Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0

Aims: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein ( GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root- colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil- borne pathogenic fungi. Methods and Results: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4- diacetylphloroglucinol ( DAPG), pyoluteorin ( PLT) and pyrrolnitrin ( PRN). The gfp[ mut3] and gfp[ AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. Conclusions: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine- tuned balance that can be affected by certain microbial and plant phenolics. Significance and Impact of the Study: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i. e. at the site of pathogen suppression.