2012 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.
Biological control offers an attractive complement to existing practices for plant disease management and therefore can contribute to future productivity and sustainability of U.S. agriculture. Unfortunately, its application in agriculture is impeded by unexplained variation in the efficacy of the biocontrol organisms. One source of variation is the inconsistent expression of biocontrol traits by antagonists that inhabit 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 produces a spectrum of secondary metabolites, including antibiotics toxic to plant-pathogenic bacteria, oomycetes, and fungi, thereby suppressing plant disease. Two of the antibiotics that contribute to biological control by Pf-5 are 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin. We previously demonstrated that phloroglucinol, an intermediate in DAPG biosynthesis, serves as a signaling molecule influencing the production of pyoluteorin by Pf-5. More recently, we constructed deletion mutants in pltZ and pltR, genes in the pyoluteorin biosynthetic gene cluster that encode transcriptional regulators, and confirmed that PltZ inhibits pyoluteorin production and PltR activates pyoluteorin production in Pf-5. We found that PltZ is involved in the inhibition of pyoluteorin production effected by high concentrations of phloroglucinol. Further, we constructed a series of 12 point mutations in pltR. The resulting pltR mutants will be tested for changes in the activation of pyoluteorin production by phloroglucinol as well as the autoinduction of pyoluteorin production by itself. We evaluated the role of phloroglucinol on the expression of genes for pyoluteorin biosynthesis and transport in Pf-5 using quantitative reverse-transcriptase polymerase chain reaction amplification. As expected, we found that the addition of high concentrations of phloroglucinol decreased expression of pyoluteorin biosynthesis genes. We optimized protocols for RNA extraction from cultures of Pf-5 strains and for mRNA enrichment, thereby obtaining mRNA of adequate quality for next-generation sequencing of RNA (RNA-Seq). We completed an RNA-Seq experiment evaluating the influence of phloroglucinol on the transcriptome of both Pf-5 wild-type and phloroglucinol-deficient strains. Following statistical evaluation of the data, we obtained a list of genes that were differentially expressed by the bacterial strains in response to phloroglucinol. The list included genes for the biosynthesis of both pyoluteorin and 2,4-diacetylphloroglucinol, as expected, indicating that our experimental methods and procedures for data analysis are appropriate. The experiment revealed a number of other genes controlled at the transcriptional level by phloroglucinol, which we are evaluating further to determine their roles in antibiotic production. We have started a second RNA-Seq experiment to further validate our methods and explore the role of phloroglucinol as a signaling molecule. The 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 research will extend this concept by evaluating the effect of antibiotics on the Pf-5 transcriptome in culture and on seed surfaces. This research was conducted in support of objective 303 2A: Fundamental Pathogen Biology.