Use of the PBS1 Decoy System to Engineer Resistance to Plant-Parasitic Nematodes

据估计，植物寄生线虫每年在全球造成超过1700亿美元的农业生产损失，仅在美国就造成了超过130亿美元的损失。2050年世界人口预计将增加20亿，为了满足预期的粮食需求，我们当务之急是减少PPN造成的作物损失。本项目的目标是利用大豆和大豆胞囊线虫(SCN)作为模式病原菌，开发基于遗传的对PPNS的抗性。植物有能力检测致病生物(病原体)，然后激活强大的防御反应，最终导致局部细胞死亡，以阻止入侵者。为了检测病原体，植物使用传感器蛋白，这些蛋白被病原体在感染过程中分泌的酶所修饰。该项目的重点是鉴定SCN分泌的感染大豆所需的酶。一旦鉴定出这样的酶，就会设计出能够在这些SCN酶修饰后激活大豆防御反应的传感器蛋白。因此，这样的系统可以在不使用昂贵的和对环境造成破坏的杀虫剂的情况下赋予对PPN感染的抵抗力，并且应该可以转移到被PPN破坏的多种重要作物上。许多植物病原体依赖于蛋白酶来感染宿主植物。Innes实验室已经开发出一种新的方法，通过检测这些特定的蛋白酶来提高对这些病原体的抗性。在拟南芥中，病原菌蛋白水解酶AvrPphB裂解宿主蛋白Pbs1激活了一种强烈的防御反应。PBS1(7个氨基酸)中的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，从而激活抵抗力。因此，通过操纵PBS1氨基酸序列，有可能工程识别许多病原体蛋白水解酶。这项提议旨在通过对北美最重要的大豆胞囊线虫(SCN)的工程抗性来将这一发现扩展到作物大豆。先前的工作表明，对大豆PBS1进行修饰，使其被大豆花叶病毒(SMV)的Nia蛋白酶切割，可以对SMV感染产生完全免疫力。为了评估SCN分泌的蛋白酶是否与毒力有关，将利用RNA干扰技术降低候选蛋白酶基因的表达，然后评估其对大豆根部侵染的影响。然后，通过使用生物素邻近标记确定它们在大豆根细胞中的靶标，并使用质谱学确定它们的切割位置，进一步分析导致感染的蛋白酶。然后，将使用CRISPR-Prime基因组编辑将SCN蛋白酶裂解位点插入到大豆PBS1基因中，从而产生抗SCN感染和不含转基因的大豆品系。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Plant-parasitic nematodes (PPNs) are estimated to cause over $170 billion in lost agricultural production globally each year, with losses of over $13 billion in the United States alone. To meet expected food demand in 2050, when the world is projected to have an additional 2 billion people, it is imperative that we reduce crop losses to PPNs. The goal of this project is to develop genetic-based resistance to PPNs using soybean and the soybean cyst nematode (SCN) as model pathosystem. Plants have the ability to detect disease-causing organisms (pathogens) and then activate a robust defense response that ultimately leads to localized cell death to stop the invader. To detect pathogens, plants use sensor proteins that are modified by enzymes that pathogens secrete during the infection process. This project focuses on identifying enzymes secreted by SCN that are required for infection of soybean. Once such enzymes are identified, sensor proteins will be designed that can activate defense responses in soybean upon modification by these SCN enzymes. Such a system would thus confer resistance to infection by PPNs without the use of costly and environmentally damaging pesticides and should be transferrable to a wide array of important crop plants that are damaged by PPNs.Many plant pathogens depend on proteases to infect host plants. The Innes laboratory has developed a novel method for engineering resistance to such pathogens based on detecting these specific proteases. In Arabidopsis, cleavage of the host kinase PBS1 by the pathogen protease AvrPphB activates a strong defense response. 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, which activates resistance. Thus, by manipulating the PBS1 amino acid sequence, it is possible to engineer recognition of many pathogen proteases. This proposal seeks to extend this discovery to the crop plant soybean by engineering resistance to its single most important pathogen in North America, soybean cyst nematode (SCN). Prior work has shown that modification of soybean PBS1 so that it is cleaved by the NIa protease of soybean mosaic virus (SMV) confers complete immunity to infection by SMV. To assess whether proteases secreted by SCN contribute to virulence, the expression of candidate protease genes will be reduced using RNA interference and then the impact on soybean root infection will be assessed. Proteases that contribute to infection will then be further analyzed by identifying their targets in soybean root cells using biotin proximity labeling, and their cleavage sites identified using mass-spectrometry. SCN protease cleavage sites will then be inserted into soybean PBS1 genes using CRISPR-Prime genome editing, thus generating soybean lines that are resistant to SCN infection and free of transgenes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.