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

该项目的目标是开发和优化新技术，以研究一类称为“霜霉病”的植物病原体的基因功能。许多霜霉病会对十字花科蔬菜、豌豆、黄瓜和葡萄等作物造成破坏性疾病。这些病原体不能用传统的遗传和分子技术有效地研究，因为它们的“专性”生活方式，病原体不能与宿主植物分开培养。因此，人们对霜霉病如何进化出对杀菌剂的抗性并克服其植物宿主的抗性知之甚少。该项目的pi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，并将推广其在不同霜霉病物种研究中的适用性。这些成果将使研究界能够克服理解霜霉病引起的植物疾病的分子机制和进化的主要障碍。此外，该研究将为开发基于rnai的生物杀菌剂防治霜霉病和其他作物病害的长期目标奠定重要的基础。该项目包括在“植物疾病RNA疫苗”专题背景下，向种植者和公众宣传用于控制植物疾病的基于RNA的生物杀菌剂。该项目还将为博士后学者和本科生研究人员提供兼收并蓄的培训经验。该项目将基于pi的两项突破，开发新的功能基因组工具，用于研究霜霉病病原体：首先，通过在霜霉孢子悬浊液中混合短链合成双链RNA (SS-dsRNAs)，可以触发针对霜霉病（DM）基因的RNA干扰（RNAi）。分离分析处理过的孢子或接种到植物上以评估病原体的毒力。这种方法非常简单，但由于dsRNA合成的高成本，对于大规模功能基因组学和该领域的应用来说，这种方法也非常昂贵。此外，该方法还需要优化（例如保护dsRNA）并推广到不同的DM物种。因此，第二个突破是在大肠杆菌的无核“迷你细胞”中生产和封装dsRNA的一步工艺的发展。由此产生的微细胞封装dsRNAs （ME-dsRNAs）不受环境降解的影响，可以长期保存，并且当作为喷雾应用于植物时，可以有效抵抗真菌病原体。这种保护体现了“喷雾诱导基因沉默”（SIGS），它有望成为研究和控制由病毒、真菌、线虫和昆虫引起的疾病的工具。然而，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，并测试这些配方与SS-dsRNA相比的RNAi功效。由此产生的方案将绕过dm -植物相互作用的基因型-表型研究的主要瓶颈，从分子到进化的尺度。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.