Time-series single-cell transcriptomics and spatial metabolomics reveal spatiotemporal tobacco leaf response to herbivory

• Integrated single-cell transcriptomics and spatial metabolomics of herbivore-stressed tobacco • Constructed a 28,318-cell atlas revealing spatiotemporal defense regulation. • Identified epidermal subpopulations as early hubs of defense signaling. • Discovered WRKY81 as a key transcription factor driving defense commitment. • Provides a cellular framework for understanding and improving pest resistance. Introduction: Plants have evolved complex defense systems to cope with herbivore attack, yet how these defense responses are coordinated across distinct cell types and time scales remains unclear. Understanding the cellular and spatial organization of such defenses is critical for elucidating the mechanisms underlying plant–insect interactions. Objectives: This study aimed to dissect the spatiotemporal regulation of tobacco leaf defense responses to herbivory by integrating time-series single-cell transcriptomics and spatial metabolomics. Methods: We generated a high-resolution single-cell atlas of 28,318 tobacco leaf cells under simulated herbivory. Transcriptional dynamics, cell–cell communication networks, and spatial metabolite distributions were analyzed using co-expression, pseudotime, and ligand–receptor inference approaches. Functional validation of key regulators was conducted through qRT-PCR and VIGS assays. Results: Our analyses revealed rapid transcriptional and metabolic remodeling following herbivore stress, with epidermal subpopulations serving as early signaling hubs. Spatial metabolomics confirmed epidermis-enriched accumulation of defense metabolites. Pseudotime and co-expression analyses identified epidermal subcluster 6 as an early-responsive population characterized by elevated WRKY81 expression. Silencing WRKY81 impaired defense activation, increasing herbivore feeding efficiency and metabolic assimilation. Conclusion: This study establishes the first single-cell–resolved dynamic defense network of tobacco leaves against herbivory. The findings uncover the central role of epidermal transcriptional reprogramming and identify WRKY81 as a critical regulator of early defense commitment, offering a mechanistic basis for the development of pest-resistant crops.