PHYTOMap: Multiplexed single-cell 3D spatial gene expression analysis in plant tissue

Abstract

Retrieving the complex responses of individual cells in the native three-dimensional tissue context is crucial for a complete understanding of tissue functions. Here, we present PHYTOMap (Plant HYbridization-based Targeted Observation of gene expression Map), a multiplexed fluorescence in situ hybridization method that enables single-cell and spatial analysis of gene expression in whole-mount plant tissue in a transgene-free manner and at low cost. We applied PHYTOMap to simultaneously analyze 28 cell type marker genes in Arabidopsis roots and successfully identified major cell types, demonstrating that our method can substantially accelerate the spatial mapping of marker genes defined in single-cell RNA-seq datasets in complex plant tissue.

Single-cell profiling of complex plant responses toPseudomonas syringaeinfection

“…Susceptibility genes are attractive targets to modify for developing disease resistant crops due to advances in genome editing technologies and decreased regulatory oversight (Garcia-Ruiz et al, 2021; Zaidi et al, 2018). Future advancements in high-resolution spatial transcriptomics enabling profiling of both plant and pathogen tissues, will facilitate investigating gene expression in a positional context in complex tissues (Nobori et al, 2022; Saarenpää et al, 2022). Detailed characterization of cellular states throughout disease development will enable a comprehensive understanding of mechanisms regulating disease progression.…”

Time-resolved single-cell and spatial gene regulatory atlas of plants under pathogen attack

“…However, it is challenging or impossible to fully capture such interactions on a 2D tissue section, and thus a 3D analysis of both plant genes and bacterial colonization is critical. Recently, we developed a technology called PHYTOMap (Nobori et al, 2022), which can spatially map dozens of genes in 3D in whole-mount plant tissues. Such a method, combined with our spatiotemporal atlas, will open a new avenue toward a comprehensive characterization of cell populations identified in this study.…”

Exploring the identity of individual plant cells in space and time

“…Markers can also be used to map different cell clusters in vivo when prior knowledge is limited, using reporter lines or in situ hybridisation, enabling generation of precise gene expression atlases. Recently, plant biologists have taken advantage of the rapidly progressing field of spatial transcriptomics to directly measure targeted (Laureyns et al ., 2021; Nobori et al ., 2023) or untargeted (Xia et al ., 2022; Liu et al ., 2023) transcript abundance in situ , removing the need of a two‐step approach. To date, most plant spatial transcriptomics studies have lacked cellular resolution, but new spatial technologies such as MERFISH (Chen et al ., 2015) that achieve cellular‐ or subcellular resolution will allow the generation of powerful atlases of cell identities in the near future.…”