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Members of the genusBurkholderiaare known for their ability to suppress soil-borne fungal pathogens by the production of various antibiotic compounds. In this study we investigated the role ofN-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) in the expression of antifungal traits. Using a quorum quenching approach, that is, by heterologous expression of theBacillussp. AiiA lactonase, we show that expression of antifungal activities is AHL-dependent in the large majority of the investigated strains belonging to variousBurkholderiaspecies. We demonstrate that in certain strains ofBurkholderia ambifaria, Burkholderia pyrrociniaandBurkholderia lata,one of the QS-regulated antifungal agents is pyrrolnitrin (prn), a common broad-spectrum antibiotic that is also produced by somePseudomonasandSerratiaspecies. To investigate the underlying molecular mechanisms of AHL-dependent prn production in better detail, we inactivated the AHL synthasecepIas well ascepR,which encodes the cognate AHL receptor protein, inB. lata383. Both QS mutants no longer produced prn as assessed by gas chromatography-mass spectrometry analysis and as a consequence were unable to inhibit growth ofRhizoctonia solani. Using fusions of thelacZgene to the promoter of theprnABCDoperon, which directs the synthesis of prn, we demonstrate that expression of prn is positively regulated by CepR at the level of transcription.