EAGER: Engineering Decoys to Detect Pathogen Proteases and Activate Host Resistance

该项目的重点是利用内源植物基因来设计对重要作物病原体的抗性。 这将通过使植物能够检测特定病原体蛋白酶的存在来实现，这些蛋白酶是像分子剪刀一样切割宿主中特定靶蛋白的酶。具体而言，将修改单个大豆基因，这将使大豆能够检测病原体蛋白酶的活性，然后激活防御反应。这种蛋白酶识别系统不需要在物种之间转移基因，因此这种转基因大豆不应被视为转基因生物，这应该简化监管部门的批准，以及消费者的接受。 如果这种方法在大豆上取得成功，它应该适用于所有作物，这将减少我们对有害环境的农药的依赖，同时提高作物产量和增加世界粮食安全。Innes实验室正在开发一种基于检测这些特定蛋白酶的工程抗病原体的新方法。 该系统源于他们对来自细菌假单胞菌的AvrPphB蛋白酶的研究。AvrPphB被注射到宿主细胞中，在那里它靶向调节基础防御反应的蛋白激酶家族，在单个位置切割它们。在拟南芥中，这种切割事件被抗病（R）蛋白RPS5检测到，然后激活防御反应。具体而言，AvrPphB对宿主激酶PBS1的切割激活了RPS5介导的抗性。Innes实验室最近表明，PBS 1中的AvrPphB识别序列（7个氨基酸）可以被其他病原体蛋白酶的识别序列取代。然后这些"诱饵"激酶可以被匹配的蛋白酶切割并激活RPS5。 因此，通过操纵PBS1氨基酸序列，可以改造RPS5以识别几乎任何病原体蛋白酶。该项目旨在将这一发现扩展到作物。 作为概念验证，将在单独的构建体中修饰大豆PBS 1直系同源物，以检测来自大豆花叶病毒和亚洲大豆锈病的蛋白酶。 由于大多数大豆品种携带检测AvrPphB蛋白酶活性的内源性抗病基因，因此预期在开花植物中高度保守的内源性大豆PBS 1蛋白的修饰将能够检测相应的蛋白酶。该项目将为在高中和大学一级代表性不足的少数群体提供参与实践研究和参与可持续农业专题的机会。

This project is focused on using endogenous plant genes to engineer resistance to important crop pathogens. This will be accomplished by enabling plants to detect the presence of specific pathogen proteases, which are enzymes that act like molecular scissors to cut specific target proteins in the host. Specifically, a single soybean gene will be modified, which should then enable soybean to detect the activity of pathogen proteases, and then activate defense responses. This protease recognition system does not require transfer of genes between species, thus such modified soybeans should not be considered GMOs, which should simplify regulatory approval, as well as consumer acceptance. If this approach is successful in soybean, it should be applicable to all crop plants, which would decrease our dependence on environmentally harmful pesticides, while boosting crop yields and increasing world food security.Many important plant pathogens depend on proteases to infect host plants. The Innes laboratory is developing a novel method for engineering resistance to such pathogens based on detecting these specific proteases. This system arises from their work on the AvrPphB protease from the bacterium Pseudomonas syringae. AvrPphB is injected into host cells, where it targets a family of protein kinases that regulate basal defense responses, cleaving them in a single position. In Arabidopsis, this cleavage event is detected by the disease resistance (R) protein RPS5, which then activates defense responses. Specifically, cleavage of the host kinase PBS1 by AvrPphB activates RPS5-mediated resistance. The Innes laboratory has recently shown that the AvrPphB recognition sequence within PBS1 (seven amino acids) can be replaced by the recognition sequence for other pathogen proteases. These 'decoy' kinases can then be cleaved by the matching protease and activate RPS5. Thus, by manipulating the PBS1 amino acid sequence, it is possible to engineer RPS5 to recognize just about any pathogen protease. This project seeks to extend this discovery to crop plants. As a proof of concept, a soybean PBS1 ortholog will be modified, in separate constructs, to detect proteases from Soybean mosaic virus and Asian Soybean Rust. Because most soybean varieties carry an endogenous disease resistance gene that detects AvrPphB protease activity, it is expected that modification of an endogenous soybean PBS1 protein, which is highly conserved among flowering plants, will enable detection of corresponding proteases. This project will provide underrepresented minorities at the high school and undergraduate levels with opportunities to participate in hands-on research, and to engage in the topic of sustainable agriculture.