PHYTOMap – multiplexed single-cell 3D spatial gene expression analysis in plant tissue

Retrieving the complex responses of individual cells in the native three-dimensional tissue context is crucial for a complete understanding of tissue functions. Researchers at The Salk Institute for Biological Studies have developed 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. The researchers applied PHYTOMap to simultaneously analyse 28 cell-type marker genes in Arabidopsis roots and successfully identified major cell types, demonstrating that their method can substantially accelerate the spatial mapping of marker genes defined in single-cell RNA-sequencing datasets in complex plant tissue.

Whole-mount spatial mapping of root tip cell-type marker genes with PHYTOMap

a, In fixed whole-mount tissue, target mRNA molecules are hybridized by pairs of DNA probes (SNAIL probes) that harbour mRNA species-specific barcode sequences (pink bars). Barcode-containing DNA probes are circularized by ligation (red star) and amplified in situ by RCA. During amplification, amine-modified nucleotides are incorporated into the DNA amplicons (RCPs) and stably cross-linked with the cellular protein matrix using a non-reversible amine cross-linker. Amplified DNA barcodes are detected by SBH chemistry through multiple rounds of imaging. b, SBH chemistry. Before each imaging round, four types of bridge probes are hybridized to a set of four DNA barcodes. Each bridge probe is then targeted by one of four fluorescent probes to be imaged. After imaging, bridge probes and fluorescent probes are stripped away, keeping RCPs in place. These steps are repeated until all the DNA barcodes are read. c, Representative images at different imaging rounds. The maximum exposure of 60 z planes of the same position in the tissue is displayed. Scale bar, 30 μm. d, Schematic representation of the root tip and UMAPs displaying root tip scRNA-seq data used in this study. In the UMAPs, cells are labelled with cell types (left) and regions (right). LRC, lateral root cap; QC, quiescent centre. e,f, Representative results from the imaging rounds 2 (e) and 3 (f). Left, UMAPs showing expression patterns of target genes. The colours of the gene name labels correspond to the colours in the images below. Middle, 3D projections (upper) and optical sections (2D, lower) of whole-mount tissue images. Right, representative cross-section views of the middle part of the samples (transition/elongation zone). Scale bar, 25 μm.

Availability – The code to analyse PHYTOMap data is available at https://github.com/tnobori/PHYTOMap.