Time-series transcriptome and metabolome profiling Uncovers WRKY6 and WRKY23 as critical regulators in tobacco response to Pseudomonas syringae infection

INTRODUCTION: Pseudomonas syringae pv. tabaci, a Gram-negative bacterial pathogen, causes devastating tobacco wildfire disease with global economic impacts. While its pathogenicity is well documented, the dynamic defense mechanisms of tobacco against infection remain poorly understood. OBJECTIVE: This study aimed to decipher phased defense mechanisms of tobacco against P. syringae infection through multi-omics integration, with emphasis on elucidating spatiotemporal coordination between transcriptional reprogramming and metabolic remodeling, and functionally validating critical regulatory modules. METHODS: Time-series transcriptomic and metabolomic profiling was integrated to reconstruct dynamic response patterns. Stage-specific regulatory modules were explored via TO-GCN and WGCNA, and the roles of WRKY6 and WRKY23 in disease resistance were validated by generating transgenic lines. RESULTS: Early infection (12-24 hpi) prioritized stress signaling and hormone pathway activation (salicylic acid/jasmonate), transitioning to cellular homeostasis regulation at late stages (48-60 hpi). WRKY, ERF, and NAC families orchestrated stage-specific gene expression. Notably, WRKY6 and WRKY23 functioned as negative regulators, with their silencing leading to a reduction in lesion area by 42-58% and pathogen load by 3.2-4.5 fold. Metabolomic analysis revealed sustained activation of phenylpropanoid metabolism, specifically regulating L-phenylalanine homeostasis and biosynthesis of its defense derivative xanthosine. Additionally, core modules involved in sphingolipid metabolism, light responses, and hormone cross-talk were also identified. CONCLUSION: We demonstrate that WRKY-mediated transcriptional reprogramming coordinates phytohormone signaling, sphingolipid dynamics, and light responses to spatiotemporally regulate secondary metabolite production. The identified WRKY6 and WRKY23 regulatory module establishes a molecular framework for engineering disease resistance, and the proposed two-phase defense model (early signaling → late metabolic remodeling) advances understanding of plant-pathogen interactions and offers targets for precision breeding.