Single-cell profiling of complex plant responses to Pseudomonas syringae infection [bulk RNA-seq]

Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We exposed Arabidopsis to Pseudomonas syringae or mock treatment and profiled the transcriptomes of 11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from Pseudomonas and mock inoculated leaves identified five distinct pathogen responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection revealed a continuum of disease progression from an immune to susceptible state. Confocal imaging of promoter reporter lines for pathogen responsive cell clusters visualized immune activated cell clusters surrounding substomatal cavities colonized by bacteria, suggesting immune clusters are sites of early pathogen invasion. Susceptibility cell clusters exhibited more general localization and were highly induced at later stages of infection. Overall, our work uncovers cellular heterogeneity within an infected leaf and provides unique insight into plant differential response to infection at a single-cell level. Overall design: 4-week old Arabidopsis thaliana leaves syringe-infiltrated with Pseudomonas syringae pv. tomato DC3000 or a mock treatment for 24 hours. After treatment, cells (Protoplasts) were isolated from leaf tissue using a tape-sandwich method to enrich for the mesophyll layer. In parallel, whole leaf tissue was also isolated. RNA was then extracted from protoplast preparations and whole leaf tissue, and processed for RNA sequencing. Three biological replicates for mock- and DC3000-treated whole leaves were prepared, and one replicate of protoplasts derived from mock- and DC3000-treated leaves were also processed.