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

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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.