A versatile Tn7transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

Investigation of plant-bacteria interactions requires quantification of in plantabacterial titers by means of cumbersome and time-consuming colony counting assays. Here, we devised a broadly applicable tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed vectors that enable Tn7transposon-mediated integration of the luxCDABEluciferase operon into a specific genomic location ubiquitously found across bacterial phyla. These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria belonging to the genera Pseudomonas, Rhizobium(Agrobacterium),and Ralstonia. Direct luminescence measurements of plant tissues inoculated with the bioluminescent Pseudomonas syringaepv. tomatoDC3000 (Pto-lux) reported bacterial titers in Arabidopsis thaliana, Solanum lycopersicum, Nicotiana benthamiana,and Marchantia polymorphaas accurately as conventional colony counting assays. We further showed the usefulness of our vectors in converting the previously generated Ptoderivatives to isogenic bioluminescent strains. Importantly, quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth, with a dynamic range of four orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated spatial patterns of Pto-lux growth in/on inoculated plant tissues. In conclusion, our vectors offer untapped opportunities to develop bioluminescence-based assays for a variety of plant-bacteria interactions.