# Plasmid pPNptGreen Expression of Green Fluorescent Protein in Pseudomonas chlororaphis Strain S1Bt23 Abrogates Biocontrol Activity Against Pythium ultimum

## TL;DR

Adding a green fluorescent protein to a bacteria used to protect plants stops it from fighting a root disease.

## Contribution

Shows that adding a fluorescent gene to a biocontrol bacterium reduces its disease-fighting ability.

## Key findings

- GFP-expressing P. chlororaphis failed to inhibit Pythium ultimum growth unlike the wild type.
- Phenazines and pyrrolnitrin, antifungal compounds, were not produced in the GFP strain.
- Gene expression for these compounds was unchanged, suggesting post-transcriptional loss.

## Abstract

Pseudomonas chlororaphis
is a highly effective plant root coloniser and biocontrol agent. To monitor the colonisation of tomato and canola roots,
P. chlororaphis
S1Bt23 was transformed with the pPNptGreen plasmid encoding for green fluorescent protein (S1Bt23‐GFP). Seedling roots inoculated with S1Bt23‐GFP were examined after 2 and 5 h using confocal laser fluorescence microscopy. Roots exposed to S1Bt23‐GFP showed pronounced biofilm formation around the root surface, and fluorescing cells were localised in the epidermis and metaxylem after 2 and 5 h of inoculation, respectively. The canola roots also showed upward active translocation of the S1Bt23‐GFP cells in xylem vessels in real time. S1Bt23‐GFP was also evaluated for antagonistic activity against
Pythium ultimum
. While S1Bt23 WT exhibited 65.70%–71.4% inhibition of radial growth of Py. ultimum, the S1Bt23‐GFP strain did not demonstrate any antagonistic effects. Thin layer chromatography and liquid chromatography mass spectrometry analyses of culture extracts of S1Bt23‐GFP did not detect phenazines or pyrrolnitrin, antifungal metabolites identified in S1Bt23 wild type. Expressions of phenazine and pyrrolnitrin genes showed no differences in S1Bt23‐GFP and wild type. This suggests that the abrogation of these metabolites occurred post‐transcriptionally, probably due to a high cellular molecular load in GFP production. This could negatively impact the ecological fitness of S1Bt23‐GFP.

Transformation of
Pseudomonas chlororaphis
S1Bt23 with plasmid pPNptGreen negates its antagonistic activity against
Pythium ultimum
due to the lack of the production of key secondary metabolites, for example phenazines.