The transcriptome landscape induced by <i>Pseudomonas syringae</i> core effectors reveals new insights into the immune network in <i>Nicotiana benthamiana</i>

The pathogen Pseudomonas syringae pv. tomato DC3000 suppresses plant immunity via type III effectors (T3Es). The majority of current research has focused on specific effectors targeting a single molecular pattern-triggered immune pathway, leaving a knowledge gap regarding a systems-level immune signaling network in plants. We employed the effectorless polymutant D36E, along with a modular library expressing 13 key T3Es individually, and performed an extensive transcriptomic analysis, integrated with genetic analysis and immune response assays, to identify D36E-induced and repressed by effector (DIRE) genes and associated pathways in Nicotiana benthamiana. The results revealed that the key T3Es collectively perturbed 19% of the N. benthamiana genome. DIRE genes were enriched for immune components, including pattern recognition receptors, kinase cascades, and transcription factors. Of 10 DIRE genes validated by qPCR, all were upregulated by D36E but suppressed by various pathogen effectors. Six of ten DIRE genes were unresponsive to flg22, elf18, or csp22, indicating that DIRE activation bypasses canonical molecular patterns. Silencing seven individual DIRE genes significantly boosted the virulence of D29E+8, a polymutant carrying minimal functional T3E repertoire. This work unveils a set of effector-targeted hubs beyond canonical pattern recognition, providing insights into layered plant defense and complex plant-pathogen signaling dynamics.