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bioRxiv · 10.1101/2021.02.11.430857

A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

Mine, A.·Matsumoto, A.·Schlueter, T.·Melkonian, K.·Takeda, A.·Nakagami, H.
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Abstract

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of colony counting assays. However, colony counting assays are cumbersome and time-consuming, and are unable to detect spatial patterns of bacterial colonization in plants. Here, to overcome these shortcomings, we devised a broadly applicable genetic engineering tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed plasmid vectors that have broad host ranges and enable Tn7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location ubiquitously found across bacterial phyla. These vectors allowed for generation of bioluminescent transformants of various plant pathogenic bacteria belonging to the genera Pseudomonas, Rhizobium (Agrobacterium), and Ralstonia. The bioluminescent transformant of Pseudomonas syringae pv. tomato DC3000 (Pto-lux) was as virulent in Arabidopsis thaliana as its parental strain. Direct luminescence measurements of Pto-lux-inoculated plant tissues reported bacterial titers in A. thaliana, Solanum lycopersicum, Nicotiana benthamiana, and Marchantia polymorpha as accurately as conventional colony counting assays. We further showed the utility of our vectors for converting the previously generated Pto derivatives 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 4 orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated spatial colonization patterns of the Pto-lux in/on inoculated plant tissues. Taken together, our vectors offer untapped opportunities for developing bioluminescence-based quantitative and spatial analysis of bacterial growth in a variety of plant-bacteria interactions. SIGNIFICANCE STATEMENTWe developed broad-host-range plasmid vectors that integrate the luciferase operon, luxCDABE, into a specific genomic location ubiquitously found across bacterial phyla. Using these vectors, we established a high-throughput method for bioluminescence-based quantitative assays of in planta bacterial growth with a dynamic range of 4 orders of magnitude and visualized spatiotemporal patterns of bacterial colonization in/on inoculated plant tissues.

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Mine, A., Matsumoto, A., Schlueter, T., Melkonian, K., Takeda, A., Nakagami, H.. 2021-02-11. A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants. https://doi.org/10.1101/2021.02.11.430857

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