Pseudomonad Cyanogenesis Figure 4. Suppression of DR5::GUS expression in A. thaliana by the indirect exposure of the pseudomonad strains (A) and cyanide (B) and the effect of exogenous IAA on cyanide mediated down regulation of DR5::GUS expression (C). The images show complete suppression of DR5::GUS expression in the Col-0 DR5::GUS transgenic seedling roots when indirectly exposed to P. aeruginosa strains PAO1 and PA14, CHAO and cyanide. The images were representative of ten independent plants imaged (A) (bar = 100 mm). The figure also shows the effect of different doses (0–700 mM) of cyanide on DR5::GUS expression. The arrows in the panel show the localized DR5 expression. doi:10.1371/journal.pone.0002073.g004 data (Figure 5B). These results clearly indicated that apart from the inhibition of plant primary root growth, pseudomonad cyanogenesis also affects other rhizospheric processes such as biofilm formation by a beneficial biocontrol PGPR such as B. subtilis. Pseudomonad cyanogenesis and cyanide down regulate Bacillus subtilis biofilm operons While pseudomonads and cyanide suppressed the ability of B. subtilis to form biofilms, it did not suppress the single cell growth. Therefore, we hypothesized that pseudomonads and cyanide may inhibit biofilm formation through limiting the induction of one (or both) of two key loci required for biofilm formation, the epsA-O and yqxM-sipW-tasA operons [40–45]. To test this hypothesis, we used the transcriptional fusions of the promoter regions for both the epsA and yqxM operons to b-galactosidase and monitored the expression profile in the presence and absence of PAO1, CHAO, PAO6344, CHAO77 and cyanide. Treatment with PAO1, cyanide and CHAO suppressed the induction of epsA by more than 40% (Figure 6B) and yqxM by about 50% (Figure 7B) when compared to the untreated control and cyanide mutants. The suppression of transcription induction in each case occurred from the late log phase and continued through the stationary phase (see Figure 6A&7A for growth), suggesting that there may be a receptor that is actively produced in the late log-phase to late stationary phase in B. subtilis that could perceive the presence of cyanide. These data clearly showed the specificity of cyanide and cyanide producing pseudomonads in down regulation of B. subtilis biofilm operons. Discussion Among the fluorescent pseudomonads, cyanogenesis in the soil bacteria P. fluorescens has been researched extensively on its role as a biocontrol agent [46]. However, the studies on the role of cyanogenesis in the virulence of P. aeruginosa are very scarce. Along similar lines, it has been reported that cyanide production in P. aeruginosa mediate paralytic killing of C. elegans in a fast killing assay [1]. To understand the role of cyanogenesis in a plant infection model system, we followed a systematic bioassay driven approach. As a first step towards this objective, we tested the effect of different pseudomonad strains (both P. fluorescens and P. aeruginosa) on the growth of A. thaliana Col-0 roots. The direct bacterial plate assay revealed that a diffusible factor produced by the pseudomo- PLoS ONE | www.plosone.org 6 April 2008 | Volume 3 | Issue 4 | e2073