2013 Annual Report
1a.Objectives (from AD-416):
1. Determine the influence of purified antibiotics on the transcriptome of P. fluorescens Pf-5.
2. Determine the influence of antibiotic-mediated cell to cell signaling on the expression of antibiotic-biosynthesis genes and biocontrol activity of P. fluorescens.
1b.Approach (from AD-416):
1. Characterize the transcriptome of P. fluorescens Pf-5 by strand-specific direct RNA sequencing, employing bioinformatic and computational tools to identify the genome-wide distribution of transcripts and small RNAs.
2. Focusing on antibiotics produced by Pf-5 and other biocontrol strains of Pseudomonas sp. (examples: 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, orfamide A, rhizoxin analogs, phenazine-1-carboxylate), evaluate the influence of purified compounds on a) the transcriptome and b) antibiotic production profiles of Pf-5 grown in culture.
3. Evaluate the influence of purified antibiotics on the transcriptome of Pf-5 on seed surfaces.
4. Determine the influence of antibiotic-producing bacterial co-inhabitants on a) the expression of biocontrol genes by Pf-5 in the rhizosphere or spermosphere of plants grown in soil, and b) biological control of Pythium damping-off. These experiments will employ a combination of well-characterized mutants and reporter gene constructs to assess gene expression and biocontrol in natural field soils.
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 microbial 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 and pyoluteorin. We previously demonstrated that phloroglucinol, an intermediate in 2,4-diacetylphloroglucinol biosynthesis, serves as a signaling molecule influencing the production of pyoluteorin by Pf-5 and other strains of Pseudomonas. This year, we completed a second experiment evaluating the influence of phloroglucinol on gene expression by Pf-5. We used protocols for RNA extraction from cultures of Pf-5 and rRNA depletion for enrichment of mRNA that were optimized last year, and obtained mRNA of adequate quality for direct sequencing of RNA (RNAseq). We determined the influence of phloroglucinol on the transcriptomes of two Pf-5 derivative strains: Pf-5phlAG (produces phloroglucinol but no 2,4-diacetylphloroglucinol) and Pf-5phlADG (lacking both phloroglucinol and 2,4-diacetylphloroglucinol production). Data analysis generated a list of hundreds of genes differentially expressed among the treatments. As expected from previous results, expression of many genes in the pyoluteorin biosynthetic gene cluster was highly regulated by phloroglucinol. This study identified a large number of other genes in the Pf-5 genome that are also regulated by phloroglucinol, which supports our hypothesis that this compound serves as a signal having broad effects on the transcriptome of Pf-5.