Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agentPseudomonas fluorescensCHA0

Abstract

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.

Characterization of PhlG, a Hydrolase That Specifically Degrades the Antifungal Compound 2,4-Diacetylphloroglucinol in the Biocontrol Agent Pseudomonas fluorescens CHA0

“…P. fluorescens strains were routinely cultivated on nutrient agar (NA), in nutrient yeast broth (NYB), and in Luria-Bertani broth (LB), at 30°C (3,31,39). Production and degradation of antifungal compounds was monitored in King's medium B broth supplemented with 0.75% (wt/vol) malt extract (KMBmalt) (3). A minimal glycerol-ammonium (OSGly) medium (3) was used for gene expression studies.…”

“…Bacterial cells were transformed with plasmid DNA by electroporation (39). PCRs were carried out according to amplification protocols described in detail previously (3,31). Nucleotide sequences of PCR-derived constructs were determined on both strands by Microsynth (Balgach, Switzerland).…”

“…DAPG is synthesized and accumulated until the early stationary growth phase, as is typical for secondary metabolites (1,3,8,31,39). Thereafter, the metabolite appears to be degraded by the producing bacterium, with MAPG temporarily accumulating as an intermediary product of the degradation process (39).…”

“…In addition, many abiotic and biotic environmental factors may modulate levels of DAPG production, including different carbon and nitrogen sources, transition metal ions and other minerals, and metabolites released by bacteria, fungi, and plants (12,27,28). For instance, DAPG biosynthesis in P. fluorescens strains CHA0 and Pf-5 is negatively affected by pyoluteorin, another antifungal compound synthesized by these bacteria (3,8,39). In the same vein, the bacterial and plant metabolite salicylate, as well as the fungal pathogenicity factor fusaric acid, strongly inhibits DAPG production (3,39).…”

“…For instance, DAPG biosynthesis in P. fluorescens strains CHA0 and Pf-5 is negatively affected by pyoluteorin, another antifungal compound synthesized by these bacteria (3,8,39). In the same vein, the bacterial and plant metabolite salicylate, as well as the fungal pathogenicity factor fusaric acid, strongly inhibits DAPG production (3,39).…”