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”的植物病原体中基因的功能。许多霜霉病种类引起包括十字花科蔬菜、豌豆、黄瓜和葡萄在内的作物的破坏性疾病。这些病原体不能有效地研究与传统的遗传和分子技术，因为他们的“专性”的生活方式，其中病原体不能培养远离其宿主植物。因此，关于霜霉病如何进化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已经开发了基于RNA干扰的新技术来检测特异性霜霉病基因。该项目将优化这些技术的效率和成本效益，并将推广其适用于不同霜霉病物种的研究。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演变的主要障碍。此外，这项研究将为开发基于RNAi的生物杀菌剂以控制霜霉病和其他作物病害的长期目标奠定重要基础。该项目包括向种植者和公众宣传用于植物病害控制的基于RNAi的生物杀真菌剂，并以“植物病害RNA疫苗”为主题。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体验。该项目将开发新的功能基因组工具，用于研究尚未充分研究的霜霉病病原体，建立在PI的两项突破基础上：首先，通过在霜霉病孢子悬浮液中混合短的合成双链RNA（SS-dsRNA），可以触发针对霜霉病（DM）基因的RNA干扰（RNAi）。将经处理的孢子隔离分析或接种到植物上以评估病原体毒力。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该领域的应用来说也过于昂贵。此外，该方法需要优化（例如，以保护dsRNA）和推广到不同的DM物种。因此，第二个突破是开发了在E.杆菌所得的小细胞包封的dsRNA（ME-dsRNA）被保护免于环境降解，可以长期搁置，并且当作为喷雾剂施用到植物时提供对真菌病原体的有效抗性。这种保护证实了“喷雾诱导基因沉默”（SIGS），该技术已显示出作为研究和控制由病毒、真菌、线虫和昆虫引起的疾病的工具的前景。然而，ME-dsRNA和SIGS均未在DM上测试。因此，本研究的目的是开发一种低成本的方法，在大肠杆菌中生产“裸”和ME-dsRNA。大肠杆菌中，并测试这些制剂与SS-dsRNA相比的RNAi功效。由此产生的协议将绕过一个主要的瓶颈，基因型表型研究DM植物相互作用，从分子到evolutionary.This规模反映了NSF的法定使命，并已被认为是值得的支持，通过评估使用基金会的智力价值和更广泛的影响审查标准。

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.