Pseudomonad Cyanogenesis Figure 6. Effect of indirect exposure of the pseudomonad strains and cyanide on the transcription of the epsA operon in B. subtilis. Strain Marburg thrC::epsA-lacZ (NRS1663) was grown in biofilm medium under biofilm formation conditions at 37uC with or without exposure to pseudomonad strains and HCN. Growth (A) and b-galactosidase activity (B) were measured at regular intervals and plotted as a function of time. Data is the mean6SD of 12 replicates and the experiment was repeated on two independent occasions. These experiments were repeated on at least 3 independent occasions and a representative plot is shown. doi:10.1371/journal.pone.0002073.g006 compartment plate assays are very important due to the fact that the microbe-synthesized cyanide is readily converted into the HCN form, diffuses into the air and exerts its effect on the flora and fauna in the soil [20]. It is reported that HCN is not required for growth, energy storage, or primary metabolism but may provide some ecological advantage to the organism [47]. The highest level of cyanide produced was observed in the late exponential and early stationary phase in both P. aeruginosa PAO1 and P. fluorescens, therefore, the present study was in accordance with earlier reports [20]. However, the slightly reduced recovery of roots from the inhibition effects in the case of cyanide mutants may be a result of the production of other virulence factors in addition to cyanide [15–17]. Whereas the effect in the case of PAO210 could be due to significantly higher hcnA expression, resulting in higher cyanide production as previously reported for mutation in rhl operon [48]. A mechanistic insight into the severe rhizotoxicity of cyanide on A. thaliana roots was envisaged by utilization of an artificial auxin responsive transgene (DR5). DR5, an artificial AuxRE containing the TGTCTC element, has increased auxin responsiveness to auxin [49]. The GUS reporter gene fused to a minimal cauliflower mosaic virus 35S promoter, and the DR5 has been used widely as a marker to monitor endogenous IAA distribution because the resulting GUS activity coincides with endogenous IAA distribution [50–51]. To gain insight into the mechanisms of cyanide-induced rhizotoxicity, we studied the involvement of the DR5 in cyanide- induced auxin expression. Transgenic A. thaliana seedlings containing the DR5-GUS reporter gene were exposed in compartment plate assays to different pseudomonad strains and HCN. Interestingly, the DR5::GUS seedlings treated with pseudomonad strains and HCN revealed a complete suppression of DR5::GUS expression at the root tip level. However, the restoration of DR5::GUS expression when supplemented with exogenous IAA further confirmed the involvement of cyanide mediated impairment of auxin biosynthesis and/or perception. A significant reduction in the seedling root growth of lettuce, barnyard grass and green foxtail was observed when treated with pseudomonad released metabolites, which was further correlated to HCN production [22]. However, there is no report on the effect of cyanide on either auxin gene expression or signaling in plant PLoS ONE | www.plosone.org 8 April 2008 | Volume 3 | Issue 4 | e2073