EAGER: Single-cell, spatial transcriptomics of plant-fungal interactions using a maskless array technology

这项提议的目标是开发新技术，以检测单个活细胞产生的RNA分子。细胞含有成千上万的rna，一个细胞产生的rna可能与邻近细胞中的rna不同。在每个细胞的基础上检测这些差异并不容易，尤其是对植物细胞而言。该项目将创造一种方法，通过保留空间信息来超越过去的技术限制，从而使区分哪些rna来自哪些细胞成为可能。这种新方法将通过鉴定受到真菌病原体感染的单个植物细胞中的rna来进行试验。这是一个高风险的项目，涉及复杂的实验设计，将具有挑战性的实施。如果成功，结果将为植物生物学家提供一个革命性的新工具来研究细胞特异性基因- rna -性状关系。这可能会导致在了解植物疾病方面的新发现，进而可能推动培育抗病植物的努力。该项目将由一名博士后科学家领导，他将接受植物生物学、显微镜学、基因组学和计算科学等多学科的培训。在植物组织切片、细胞类型甚至单细胞中，已经建立了几种转录分析方法。这些方法通常需要细胞分选、转基因植物、原生质体生产或其他有害或费力的过程。大多数这些技术在实施过程中失去了大部分或全部的空间分辨率，而那些为RNA定位提供高空间分辨率的技术在表征转录本的数量上缺乏广度，特别是对于像小RNA这样更具挑战性的RNA。与其他生物相比，植物小rna在更多的途径中发挥作用，然而，除了荧光原位杂交显微镜（fluorescence in situ hybridization microscopy）之外，没有其他技术可以对它们进行单细胞分析，通常一次只对一个或几个小rna（或mrna）进行分析。一种新的方法，空间转录组学，已经被描述，克服了许多这些限制的mRNA分析；RNA-seq文库是在切片材料中使用具有高位置分辨率的微阵列制作的。然而，空间转录组学尚未开发用于小rna或裂解mrna，并且迄今为止描述的实现仍然存在各种缺点。该项目将极大地推进该方法，使用基于无掩模阵列合成仪器的高分辨率阵列（14微米网格和1微米线性间隙），实现对植物和其他潜在生物中多种类型rna的近单细胞分析。这项工作的试验台是一个系统，在这个系统中，这种高分辨率的分析将提供大量信息，即植物与病原体的相互作用，将测量局部和系统的转录反应。该奖项由生物科学理事会分子和细胞生物科学部的遗传机制和细胞动力学与功能项目共同资助。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 proposal is to develop new technology to allow detection of RNA molecules produced by single, living cells. Cells contain thousands of RNAs, and the RNAs produced from one cell may differ from the RNAs in a neighboring cell. Detecting these differences on a cell-by-cell basis is not easy, especially for plant cells. This project will create a method that transcends past technical limitations by preserving spatial information, thereby making it possible to tell which RNAs come from which cells. The new method will be put to the test by identifying RNAs in single plant cells that are undergoing infection by a fungal pathogen. This is a high-risk project that involves a complicated experimental design that will be challenging to implement. If successful, the results will provide plant biologists with a revolutionary new tool for studying cell-specific gene-to-RNA-to-trait relationships. This could lead to new discoveries in understanding plant diseases, which in turn could advance efforts to breed disease resistant plants. The project will be led by a postdoctoral scientist, who will receive multidisciplinary training in plant biology, microscopy, genomics, and computational science. Several methods have been established in plants for transcriptional analysis in tissue sections, cell types or even single cells. These methods typically require cell sorting, transgenic plants, protoplast production, or other damaging or laborious processes. Most of these technologies lose most or all spatial resolution during implementation, while those that offer high spatial resolution for RNA localization lack breadth in the number of transcripts characterized, particularly for more challenging RNAs like small RNAs. Plant small RNAs function in many more pathways than other organisms, yet there are no techniques for their single- cell analysis other than fluorescent in situ hybridization microscopy typically performed with just one or a few small RNAs (or mRNAs) at a time. A new approach, spatial transcriptomics, has been described that overcomes many of these limitations for mRNA analysis; RNA-seq libraries are made using microarrays with high positional resolution within sectioned materials. Yet, spatial transcriptomics has not been developed for small RNAs or cleaved mRNAs, and the implementations described to date still suffer from various shortcomings. This project will substantially advance this method, using high-resolution arrays (14 micron grids with 1 micron linear gaps) built on a maskless array synthesis instrument, to enable near single-cell analysis of diverse types of RNAs in plants and potentially other organisms. The test bed for this work is a system in which such high-resolution analyses would be highly informative, namely plant-pathogen interactions, for which localized and systemic transcriptional responses will be measured. This award is jointly funded by the Genetic Mechanisms and the Cellular Dynamics and Function programs in the Division of Molecular and Cellular Biosciences in the Biological Sciences Directorate.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.