supernatant was then estimated by using ISM-146CN; C (micro combination ion cyanide electrode) procured from Lazar research Laboratories, Inc, Los Angeles, CA-90038, USA, by following a previously reported protocol [36]. The micro combination ion cyanide electrode is a combination electrode not requiring a separate reference electrode and can measure in volumes as low as ,10 ml. Measurements were made by connecting electrode to a Mettler-Toledo MP220 pH meter set to read on mV. The cyanide content was calculated using a standard curve developed using standard KCN and data was presented as mM of cyanide ions produced. All the treatments had three replicates and the experiment was completed on two independent occasions. Cyanide sensitivity assay on the growth of A. thaliana Col-0 roots The effect of cyanide on Arabidopsis thaliana Col-0 root growth was studied by using both the direct plate assay and the compartment plate assay described above except that the bacterial culture was replaced with KCN (0–700 mM). The filter disc soaked in a required amount of KCN from the stock was directly placed at a distance of 1.5 cm below the growing root tip of A. thaliana Col-0 plants. The indirect effect of cyanide was studied using compartment plates by soaking the sterile filter discs with KCN (0–700 mM KCN). A control filter disc soaked with sterile water used in both the assays. Further, 3–4 day old Arabidopsis seedlings were placed vertically on MS solid medium in the opposite compartment. The plates were incubated for five days, 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. Histochemical assay for b-glucouronidase (GUS) activity Three-day old seedlings of A. thaliana Col-0 transgenic line stably expressing a DR5::GUS fusion were used to study the indirect effect of pseudomonad metabolites and cyanide on auxin responsive gene expression in Arabidopsis. The experimental set up was similar to that of the compartment plate assay conducted to study the effect of indirect exposure of the pseudomonad strains and cyanide except Col-0-DR5::GUS seedlings were used instead of wild type Col-0. Other treatment sets such as culturing DR5::GUS seedlings on MS solid medium containing IAA (1 mg L21), IAA+cyanide, transferring back 48 hr cyanide exposed plants back to IAA medium, were also included to study the effect of exogenously supplied IAA on DR5 expression. At the end of 5 days of incubation, the roots were stained for DR5 driven GUS expression using a GUS staining kit procured from Sigma, USA. The staining was performed according to the manufacturer’s instructions. The stained roots were imaged using a stereo- microscope (Zeiss Axioskop-2). The resulting images were captured and processed using the Axiovision software. Effect of Pseudomonad Cyanide on B. subtilis FB17 Colonization and Biofilm Formation on A. thaliana Col-0 roots Three-day-old seedlings of A. thaliana Col-0 germinated on MS solid medium with 3% sucrose were transferred to 4 wells of the six well plates containing 4 ml MS liquid medium with 1% sucrose. The other two wells were filled with LB solid medium. The plants were grown for an additional 9 days; on the 9th day the wells with LB solid medium were inoculated with a 10 ml of 0.02 OD600 cultures each of the different strains used in the study, each strain to separate plates. The cultures were allowed to grow for one more day and on the 10th day the wells with the plants were Pseudomonad Cyanogenesis supplemented with 5 ml of 0.02 OD600 overnight grown cultures of B. subtilis FB17. Another set of plates were also set up where the two wells without the plants were used to generate cyanide gas by adding 20 ml each of 0.1 mM KCN and 0.1 mM HCl to the sterile filter discs placed in the wells. The plates were made airtight by sealing with parafilm, incubated on a rotary shaker set at 90 rpm, 24 hour post-inoculated roots were fixed in 4% para-formaldehyde and used for visualization and imaging for biofilm formation using confocal scanning laser microscope (CSLM). b-Galactosidase assay To study the effect of pseudomonad cyanogenesis on the transcription profile of either the yqxM and epsA promoters under biofilm formation conditions, B. subtilis strain Marburg carrying either the epsA-lacZ fusion (NRS1663) or yqxM-lacZ fusion (NRS1531) were utilized. The B. subtilis strains were grown to mid-late exponential phase in biofilm medium [37] from a freshly streaked LB plate and subsequently diluted to an OD600 of 0.02 in fresh biofilm medium. 100 ml of the diluted culture was transferred to a flat-bottomed 96-well microtitre plate. Two rows on either side of the B. subtilis culture were supplemented with 100 ml 0.2 OD600 culture of different pseudomonad strains, each in a separate microtitre plates. A separate set of plates was also prepared with generated HCN instead of pseudomonad cultures in the two rows on either side of the B. subtilis culture by adding 30 ml each of 0.1 mM KCN and 0.1 mM HCl. The control plates had 100 ml of LB liquid medium in the wells of two rows on either side of the row containing B. subtilis. The microtitre plates were made airtight by sealing with parafilm and were incubated at 37uC in an incubator shaker. During incubation, the samples were collected at 3hr time intervals from 3–12 hrs and the OD600 recorded at each time point was used as an indication of cell growth. The b- galactosidase units produced per minute were estimated using the following protocol. Briefly, about 300 ml of the culture drawn at each time point were centrifuged at 14000 rpm at 4uC for 20 min. The pellet was then re-suspended in 0.5 ml Z buffer (0.06 M Na2HPO4.7H2O, 0.04 M NaH2PO4.H2O, 0.01 M KCl, 0.001 M MgSO4.7H2O), pH adjusted to 7.0 (the buffer was supplemented with 2.9 ml ml21 (,0.05M) of b-mercapto-ethanol prior to use) and also a drop of toluene was added. The re-suspended pellet and buffer solution was then vortexed for 5 min to lyse the cells. To the lysate, 0.2 ml of ONPG (O-nitrophenyl- b-D-galactopyranoside) (4 mg ml21 in Z-buffer) was added. The reaction was stopped by adding 0.5 ml 1M NaCO3 and the time length of the reaction was recorded. The supernatant was collected by spinning down the cell debris at 14000 rpm for 5 min and the OD420 was recorded. The specific activity of b-galactosidase in terms of Miller units produced per minute was calculated by using the formula; specific activity (Miller units) = 10006[OD420/(t6v6OD600) where, t = time in minutes; v = volume of the cells assayed. The b- galactosidase activity values were plotted against time. A separate graph showing the growth of the culture at different time intervals was also plotted as OD600 against time. The experiment was repeated on at least 3 independent occasions and a representative plot is shown. Microscopy In order to view adherent B. subtilis FB17 cells and biofilm on the root surface by confocal scanning laser microscopy (CSLM), the roots were stained with SYTOH13 (Invitrogen, Molecular Probes, Eugene, OR). Images were captured with a 106 objective on a Zeiss LSM 510 NLO attached to an Axiovert 200M with an automated stage microscope equipped with Zeiss LSM 510 PLoS ONE | www.plosone.org 3 April 2008 | Volume 3 | Issue 4 | e2073