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.
Biological control offers an attractive complement to existing practices for management of plant disease and therefore can contribute to the productivity and sustainability of U.S. 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 fluorescens 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 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 also consulted with ARS on methods for direct RNA sequencing (RNAseq). One RNAseq experiment has been completed to date. We analyzed the data from this experiment and provided ARS cooperators with a list of genes that were significantly regulated by phloroglucinol in Pf-5 and a mutant (phlD) deficient in phloroglucinol 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 (G-Browse) to enable ARS cooperators to view the data in a graphic format. The results of the first RNAseq experiment were promising, indicating that the methods for sequencing and sequence analysis were appropriate and are providing high quality data. We worked with ARS cooperators to design a second experiment that will more fully test the hypothesis that phloroglucinol is a signaling molecule in P. fluorescens. We are currently awaiting the samples for the second RNAseq experiment. This project builds upon a body of work establishing that the expression of specific biocontrol traits by P. fluorescens is influenced by compounds produced by its co-inhabitants in the rhizosphere. The proposed research will extend this concept by evaluating the effect of antibiotics on the genome-wide expression profiles of Pf-5 in culture and on seed surfaces. This research was conducted in support of objective 2A: Fundamental Pathogen Biology.