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

这个项目的目标是开发和优化新的技术来研究一类被称为霜霉病的植物病原体的基因功能。许多霜霉病会导致十字花科蔬菜、豌豆、黄瓜和葡萄等农作物的破坏性疾病。这些病原体不能用传统的遗传和分子技术进行有效的研究，因为它们的“专属”生活方式使病原体不能离开它们的宿主植物进行培养。因此，关于霜霉病如何在植物寄主中进化出对杀菌剂的抗药性并克服抗药性，人们知之甚少。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，并推广它们在研究不同霜霉病品种方面的适用性。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，这项研究将为开发基于RNAi的生物杀菌剂以防治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以RNAi为基础的生物杀菌剂用于植物病害控制，框架是在“植物病害RNA疫苗”的主题背景下进行的。该项目还将为博士后学者和本科生研究人员提供折中的培训经验。该项目将开发新的功能基因组工具，用于未被研究的霜霉病病原菌，建立在PI的两个突破的基础上：第一，RNA干扰(RNAi)可以通过在霜霉菌孢子悬浮液中混合短的、合成的、双链的RNAs(SS-dsRNAs)来触发对霜霉病(DM)基因的干扰。处理过的孢子被分离分析或接种到植物上，以评估病原菌的毒力。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，这种方法出人意料地简单，但也昂贵得令人望而却步。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一步法生产dsRNA，并将其封装在大肠杆菌的无核“微型细胞”中。由此产生的微细胞包裹的dsRNAs(ME-dsRNAs)可以防止环境退化，可以长期搁置，并在作为喷雾应用于植物时提供对真菌病原体的有效抗性。这种保护是“喷雾诱导基因沉默”(SIGS)的例证，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。然而，ME-dsRNAs和Sigs都没有在DM上进行测试。因此，这项建议的目的是开发低成本的程序，在大肠杆菌中生产“裸”和ME-dsRNA，并测试这些制剂与SS-dsRNA相比的RNAi效果。由此产生的协议将在从分子到进化的范围内绕过DM-植物相互作用的基因型-表型研究的主要瓶颈。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.