Lay summary
The root-associated bacterium Pseudomonas fluorescens CHA0 protects crop plants against diseases caused by fungal pathogens. Antifungal compounds, notably 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) are key determinants of biocontrol activity. The present project aims at identifying regulatory mechanisms that help the biocontrol agent control the balance of DAPG and PLT production in response to plant-derived signals. In a survey of phenolic compounds, we have identified indoleacetic acid (IAA) as an important plant signal stimulating DAPG and PLT gene expression. To identify regulatory loci involved in signal response, we have screened a mutant library of strain CHA0 for candidates displaying IAA-hypersensitive or -insensitive phenotypes with respect to stimulation of DAPG gene expression. Two of the hypersensitive mutants were of particular interest since they were defective in loci that share homology with genes encoding regulators of virulence and stress response functions in pathogenic bacteria, among them notably a member of the MarR family of transcriptional regulators that is currently being analyzed in some detail. Not only the plant, but also the root-commensal pseudomonads themselves may manipulate their rhizosphere environment and traits that help them utilize components of the root exudates may be particularly relevant. We are currently focusing on the contribution of enzymes involved in initial steps of glucose metabolism to microenvironment manipulation and antifungal activity by P. fluorescens. To study antifungal gene expression in situ, we have developed a set of reporter fusions that are based on the green fluorescent protein GFP and on the cherry-red fluorescent protein mCherry that can be used in dual-colour applications. Using these reporters, we have set up plant assays in which we monitored and quantified bacterial growth and alterations in DAPG and PLT gene expression on roots in response to the plant species and to attack by leaf and root pathogens, applying a flow cytometric approach that relies on fluorescence-activated cell sorting (FACS). The GFP- and mCherry-based reporters proved useful not only for studying interactions of P. fluorescens with host plants, but also with potential predators in the rhizosphere, in particular nematodes and protozoa. Ultimately, an improved understanding of how root-associated pseudomonads adapt the expression of key biocontrol traits to changes in their rhizosphere habitat, in particular following attack of the host plant by fungal pathogens, may serve in the choice of strategies aimed at optimizing plant protection by beneficial bacteria.