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

使用 PBS1 诱饵系统来设计对植物寄生线虫的抗性

• 批准号: 2017314
• 负责人: Roger Innes
• 金额: $ 30万
• 依托单位: Indiana University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2017314&HistoricalAwards=false
• 关键词: Use; PBS1; Decoy; System; Engineer

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.

据估计，植物寄生线虫（PPN）每年在全球造成超过1700亿美元的农业生产损失，仅在美国就损失超过130亿美元。为了满足2050年的预期粮食需求，预计世界人口将增加20亿，我们必须将作物损失减少到PPN。 本项目的目标是利用大豆和大豆胞囊线虫（SCN）作为模式病理系统，开发基于遗传的抗PPN。 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，植物使用被病原体在感染过程中分泌的酶修饰的传感器蛋白。本项目的重点是鉴定SCN分泌的感染大豆所需的酶。一旦这种酶被鉴定，传感器蛋白将被设计成在被这些SCN酶修饰后可以激活大豆中的防御反应。 因此，这样的系统将赋予对PPN感染的抗性，而不使用昂贵且对环境有害的杀虫剂，并且应该可转移到被PPN损害的多种重要作物植物中。Innes实验室已经开发出一种新的方法，基于检测这些特定的蛋白酶来设计对这些病原体的抗性。在拟南芥中，病原体蛋白酶AvrPphB对宿主激酶PBS 1的裂解激活了强烈的防御反应。PBS 1内的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。这些“诱饵”激酶可以被匹配的蛋白酶切割，从而激活抗性。 因此，通过操纵PBS 1氨基酸序列，可以工程化识别许多病原体蛋白酶。该提案试图通过对大豆在北美最重要的病原体大豆胞囊线虫（SCN）的工程抗性将这一发现扩展到作物大豆。先前的工作已经表明，大豆PBS 1的修饰使得其被大豆花叶病毒（SMV）的NIa蛋白酶切割，从而赋予对SMV感染的完全免疫力。为了评估SCN分泌的蛋白酶是否有助于毒力，将使用RNA干扰降低候选蛋白酶基因的表达，然后评估对大豆根感染的影响。然后，通过使用生物素邻近标记鉴定大豆根细胞中的蛋白酶靶标，并使用质谱法鉴定其切割位点，进一步分析有助于感染的蛋白酶。通过CRISPR-Prime基因组编辑技术，将SCN蛋白酶切割位点插入大豆PBS 1基因中，从而培育出抗SCN感染且不含转基因的大豆品系。该奖项体现了NSF的法定使命，通过使用基金会的智力价值和更广泛的影响力评审标准进行评估，被认为值得支持。