化背景下在作物中实施耐旱胁迫的新方法，在这种背景下，干旱事件的发生率可能会影响到世界各地的几种主要作物，包括马铃薯。另一项成果将是我们使用蛋白质设计和功能蛋白质组学的新工具，合理控制转基因作物中外来蛋白质预期和意外影响之间的精细平衡的能力的概念证明。

Protease inhibitors have long been recognized as potential biopesticides for the development of pest-resistant crops. In recent years, studies have also described indirect effects for these proteins on the plant's own physiology, potentially useful to introduce such new traits of agronomical value as pathogen, cold or drought tolerance. Taking advantage of recent developments in functional proteomics and protein engineering, this project aims at further understanding the effects of recombinant protease inhibitors in plants, and at expanding their usefulness to protect plants in drought conditions. Protease inhibitors of the plant cystatin protein family will be used as model for the project, given the number of recent reports suggesting their positive impact on plants under stress conditions. Candidate cystatins with different affinities for plant proteases will first be isolated among a collection of cystatin variants designed based on a novel protein design approach developed during the last funding cycle of this Discovery program. The selected variants will then be used for expression studies to test the feasibility of triggering variable effects in the plant depending on each variant's target proteases. Finally, the most promising variants will be used to implement drought tolerance in potato selected as a drought-susceptible crop model of major agronomic significance. A first output of this project will be a novel way to implement drought stress tolerance in crop plants, in the current climate change context where an increased incidence of drought episodes is likely to affect several major crops worldwide, including potato. Another output will be a proof-of-concept of our ability to rationally controlling the fine balance between intended and unintended effects of foreign proteins in genetically modified crops using the new tools of protein design and functional proteomics.