Bioengineering plant resilience to viral infection

据估计，到2050年，粮食产量需要增加高达70%，才能养活未来的人口。这一挑战的核心将是发现可持续的方法来保护农作物免受疾病，包括病毒感染。我的NSERC研究的长期目标是了解病毒蛋白酶在病毒复制和免疫逃避中的作用，并利用所获得的知识为动植物开发抗病毒策略。最近，我们开发了产生基于蛋白质的病毒蛋白酶抑制剂的方法。这些抑制剂是泛素(UbV)的氨基酸变体，与这些酶紧密结合并特异地结合。我的短期目标是开发和鉴定UBV抑制剂，以阻断一些致病植物病毒的蛋白酶活性，并检验我的假设，即表达这些UBV的转基因作物对病毒感染具有弹性。OBJ 1：植物病毒蛋白水解酶的生化特性及其紫外线的抑制作用。来自7种植物病毒(代表5个主要病毒属)的蛋白酶已被用于UBV抑制剂的开发。阻断其活性的UV将从泛素变体的噬菌体展示文库中识别出来。紫外线的抑制效力将通过体外结合试验进行评估。将通过测量UBV与来自拟南芥的一组功能相关的植物细胞蛋白酶的结合能力来评估UBV对其目标蛋白酶的特异性。OBJ 2：植物病毒蛋白酶活性的结构基础和紫外线的抑制。关于植物病毒蛋白如何参与病毒复制或它们如何识别泛素的结构基础，人们知之甚少。我们将通过测定这些酶及其与紫外线形成的复合体的X射线结构来阐明植物病毒蛋白水解酶活性的分子基础及其对紫外线的抑制作用。OBJ 3：植物中UBV抑制活性：从原生质体到转基因植物。来自OBJ 1的UBV将被验证为它们有能力阻止活的植物原生质体中的蛋白酶活性。那些具有最大效力的植物将被用于改造植物，以应对随后的病毒挑战。抗芜菁黄花叶病毒和玉米Rayado Fino病毒蛋白水解酶的UBV已经问世。与马尼托巴大学(UM)和美国农业部(Fargo，ND)的植物生物学家合作，我们将确定UBV在拟南芥和油菜中的表达是否能保护这些植物免受芜菁黄花叶病毒的侵染，以及UBV在玉米中的表达是否能保护玉米免受雷诺病毒的侵染。这次OBJ的结果将检验我的假设，即UbV可以预防病毒感染。我提出了一项战略，可以可持续地保护许多经济作物免受病毒性疾病的侵袭，从而对全球粮食安全产生积极影响。这项研究的多学科性质将对受训者具有极大的吸引力，使他们能够在开发突破性技术的同时，获得在学术界、行业或政府从事职业所需的技能。

It is estimated that food production will need to increase up to 70% by 2050 to sustain future populations. Central to this challenge will be the discovery of sustainable ways to protect crops from disease, including viral infections. The long-term goal of my NSERC research is to understand the role of viral proteases in virus replication and immune evasion, and to use the knowledge gained to develop antiviral strategies for plants and animals. Recently, we have developed the means to generate protein-based inhibitors of viral proteases. The inhibitors are amino acid variants of ubiquitin (UbVs) that bind tightly and specifically to these enzymes. My short-term goal is to generate and characterize UbV inhibitors that block the activity of proteases from a number of pathogenic plant viruses, and to test my hypothesis that transgenic crops that express these UbVs are resilient to viral infection. Obj 1: Biochemical characterization of plant virus proteases and their inhibition by UbVs. Proteases from 7 plant viruses (representing 5 major virus genera) have been targeted for UbV inhibitor development. UbVs that block their activity will be identified from a phage display library of ubiquitin variants. The inhibitory potency of the UbVs will be assessed using in vitro binding assays. Specificity of the UbVs for their target proteases will be assessed by measuring their ability to bind a panel of functionally related plant cell proteases from Arabidopsis thaliana. Obj 2: Structural basis of plant virus protease activity and inhibition by UbVs. Little is known about the structural basis for how plant virus protases participate in virus replication or how they recognize ubiquitin. X-ray structures of the proteases and the complexes they form with UbVs will be determined to elucidate the molecular basis of plant virus proteases activity and their inhibition by UbVs. Obj 3: UbV inhibitor activity in planta: from protoplasts to transgenic plants. UbVs from Obj 1 will be verified for their ability to block protease activity in live plant protoplasts. Those with greatest potency will be used to transform plants for subsequent virus challenge. UbVs against Turnip Yellow Mosaic Virus and Maize Rayado Fino virus proteases are in-hand. Working with plant biologists at U of Manitoba (UM) and the USDA (Fargo, ND), we will determine if UbV expression in Arabidopsis and canola protects these plants from Turnip Yellow Mosaic Virus, and if UbV expression in maize protects against Maize Rayado Fino Virus.  Results from this Obj will test my hypothesis that UbVs protect against viral infection. I propose a strategy that could sustainably protect numerous cash crops from viral disease and thus positively impact global food security. The multidisciplinary nature of the research will be highly engaging for trainees and will allow them to develop a ground breaking technology while they acquire the skills they need to pursue careers in academia, industry or government.