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

Plant leaf intercellular space provides a nutrient-rich and heterogeneous niche for microbes that have a critical impact on plant health. However, how individual plant cells respond to heterogeneous microbial colonization remains largely elusive. Here, by time-resolved simultaneous single-cell transcriptome and epigenome profiling of plants (Arabidopsis thaliana) infected by virulent and avirulent bacterial pathogens (Pseudomonas syringae), we present an atlas of gene regulatory logic involving transcription factors, potential cis-regulatory elements, and target genes associated with disease and immunity. We also identify previously uncharacterized cell populations with distinct immune gene expression within major developmental cell types. Furthermore, we employ time-resolved spatial transcriptomics to reveal spatial heterogeneity of plant immune responses linked to pathogen distribution. Integration of our single-cell multiomics and spatial omics data enables spatiotemporal mapping of defense gene regulatory logic. Overall, this study provides a molecularly-defined spatiotemporal map of plant-microbe interaction at the single-cell resolution. Overall design: Arabidopsis thaliana Col-0 were grown in a chamber at 22°C with a 12-h light period and 60-70% relative humidity for 30-31 days. Bacterial stains were cultured in the King's B liquid medium with antibiotics (Rifampicin and Tetracyclin) at 28°C. Three bacterial strains–Pseudomonas syringae pv tomato DC3000 (Pto DC3000) carrying empty vector (pLAFR3), avrRpt2 (pLAFR3), and avrRpm1 (pLAFR3)–were described previously (Whalen et al. 1991; Kunkel et al. 1993). Bacteria were harvested by centrifugation and resuspended in sterile water to an OD600 of 0.001 (approximately 5 x 105 CFU ml-1). In total, 20 A. thaliana leaves (four fully-expanded leaves per plant) were syringe-inoculated with bacterial suspensions using a needleless syringe. For each strain, four time points (4, 6, 9, and 24 h) were sampled at the same time by infiltrating bacteria at different times. The 20 infected leaves were harvested with forceps and immediately processed for nuclei extraction. For the mock condition, water-infiltrated leaves were harvested after 9 h.