Bioluminescence-Based Growth Quantification of the Phytopathogenic Bacterium Pseudomonas syringae pv. tomato DC3000

Bioluminescence tagging has gained popularity as an effective tool for investigating infection processes of phytopathogenic bacteria. A critical consideration in employing this approach is to minimize the impact of the genetically introduced luciferase genes on bacterial fitness, while maximizing the intensity and stability of bioluminescence. Recently, the pBJ vector series was developed as all-in-one system for bioluminescence tagging in a wide range of Pseudomonadota, including the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pto). The pBJ vectors enable inducible transposition of the luxCDABE luciferase operon, derived from Photorhabdus luminescens, into a specific and neutral genomic location via Tn7 transposon. The resulting bioluminescent Pto strain, termed Pto-lux, emits stable and strong bioluminescence while maintaining bacterial fitness during plant infection. Moreover, bioluminescence-based assays using Pto-lux enable accurate quantification of in planta bacterial titers across diverse host plants, with a dynamic range of four orders of magnitude. In this chapter, we present detailed protocols for the bioluminescence-based bacterial growth assays in Arabidopsis thaliana and Marchantia polymorpha.