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到特定病原体蛋白酶的存在来实现，蛋白酶是一种像分子剪刀一样切割宿主中特定目标蛋白的酶。具体来说，一个大豆基因将被修改，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进行基因转移，因此这种转基因大豆不应被视为转基因生物，这将简化监管审批和消费者接受程度。如果这种方法在大豆上取得成功，它应该适用于所有作物，这将减少我们对对环境有害的农药的依赖，同时提高作物产量，增加世界粮食安全。许多重要的植物病原体依靠蛋白酶来感染寄主植物。Innes实验室正在开发一种新的方法，通过检测这些特定的蛋白酶来设计对这些病原体的抗性。这个系统源于他们对来自丁香假单胞菌的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，将它们切割在一个单一的位置。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。具体来说，AvrPphB切割宿主激酶PBS1激活rps5介导的抗性。Innes实验室最近表明，PBS1（7个氨基酸）内的AvrPphB识别序列可以被其他病原体蛋白酶的识别序列所取代。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，可以设计RPS5来识别几乎任何病原体蛋白酶。该项目试图将这一发现推广到农作物上。作为概念的证明，大豆PBS1同源基因将在不同的结构中进行修饰，以检测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都携带一种内源抗病基因，可以检测AvrPphB蛋白酶的活性，因此我们希望通过对一种在开花植物中高度保守的内源大豆PBS1蛋白进行修饰，可以检测到相应的蛋白酶。该项目将为高中和本科阶段未被充分代表的少数民族提供参与实践研究的机会，并参与可持续农业的主题。

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.