Chromoprotein-modified plant pathogenic bacteria: tools for experimental tracking and visualization

Microbiology research often requires tracking of specific bacterial strains within a host infection system or in the environment, as well as differentiation of strains in a co-infection or microbe-microbe interaction scenario. Various tools are used for this purpose, including antibiotic resistance marker genes, fluorescent proteins, DNA sequence-based methods, and phenotypic markers. Chromoproteins produce intense pigmentation visible in ambient light and are a unique option for bacterial tracking that does not require use of antibiotics, specialized equipment, or DNA sequencing. Development of traceable bacterial strains across a wide range of species is important to facilitate the investigation of challenging research questions and expand our understanding of microbial dynamics in complex environments. In this study, different species of plant pathogenic bacteria (<i>Xylella fastidiosa</i>, <i>Pantoea stewartii</i>, <i>Pseudomonas syringae</i>, and <i>Xanthomonas campestris</i>) were modified with a set of chromoproteins and tested in plant infection assays to evaluate chromoprotein stability and impact on bacterial pathogenicity. The primary goal of this study was to develop chromoprotein-modified strains of <i>X. fastidiosa</i> using chromosomal insertion, which was highly successful and stable during infection in grapevines. Plasmid-based expression of chromoproteins in <i>P. stewartii</i>, <i>P. syringae,</i> and <i>X. campestris</i> had mixed results depending on the specific species-chromoprotein combination. Overall, these results provide some successful chromoprotein-modified plant pathogen strains for use by the research community, as well as insight into which chromoproteins might be best utilized in different bacterial species.IMPORTANCEInvestigation of challenging research questions in plant health requires availability of the necessary microbial tools. This study adapts chromoprotein modification for bacterial tracking of plant pathogens during host infection and plant-plant transmission. Successful development of visibly colored strains of <i>Xylella fastidiosa</i>, <i>Pantoea stewartii</i>, and <i>Pseudomonas syringae</i> provides a useful resource for research and educational purposes, as well as insight into optimization of chromoprotein expression in a range of plant pathogen species.