Characterization of the Pseudomonas sp. DF41 quorum sensing locus and its role in fungal antagonism

Pseudomonas sp. DF41 is able to suppress the fungal pathogen Sclerotinia sclerotiorum through production of a lipopeptide called sclerosin. The aim of this study was to isolate the DF41 QS locus and characterize its role in fungal antagonism. Through screening of a fosmid library, one clone was selected that tested positive for AHL production. Sequence analysis revealed the presence of two QS genes: pdfR and pdfI, encoding a LuxR transcriptional activator and an AHL synthase, respectively. Downstream of pdfI lays a gene encoding a transcriptional activator called RfiA followed by pdfC, comprising part of an efflux locus. Characterization of an AHL-deficient strain revealed it to be phenotypically identical to the wild type. Conversely rfiA, which is co-transcribed with pdfI, is essential for both AF activity and sclerosin produc- tion. Using a pdfI-lacZ fusion analysis, we discovered that pdfI is positively autoregulated. Additionally, pdfI expression was markedly increased in the rfiA mutant and quantification of AHL levels revealed ele- vated intracellular signal accumulation. We hypothesize that RfiA is a positive activator of the down- stream efflux pump which serves to export both sclerosin and AHL signals. In a gacS mutant, pdfI-lacZ activity was decreased; however, plasmid-borne rsmZ was able to restore expression. Collectively, our findings indicate that: (i) QS indirectly controls DF41 suppression of Sclerotinia through RfiA; and (ii) pdfI expression and AHL signal production are positively regulated by the Gac-Rsm system. Identification of the PdfRI QS system, RfiA and RsmZ add to the increasingly complex network overseeing expression of DF41 biocontrol factors.