these foliar examples, there are no reports of secretion of cyanide into the rhizosphere through plant roots. However, P. fluorescens, when associated with weed seedlings, are known to produce toxic levels of cyanide, causing considerable inhibition of root growth [28–30]. Although the potential of cyanogenesis by different strains of rhizobacteria such as P. fluorescens and P. aeruginosa, for suppression of weed growth or allelopathy has been examined, few of these studies have attempted to investigate the rhizotoxic effect of bacterial cyanogenesis [30–31]. Auxin is the key component which stimulates cell extension and growth in plant roots [32]. Although, the intervention of secondary metabolites such as flavonoids with auxin activity is well investigated [33], the effect of cyanide on auxin perception and biosynthesis still awaits elucidation. The effects of cyanide on the processes of auxin perception or biosynthesis cascade at the root tips are worth considering owing to its role in gibberellic acid mediated root growth and elongation [34]. Auxin plays a key role in the regulation of nuclear export and distribution of DELLA protein RGA which suppresses root growth [34]. To our knowledge, no study has attempted to elucidate the mechanism of inhibition of plant root growth by cyanogenesis in the opportunistic pathogen P. aeruginosa and its effect on the colonization of beneficial plant growth promoting rhizobacteria (PGPR) such as Bacillus subtilis on the plant roots. In the present study, we systematically examined cyanogenesis in different strains of P. aeruginosa and P. fluorescens for its phytotoxicity by following a bioassay driven approach using the model plant Arabidopsis thaliana. We demonstrated that both indirect and direct exposure of pseudomonads and cyanide causes inhibition of Arabidopsis primary root growth. Further, we show that mechanistically, cyanide completely suppresses the auxin signalling mechanism, causing inhibition of the primary root growth. In addition, we show that pseudomonad cyanogenesis also causes suppression of beneficial rhizospheric processes such as colonization and biofilm formation by the biocontrol bacteria Bacillus subtilis in the A. thaliana-B. subtilis model system. Materials and Methods Plant material and chemicals Arabidopsis thaliana wild type cultivar Columbia (Col-0) seeds were procured from Lehle Seeds (Round Rock, TX, USA). The Arabidopsis line stably expressing the DR5: GUS reporter fusion was obtained from Dr. Thomas Guilfoyle, University of Missouri, Columbia, MO 65211. KCN was obtained from Fluka, Germany. Culture conditions Seeds were washed in double distilled water three times and surface sterilized using 50% commercial bleach (sodium hypo- chlorite) for 3–5 min followed by 3–4 washes in sterile distilled water. The seeds were cultured on Murashige and Skoog’s [35] (MS) solid medium with 3% sucrose and allowed to germinate for 3–4 days by incubating at 2362uC under 16 hr light and 8 hr dark. The plates were illuminated with cool fluorescent light with an intensity of 24 mmol m22 s21. Microbial strains and culture conditions The P. aeruginosa strains PAO1, PA14 (obtained from Dr. Frederick Ausubel, Department of Molecular Biology, Massachu- setts General Hospital, Boston, MA) were grown on Luria broth agar (LB) plates with 20 mg ml21 rifampicin. The P. fluorescens strains CHAO, CHAO77 (cyanide synthase (DhcnABC) mutant), P. aeruginosa cyanide synthase mutant (DhcnB) PAO6344 and quorum sensing mutant PAO210 derived from PAO1 parent strain Pseudomonad Cyanogenesis (obtained from Dr. Dieter Haas, Department of Fundamental Microbiology, Universite de Lusanne, Switzerland) were grown on LB plates. The B. subtilis wild type strain FB17 (obtained from Dr. Ray Fall, University of Colorado, Boulder) and strains with biofilm operon-lacZ transcription fusions, Marburg thrC::yqxM-lacZ (NRS1531) and Marburg thrC::epsA-lacZ (NRS1663) (obtained from Dr. Nicola R. Stanley-Wall, Division of Applied and Environmental Biology, School of Life Sciences, University of Dundee, Dundee, Scotland ) were grown on LB plates supplemented with 0.5 mg ml21 erythromycin. A single colony from a freshly streaked plate (from glycerol stocks maintained at 280uC) with or without antibiotic selection of each of the cultures was used to grow overnight cultures from which approximately 0.02–0.05 OD600 culture was prepared and used in all the experiments. Vertical plate assay to study the direct effect of pseudomonad strains on the growth of A. thaliana Col-0 roots MS solid medium (with 3% sucrose) plates containing a strip of LB solid medium were prepared by cutting out a strip of MS medium from pre-made MS plates and replacing it with LB solid medium. The LB strip was spotted with 10 ml of 0.02–0.05 OD600 cultures of different strains (of P. aeruginosa, P. fluorescens) and grown overnight in LB liquid medium with or without antibiotic selection (prepared by inoculating a single colony from a freshly streaked plate). The plates were incubated overnight at 37uC and the next day the plates were cultured with 3–4 day old A. thaliana Col-0 plants by laying them approximately 1.5 cm above the bacterial colony and the plates were incubated vertically at 2362uC. The plant growth in terms of primary root length was measured on 5th day and the data was expressed as total primary root length in centimeters. Compartment plate assay to study the indirect effect of pseudomonad strains on the growth of A. thaliana Col-0 roots The indirect effect of pseudomonad strains on A. thaliana Col-0 roots was studied by using compartment plates. Of the two opposite compartments, one was filled with MS solid medium with 3% sucrose and the other was filled with LB solid medium. The compartment with LB medium was inoculated by spotting 10 ml of 0.02–0.05 OD600 cultures of different strains (of P. aeruginosa, P. fluorescens) grown in LB liquid medium with or without antibiotic selection. The plates were incubated overnight at 37uC and the next day the opposite compartment with MS solid medium was cultured with 3–4 day old A. thaliana Col-0 plants. Plants were added by laying them vertically, opposite to the compartment with the bacterial colony. The plates were made airtight by sealing with parafilm and incubated vertically at 2362uC. The plant growth in terms of primary root length was measured on 5th day and the data was expressed as total primary root length in centimeters. Kinetics of cyanide production in different pseudomonad strains The kinetics of cyanide production at different time points (0– 36 hr) was studied by growing the pseudomonad strains in 10 ml of LB broth. The culture was initiated by adding 2 ml culture (0.02 OD600) of each strain, prepared from overnight grown cultures. The cultures were incubated in a shaking incubator maintained at 37uC and set at 220 rpm. To estimate the cyanide content, a set of three culture flasks were removed at each time point and centrifuged to pellet out the cells. The cyanide content in the PLoS ONE | www.plosone.org 2 April 2008 | Volume 3 | Issue 4 | e2073