1a.Objectives (from AD-416):
1. Determine the influence of phloroglucinol and other metabolites of Pf-5 on the transcriptome and antibiotic-production profiles of the bacterium
2. Evaluate the role of phloroglucinol in cell-to-cell communication between strains of P. fluorescens.
1b.Approach (from AD-416):
Global patterns of gene expression by P. fluorescens will be assessed using RNA-Seq, and statistical methods employed to identify genes whose expression is altered by exogenous sources of phloroglucinol or other metabolites. Metabolic profiles will be generated by HPLC analysis of extracts from cultures of P. fluorescens.
This research was conducted in support of NP303 objective 2 "Identify plant germplasm and cultivars of small fruits resistant to economically-important soilborne diseases" of the parent project. Biological control offers an attractive complement to existing practices for management of plant disease and therefore can contribute to the productivity and sustainability of US agriculture in the future. Unfortunately, its widespread use in agriculture is impeded by unexplained variations in the efficacy of biocontrol organisms. One source of this variation is in the inconsistent expression of biocontrol traits by antagonists inhabiting soil or plant surfaces where they suppress target plant pathogens. The goal of this project is to identify factors controlling antibiotic production, a predominant trait contributing to biological control, in the soil bacterium Pseudomonas protegens Pf-5. Strain Pf-5 suppresses plant diseases caused by certain soilborne pathogens and produces a spectrum of secondary metabolites, including antibiotics toxic to plant pathogenic bacteria, Oomycetes, and fungi. Two of the antibiotics that contribute to biological control are 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin. Phloroglucinol, an intermediate in DAPG biosynthesis, is known to influence the production of pyoluteorin by Pf-5 and other strains of Pseudomonas. In this study, we are exploring the potential role of phloroglucinol as a signal having broad effects on the transcriptome of Pf-5. During the past year, we again oversaw the sequencing of mRNA that was isolated by ARS cooperators from cultures of Pf-5 and derivative strains that were amended with phloroglucinol. We analyzed the data from this RNA sequencing experiment and provided ARS cooperators with a list of genes that were significantly regulated by phloroglucinol in Pf-5 and several mutants deficient in phloroglucinol and/or DAPG production. We provided data as to the fold change differences between treatments for all significantly regulated genes. We also entered the data into a genome browser to enable ARS cooperators to view the data in a graphic format. We generated heat maps to display the experimental results in a visual way and are actively involved in discussions of the experimental results and their interpretation. Finally, we quantified secondary metabolites in Pf-5 culture extracts that were provided to us by ARS cooperators, which allowed our cooperators to make correlations between gene expression and metabolite production by Pf-5. This project builds upon a body of work establishing that the expression of specific biocontrol traits by P. protegens is influenced by compounds produced by its co-inhabitants in the rhizosphere.