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.

该项目的目的是开发和优化新技术，以研究一类称为“唐尼米尔夫”的植物病原体中基因的功能。许多淡淡的千毫升物种会引起农作物的破坏性疾病，包括十字花科蔬菜，豌豆，黄瓜和图形。这些病原体不能用常规的遗传和分子技术有效地研究，因为它们的“义务”生活方式无法与宿主植物分开培养病原体。因此，对于淡淡的米勒斯如何发展对杀菌剂的抗性并克服植物宿主的耐药性知之甚少。该项目的PI开发了基于RNA干扰的新技术，以使特定的倾斜基因失活。该项目将优化这些技术的效率和成本效益，并将其推广到研究潜水少年物种的研究。可交付成果将使研究界能够克服理解唐尼米勒斯引起的植物疾病的分子机制和进化的主要障碍。此外，这项研究将为开发基于RNAi的生物杀菌剂来控制倾向米勒斯和其他作物疾病的长期目标为重要的基础。该项目包括向种植者和公众宣传基于RNAi的生物膜剂，以控制植物性疾病，这是在“植物疾病的RNA疫苗”的局部背景下构建的。该项目还将为博士后学者和本科研究人员提供一种培训培训经验。该项目将开发新的功能基因组工具，以理解Millew病原体，以PIS的两种突破为基础：首先，RNA干扰（RNAI）可以通过与Down，Doup doup down toup doup doup doup doup doup doupds-nilth，santth Minthere的基因（RNAI）（RNAI）（RNAI）触发。孢子悬浮液。分别分析处理过的孢子或将其接种到植物上以评估病原体病毒。这种方法令人惊讶地简单，但由于DSRNA合成的高成本，对于大规模功能基因组学和现场的应用也很昂贵。此外，该方法需要优化（例如，保护DSRNA）和对潜水DM物种的概括。根据以下内容，第二个突破是开发了大肠杆菌Anuclete“迷你细胞”中DSRNA生产和封装的一步过程。所得的微型元素封装的DSRNA（ME-DSRNA）受到保护免受环境降解的保护，可以长期搁置，并在将其作为喷雾剂施加到植物上时对真菌病原体的有效抗性。该保护示例为“喷雾诱导的基因沉积”（SIGS），该保护表现出了有望作为研究和控制病毒，真菌，线虫和昆虫的疾病的工具。但是，在DMS上均未对ME-DSRNA和SIGS进行测试。因此，该提案的目的是开发出低成本的程序，用于在大肠杆菌中生产“裸”和Me-dsRNA，并与SS-DSRNA相比测试这些公式的RNAi效率。在从分子到进化的量表上，最终的方案将避免针对DM植物相互作用的基因型 - 表型研究的主要瓶颈。该奖项反映了NSF的法定任务，并被认为是通过基金会的知识分子和更广泛影响的审查审查标准来通过评估来诚实地通过评估来诚实的。