Figure 7. Effect of indirect exposure of the pseudomonad strains and cyanide on the transcription of the yqxM operon in B. subtilis. Strain Marburg thrC::yqxM-lacZ (NRS1531) 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. These experiments were repeated on at least 3 independent occasions and a representative plot is shown. Data is the mean6SD of 12 replicates and the experiment was repeated on two independent occasions. doi:10.1371/journal.pone.0002073.g007 roots. Interestingly, it has been observed that the phytotoxic effect of the auxin herbicide quinclorac on shoot growth was mediated through the accumulation of phytotoxic levels of cyanide [52]. Further, this accumulation was caused through the induction of ACC synthase (1-aminocyclopropane-1-carboxylic acid) activity in the tissues, which produce cyanide as a co-product of ethylene [52,53]. Plant respiration rates are usually amended in response to both biotic and abiotic stress [54]. Cyanide has been reported to play an important role in modulating the respiratory electron transport in aerobic respiration in all plants, many fungi and some protozoa [54]. Conversely, decrease in respiration rates, because of abiotic stress like cyanide exposure results in cell death [54]. This mortality effect is due to the inhibition of cytochrome c oxidase (COX) which prevents electron transport from COX to O2, resulting in impedance of proton motive force that drives ATP synthesis [55–57]. Additionally, it is known that inhibition of ATP synthesis affects auxin biosynthesis in terms of its transport and localization in plants [58]. It is reported that auxin can stimulate cell elongation in plant roots, which is usually accompanied by promotion of respiration [59,60]. However, it is uncertain if respiration is promoted when auxin induces cell elongation. Our studies showed the cyanide mediated impairment of auxin signaling. It could be argued that our observation relates to an indirect effect of the impeded respiration post-cyanide treatment on auxin biosynthesis. Inter- Pseudomonad Cyanogenesis estingly, a report on other respiration inhibitors (monoiodoacetate, malonate and 2,4-dinitrophenol) shows elimination of the auxin effect on respiration and cell division [61]. Based on previous studies and our findings, it can be concluded that promotion of respiration is a common auxin effect in plants [61] and impeding auxin biosynthesis could lead to respiration inhibition and cell death. In the future, it would be interesting to study the relationship between cyanide-mediated suppression of auxin induced gene expression and the reported mechanism of cyanide mediated inhibition of mitochondrial respiration [62]. Apart from its effect on root growth, the cyanide produced by pseudomonad strains in the soil has been reported to cause inhibitory effect on pathogenic fungi and bacteria [63,64]. Except for this ecological role, there are no reports available which demonstrate the effects of pseudomonad cyanogenesis on rhizosphere microorganisms. Our study shows the effect of cyanide on the B. subtilis colonization and biofilm formation on the roots of A. thaliana. Our recent reports suggest that B. subtilis FB17 forms a complete and mature biofilm on A. thaliana roots [39,65]. The biofilm formation ability of B. subtilis is related to its biocontrol activity against root borne pathogens [39]. Our results indicated a suppression of B. subtilis FB17 colonization by the indirect exposure of the pseudomonad strains and cyanide without affecting the B. subtilis single cell growth. Further, the suppression of the B. subtilis biofilm operons epsA and yqxM by indirect exposure of pseudomonads and cyanide supported this observa- tion. These results suggest that the effect is brought about through the down regulation of key biofilm operons in B. subtilis. This intriguing result signifies the plausible role of cyanogenesis in regulating various rhizospheric interactions. However, another possibility for the observed non-lethality of released cyanide on the single cell growth of B. subtilis maybe due to partial degradation of the released cyanide, keeping bacterial exposure below the lethal dose. A cyanide dihydratase capable of degrading the cyanide has been reported in Bacillus pumilis [66]. In summary, the present report established that pseudomonad cyanogenesis affects A. thaliana Col-0 primary root growth by inhibiting auxin synthesis/perception. Additionally, this study established that cyanogenesis also affected one of the multitrophic rhizospheric processes; in particular, B. subtilis biofilm formation on Arabidopsis roots in vitro. Therefore, this study highlights the multifaceted attributes of a bacterial virulence factor, capable of harming its host through more than one mechanism and also implicates cyanogenesis as a multi-host virulence factor. Since these studies are conducted, in vitro, future studies would benefit from testing other host and testing the infection processes in host’s microcosm. It would also be interesting to study the effects of cyanogenesis in other root- rhizospheric microbial interactions and its effect on the root exudation profile as a marker for perturbation in overall plant defense. Acknowledgments The authors thank all the researchers who provided various materials for this study. The authors also thank Dr. Kirk Czymmek and the faculty, Bio- imaging center, Delaware Biotechnology Institute for their help with microscopic studies. Author Contributions Conceived and designed the experiments: HB TR. Performed the experiments: TR RS MB. Analyzed the data: HB TR. Contributed reagents/materials/analysis tools: TR. Wrote the paper: HB TR. PLoS ONE | www.plosone.org 9 April 2008 | Volume 3 | Issue 4 | e2073