Understanding plant pathogen interactions using spatial and single-cell technologies

Abstract

Plants are in contact with diverse pathogens and microorganisms. Intense investigation over the last 30 years has resulted in the identification of multiple immune receptors in model and crop species as well as signaling overlap in surface-localized and intracellular immune receptors. However, scientists still have a limited understanding of how plants respond to diverse pathogens with spatial and cellular resolution. Recent advancements in single-cell, single-nucleus and spatial technologies can now be applied to plant-pathogen interactions. Here, we outline the current state of these technologies and highlight outstanding biological questions that can be addressed in the future.

Fig. 1. A schematic workflow of single-cell RNA sequencing and spatial transcriptomics.

For scRNA-seq, individual cells are isolated through enzymatic digestion from intact tissue to generate protoplasts. Microfluidics are used to separate and encapsulate single protoplasts in droplets with barcoded beads. Each barcoded bead is coated with DNA probes, which contain poly (dT) to capture mRNAs, a unique molecular identifier (UMI) and a cell-specific barcode. Cell lysis then occurs, and mRNAs are hybridized to probes and reverse transcribed on beads within droplets. Finally, a library of barcoded cDNA from thousands of single cells is sequenced to generate single-cell transcriptomes. For spatial transcriptomics, freshly frozen plant tissue is sectioned onto a spatial transcriptomics slide. Capture zones on the slide contain thousands of spots. Each spot consists of probes containing a unique spatial barcode, unique molecular identifier (UMI), and poly (dT) region to capture transcripts. Transcripts are released through permeabilization, hybridized to probes and reverse transcribed to incorporate spatial barcodes. The generated cDNAs carrying spatial information are cleaved from the slide and used to prepare a library for sequencing. The sequenced reads are spatially mapped based on spatial barcodes. Created with BioRender.com.

Fig. 2. Application of cellular and spatial profiling technologies in plant-microbe interactions.

A schematic diagram showing representative research areas that can be investigated by single-cell, single-nucleus and spatial profiling. A combination of different approaches will lead to a more comprehensive understanding of plant-microbe interactions. Created with BioRender.com.