EDGE FGT: RNAi-based tools to unlock functional genomics of obligate oomycete plant pathogens

EDGE FGT：基于 RNAi 的工具，用于解锁专性卵菌植物病原体的功能基因组学

• 批准号: 2319757
• 负责人: John McDowell
• 金额: $ 40万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2319757&HistoricalAwards=false
• 关键词: EDGE; FGT; RNAi; based; tools

The goal of this project is to develop and optimize new techniques to investigate the function of genes in a class of plant pathogens called “downy mildews”. Many downy mildew species cause destructive diseases of crops that include cruciferous vegetables, peas, cucumbers, and grapes. These pathogens cannot be studied efficiently with conventional genetic and molecular techniques, because of their “obligate” lifestyle in which the pathogens cannot be cultured apart from their host plants. Therefore, little is known about how downy mildews evolve resistance to fungicides and overcome resistance in their plant hosts. The PIs of this project have developed novel techniques, based on RNA interference, to inactivate specific downy mildew genes. This project will optimize the efficiency and cost-effectiveness of these techniques and will generalize their applicability for study of diverse downy mildew species. The deliverables will enable the research community to overcome a major obstacle for understanding the molecular mechanisms and evolution of plant diseases caused by downy mildews. In addition, this research will lay important groundwork for the long-term goal of developing RNAi-based biofungicides to control downy mildews and other crop diseases. The project includes outreach to growers and the public about RNAi-based biofungicides for plant disease control, framed in the topical context of “RNA vaccines for plant diseases”. The project will also provide an eclectic training experience for postdoctoral scholars and undergraduate researchers.The project will develop new functional genomic tools for understudied downy mildew pathogens, building on two breakthroughs by the PIs: First, RNA interference (RNAi) can be triggered against downy mildew (DM) genes by mixing short, synthetic, double-stranded RNAs (SS-dsRNAs) in downy mildew spore suspensions. Treated spores are analyzed in isolation or inoculated onto plants to assess pathogen virulence. This approach is surprisingly simple but also prohibitively expensive for large-scale functional genomics and for applications in the field, due to high costs of dsRNA synthesis. Moreover, the approach needs optimization (e.g., to protect dsRNA) and generalization to diverse DM species. Accordingly, the second breakthrough is development of a one-step process for production and encapsulation of dsRNA in anucleate “mini-cells” of E. coli. The resultant minicell-encapsulated dsRNAs (ME-dsRNAs) are protected from environmental degradation, can be shelved for long periods, and provide effective resistance to fungal pathogens when applied as a spray to plants. This protection exemplifies “Spray-Induced Gene Silencing” (SIGS) which has shown promise as a tool for research and control of diseases caused by viruses, fungi, nematodes, and insects. However, neither ME-dsRNAs nor SIGS have been tested on DMs. Therefore, the aims of this proposal are to develop low-cost procedures for production of “naked” and ME-dsRNA in E. coli and test the RNAi efficacy of these formulations compared to SS-dsRNA. The resultant protocols will circumvent a major bottleneck for genotype-phenotype research on DM-plant interactions, at scales ranging from molecular to evolutionary.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.

该项目的目标是开发和优化新技术，以研究“霜霉病”一类植物病原体的基因功能，许多霜霉病物种会导致十字花科蔬菜、豌豆、黄瓜和葡萄等作物的破坏性病害。这些病原体无法通过传统的遗传和分子技术进行有效的研究，因为它们的“专一”生活方式使得它们无法脱离宿主植物进行培养，因此，人们对霜霉病如何进化知之甚少。该项目的主要负责人开发了基于 RNA 干扰的新技术，以灭活特定的霜霉病基因。该项目将优化这些技术的效率和成本效益，并将推广其技术。这些成果将帮助研究界克服了解霜霉病引起的植物病害的分子机制和进化的主要障碍。基于 RNAi 的生物杀菌剂控制霜霉病和其他作物病害的长期目标 该项目包括向种植者和公众宣传基于 RNAi 的生物杀菌剂用于植物病害控制，并在“植物病害 RNA 疫苗”的主题背景下进行开发。该项目还将为博士后学者和本科生研究人员提供不拘一格的培训经验。该项目将以 PI 的两项突破为基础，为尚未研究的霜霉病病原体开发新的功能基因组工具：首先，可以通过在霜霉病孢子悬浮液中混合短的合成双链 RNA (SS-dsRNA) 来触发针对霜霉病 (DM) 基因的 RNA 干扰 (RNAi)，对经过处理的孢子进行分离分析或接种到植物上进行评估。这种方法非常简单，但由于 dsRNA 合成成本高昂，对于大规模功能基因组学和现场应用来说也非常昂贵。此外，该方法需要优化。 （例如，保护 dsRNA）并推广到不同的 DM 物种。因此，第二个突破是开发一种在大肠杆菌无核“微型细胞”中生产和封装 dsRNA 的一步法。 dsRNA (ME-dsRNA) 可以免受环境退化的影响，可以长期保存，并且当喷洒到植物上时，可以有效抵抗真菌病原体。保护的例子是“喷雾诱导基因沉默”（SIGS），它已显示出作为研究和控制病毒、真菌、线虫和昆虫引起的疾病的工具的前景，但是，ME-dsRNA 和 SIGS 都没有在 DM 上进行过测试。因此，本提案的目的是开发在大肠杆菌中生产“裸”和 ME-dsRNA 的低成本程序，并测试这些制剂与传统制剂相比的 RNAi 功效。由此产生的方案将在从分子到进化的尺度上绕过DM-植物相互作用的基因型-表型研究的主要瓶颈。该奖项是法定使命，并且通过使用基金会的智力价值和评估进行评估，被认为值得支持。更广泛的影响审查标准。