A rapid and non-destructive method for spatial–temporal quantification of colonization by Pseudomonas syringae pv. tomato DC3000 in Arabidopsis and tomato

Background

The bacterial leaf pathogen Pseudomonas syringae pv tomato (Pst) is the most popular model pathogen for plant pathology research. Previous methods to study the plant-Pst interactions rely on destructive quantification of Pst colonisation, which can be labour- and time-consuming and does not allow for spatial–temporal monitoring of the bacterial colonisation. Here, we describe a rapid and non-destructive method to quantify and visualise spatial–temporal colonisation by Pst in intact leaves of Arabidopsis and tomato.

Results

The method presented here uses a bioluminescent Pst DC3000 strain that constitutively expresses the luxCDABE operon from Photorhabdus luminescens (Pst::LUX) and requires a common gel documentation (Gel Doc) system with a sensitive CCD/CMOS camera and imaging software (Photoshop or Image J). By capturing bright field and bioluminescence images from Pst::LUX-infected leaves, we imaged the spatiotemporal dynamics of Pst infection. Analysis of bioluminescence from live Pst bacteria over a 5-day time course after spray inoculation of Arabidopsis revealed transition of the bacterial presence from the older leaves to the younger leaves and apical meristem. Colonisation by Pst:LUX bioluminescence was obtained from digital photos by calculating relative bioluminescence values, which is adjusted for bioluminescence intensity and normalised by leaf surface. This method detected statistically significant differences in Pst::LUX colonisation between Arabidopsis genotypes varying in basal resistance, as well as statistically significant reductions in Pst::LUX colonisation by resistance-inducing treatments in both Arabidopsis and tomato. Comparison of relative bioluminescence values to conventional colony counting on selective agar medium revealed a statistically significant correlation, which was reproducible between different Gel Doc systems.

Conclusions

We present a non-destructive method to quantify colonisation by bioluminescent Pst::LUX in plants. Using a common Gel Doc system and imaging software, our method requires less time and labour than conventional methods that are based on destructive sampling of infected leaf material. Furthermore, in contrast to conventional strategies, our method provides additional information about the spatial–temporal patterns of Pst colonisation.