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Title: Saccharomyces cerevisiae genome-wide mutant screen for sensitivity to 2,4-diacetylphloroglucinol, a biocontrol antibiotic produced by Pseudomonas fluorescens

Author
item KWAK, YOUN-SIG - Washington State University
item HAN, SANG JO - Washington State University
item Thomashow, Linda
item Topham, Jennifer
item Paulitz, Timothy
item KIM, DONGSUP - Washington State University
item Weller, David

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2010
Publication Date: 3/1/2011
Citation: Kwak, Y., Han, S., Thomashow, L.S., Topham, J., Paulitz, T.C., Kim, D., Weller, D.M. 2011. Saccharomyces cerevisiae genome-wide mutant screen for sensitivity to 2,4-diacetylphloroglucinol, a biocontrol antibiotic produced by Pseudomonas fluorescens. Applied and Environmental Microbiology. 77(5):1770-1776.

Interpretive Summary: Biological control is the application or stimulation of antagonistic microorganisms for the control of plant pathogens. Some of the most effective biocontrol agents of root diseases caused by soilborne pathogens are strains of beneficial bacteria that produce the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). 2,4-DAPG has broad spectrum activity against fungi, bacteria and nematodes. Although 2,4-DAPG plays a key role in plant defense in many different agroecosystems, little is know about its mode of action. As a first step to elucidating the mechanism of action, we screened a deletion library of the yeast Saccharomyces cerevisiae in broth and agar media supplemented with 2,4-DAPG. Yeasts are widely used are model systems for studying the mode of action of antibiotics and pharmaceuticals. Our study showed that 2,4-DAPG acts on multiple basic cellular processes. Thus, it is not likely that resistance will develop in plant pathogens to this important biocontrol metabolite. Producers are responsible for the natural biocontrol of take-all disease known as take-all decline that occurs when wheat is grown continuously in a field. Previous studies have shown that 2,4-DAPG produced by P. fluorescens inhibits the take-all pathogen, Gaeumannomyces graminis var. tritici (Ggt) on roots. In this study, we determined the sensitivity to 2,4-DAPG of Phialophora spp. that co-exist with the take-all pathogen on wheat roots. We found that Phialophora was significantly more tolerant to 2,4-DAPG than the take-all pathogen and that Phialophora suppressed take-all when it was co-inoculated with Ggt. The results of this research are important because they demonstrated that Phialophora is enhancing the suppression of take-all that is mediated by 2,4-DAPG producers.

Technical Abstract: 2,4-diacetylphloroglucinol (2,4-DAPG) is an antibiotic produced by Pseudomonas fluorescens that plays a key role in the ability of the bacterium to suppress phytopathogenic fungi. 2,4-DAPG has broad antibiotic activity, affecting organisms ranging from bacteria to higher plants. The biosynthesis and regulation of 2,4-DAPG in P. fluorescens have been well-described, but the mode of action against target fungi is poorly understood. As a first step to elucidating the mechanism, we screened a deletion library of Saccharomyces cerevisiae in broth and agar media supplemented with 2,4- DAPG. We identified 231 mutants that showed increased sensitivity to 2,4-DAPG under both conditions, including 22 multi-drug resistance-related mutants. Three major physiological functions correlated with increase in sensitivity to 2,4-DAPG: membrane function, reactive oxygen regulation and cell homeostasis. Physiological studies with wild-type yeast validated the results of the mutant screens. The chemical-genetic fitness profile of 2,4-DAPG resembled those of menthol, sodium azide and hydrogen peroxide determined in previous high-throughput screening studies. Collectively, these findings suggest that 2,4-DAPG acts on multiple basic cellular processes.