Macroscopic and microscopic visualization of Pseudomonas syringae pv. tabaci infection using complementary bioluminescent and fluorescent reporter systems

Abstract

Abstract The development of bioluminescent and fluorescent reporter systems has enabled visualization of plant-pathogenic bacterial infection dynamics, providing insights into detailed mechanisms of infection and potential applications for disease control. Pseudomonas syringae pv. tabaci 6605 ( Pta ) possesses quorum sensing systems and produces fluorescent pigments, which make it difficult to directly apply commonly used reporter systems. In this study, we first constructed a homologous recombination-based genomic insertion system for stable chromosomal integration of reporter genes in Pta . Using this system, we established a LuxCDABE bioluminescent system driven by the nptII promoter, which successfully enabled macroscale tracking of bacterial populations during infection. We then attempted to construct an sGFP fluorescent reporter using the same nptII promoter. However, the endogenous autofluorescence of Pta masked the sGFP signal, making quantitative detection difficult. To overcome this autofluorescence problem, we screened constitutively highly expression promoters based on transcriptome data and identified two effective promoters suitable for fluorescent monitoring of Pta infection. Together with the LuxCDABE system, this integrated platform provides comprehensive spatiotemporal analysis of virulence mechanisms and plant defense responses in the Pta pathosystem, with potential applicability to other plant-pathogenic bacteria.