From macro to micro: a combined bioluminescence‐fluorescence approach to monitor bacterial localization

Abstract

Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro-and micro-scale imaging techniques. We demonstrate that a dual bioluminescencefluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro-and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.

AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity

“…Colony‐PCR was performed with primers OXRS_F4 and OXRS_R4 (Table S1) to validate the assembly. The lux operon, under the constitutive nptII promoter, was amplified with primers OXRS_F5 and OXRS_R5 (Table S1) from pRS p nptII ::lux (Soldan et al ., 2020). The PCR product was ligated into Sma I‐digested pRS01, generating plasmid pRS02.…”

“…Plasmid pRS02 was sequenced with primers OXRS_F4 and OXRS_R4 (Table S1) to validate the assembly. The λT0 terminator was amplified with primers OXRS_F6 and OXRS_R6 (Table S1) from pRS p nptII ::lux‐p A1/04/03 ::eYFP (Soldan et al ., 2020), and ligated into Sma I‐digested pRS02. The resulting plasmid contains pgl , p nptII ::lux operon with the λT0 terminator and ORF1 of picA .…”

“…Bacterial bioluminescence has been used to detect bacterial plant pathogens and symbionts in plants and to characterize the effect of plant immune responses on bacteria (Fan et al ., 2008; Fleiss and Sarkisyan, 2019; Soldan et al ., 2020; Yeh and Ai, 2019). Bioluminescent Agrobacterium has also been generated, e.g.…”

Sensor-based phenotyping of above-ground plant-pathogen interactions

“…A recent study generated bioluminescent and fluorescent Pseudomonas syringae pv. phaseolicola [ 34 ]. The bioluminescence enabled detection of the bacteria at the leaf scale in a dedicated imaging chamber.…”

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

“…AttTn 7 is located downstream of the glmS gene, which encodes an essential glucosamine-fructose-6-phosphate aminotransferase ( Peters and Craig, 2001 ) and is found across bacterial phyla, including Proteobacteria, Firmicutes, and Bacteroidetes ( Wiles et al., 2018 ). Previous reports have shown that Tn 7 is active in the genera Pseudomonas , Xanthomonas , Rhizobium , Ralstonia , Burkholderia , Erwinia , Pectobacterium , and Pantoea to which many, if not all, phytopathogenic bacteria belong ( Choi et al., 2006 ; Choi and Schweizer, 2006 ; Jittawuttipoka et al., 2009 ; Kernell; Kovács et al., 2009 ; Kernell Burke et al., 2015 ; Romero-Jimenez et al., 2015 ; Zhang et al., 2021 ; Riccardo et al., 2021 ). Thus, Tn 7 -based genetic manipulation tools should be broadly applicable to studies on plant-bacterial pathogen interactions.…”