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

Abstract

Investigation of plant-bacteria interactions requires quantification of in planta bacterial 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 Tn 7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location found ubiquitously across bacterial phyla. These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria from the genera Pseudomonas , Rhizobium ( Agrobacterium ), and Ralstonia . Direct luminescence measurements of plant tissues inoculated with bioluminescent Pseudomonas syringae pv. tomato DC3000 ( Pto -lux) reported bacterial titers as accurately as conventional colony-counting assays in Arabidopsis thaliana , Solanum lycopersicum , Nicotiana benthamiana , and Marchantia polymorpha . We further showed the usefulness of our vectors in converting 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 four orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated the 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.

Single-cell profiling of complex plant responses toPseudomonas syringaeinfection

“…Moreover, different stages of pathogen infection are often observed within a leaf (Fantozzi et al, 2021; Haueisen et al, 2019). Heterogeneity in pathogen distribution and likely, the plant response, impacts symptom development (Matsumoto et al, 2022). Strains of the bacterial pathogen, Pseudomonas syringae , have a broad host range and can infect many economically important plant species, causing a variety of foliar symptoms (Katagiri et al, 2002; Xin et al, 2018).…”

Contrasting and conserved roles of NPR pathways in diverged land plant lineages

“…AtNPR1 positively regulates SA-dependent defense gene expression, and thus At npr1 mutants display increased susceptibility to various bacterial pathogens [7,36,55]. Therefore, we investigated whether and how MpNPR contributes to resistance against bacterial pathogens using recently established pathosystems [56, 57]. Two-week-old thalli of Tak-1 and Mp npr mutants were inoculated with bioluminescent Pseudomonas syringae pv.…”

“…Bacterial quantification in infected thallus was carried out as described before [57]. Briefly, M. polymorpha were grown on autoclaved cellophane disc on half-strength GB5 media for 2 weeks.…”

A bioluminescence-basedex vivoburn wound model for real-time assessment of novel antibacterial compounds

“…Theoretically, this system allows insertion in most bacterial species as the integrative attP sites are present in virtually all bacteria. However, only Gram-negative bacteria have been reported to be site-specifically tagged with this system (Choi et al 2005; Matsumoto et al 2021; Shivak et al 2016). This system can be used in eukaryotes as well as for Saccharomyces cerevisiae, which enables the expansion of the model towards fungal infections (Bachman et al 2002).…”

“…With the various genome editing tools currently available, genetic insertion of a set of genes has become increasingly efficient, resulting in a stable copy of this operon into the genome of a desired host. While BLI was already widely used in animal studies to visualize bacterial load (Chauhan et al 2012; Li et al 2021), this approach recently gained more attention in the microbiology field to replace cumbersome and time-consuming replica plating (Andersson et al 2021; Matsumoto et al 2021).…”