Imaging-based resistance assay using enhanced luminescence-tagged Pseudomonas syringae reveals a complex epigenetic network in plant defense signaling pathways

High-throughput resistance assays in plants have a limited selection of suitable pathogens. In this study, we developed a Pseudomonas syringae strain chromosomally tagged with the NanoLuc (NL) luciferase from the deep-sea shrimp Oplophorus gracilirostris, a bioluminescent marker significantly brighter than the conventional firefly luciferase. Our reporter strain tagged with NL was over 100 times brighter than P. syringae tagged with the LuxCDABE operon from Photorhabdus luminescens, one of the existing luciferase-based strains. In planta-imaging was improved by using the surfactant Silwet L-77, particularly at a lower reporter concentration. Using this imaging system, over 30 epigenetic mutants were analyzed for their resistance traits since the defense signaling pathway is known to be epigenetically regulated. SWC1, a defense-related chromatin remodeling complex, was found to be a positive defense regulator, which supported one of two earlier conflicting reports. Compromises in DNA methylation in the CG context led to enhanced resistance against virulent Pst. DCLs and AGOs, important in the biogenesis and exerting the effector function of small RNAs, respectively, showed modest but distinct requirements for ETI and basal resistance to Pst. In addition, the transcriptional expression of an epigenetic component was found to be a significant predictor of its immunity contribution. In summary, this study showcased how a high-throughput resistance assay enabled by a pathogen strain with an improved luminescent reporter could provide insightful knowledge on complex defense signaling pathways.