Figure 2. Kinetics of cyanide synthesis and accumulation in the different pseudomonad strains. The data shows highest cyanide accumulation in CHAO and PAO1 with an increased synthesis during late log phase and early stationary phase. The data is the mean6SD of 3 replicates and the experiment was repeated on two independent occasions. doi:10.1371/journal.pone.0002073.g002 produced by the pseudomonad strains is responsible for primary root growth inhibition, exogenously supplied cyanide should also inhibit the A. thaliana Col-0 root growth. Consistent with our hypothesis, the plants exposed with both direct KCN and indirect (HCN) showed significant (P,0.05; n = 12) inhibition of the primary root growth of A. thaliana Col-0 seedlings (Figure 3A&B). The plot shows the linearity of the relationship between the Figure 3. Direct and indirect effect of different doses (0– 700 mM) cyanide on the growth of A. thaliana Col-0 seedling. The data shows the linear regression plots (A&B) shows the predicted (line) and experimental (dots) values of primary root length at different concentrations of KCN(B; r2 = 0.8581) and HCN (D; r2 = 0.779). Data is the mean6SD of 12 replicates and the experiment was repeated on two independent occasions. doi:10.1371/journal.pone.0002073.g003 Pseudomonad Cyanogenesis predicted (line) and experimental (dots) values of primary root length and cyanide concentration (Figures 3A&B). Thus, all these results together conclusively established that the inhibition of A. thaliana Col-0 primary root growth by different strains of pseudomonads especially P. aeruginosa PAO1, PA14 and P. fluorescens CHAO is due to the production and release of cyanide. This result was also confirmed by the reduced inhibition of primary root growth by cyanide mutants of P. aeruginosa PAO6344 and P. fluorescens CHAO77 (Figures 1A&B). Pseudomonad cyanogenesis and cyanide down-regulate the expression of the auxin responsive promoter DR5 Auxin is an important plant hormone which controls primary root growth by regulating cell proliferation and enlargement [32]. Since all of our results with pseudomonad strains and cyanide showed severe inhibition of primary root growth in A. thaliana Col- 0, we further speculated that this may affect the auxin biosynthesis/perception at the root tip. To examine this interesting speculation, we conducted compartment plate assays using an A. thaliana Col-0 transgenic line stably expressing a GUS reporter gene fusion to the auxin responsive promoter DR5 (DR5::GUS). The results presented in Figure 4A show a complete suppression of the DR5 expression when co-inoculated with P. aeruginosa strains PAO1/PA14, P. fluorescens strain CHAO and cyanide (KCN) treatment. All of the other bacterial treatments such as cyanide mutants PAO6344 and CHAO77, showed stable expression of DR5::GUS similar to the control untreated plants. The dose response experiments performed using HCN (0–700 mM KCN+0.1 M HCl) showed complete suppression of DR5::GUS expression at $100 mM (Figure 4B). Further, the DR5::GUS expression was not affected when exogenous IAA (1 mg l21) was included in the assay plates and the DR5::GUS expression was restored in the plants pre-treated with cyanide and subsequently exposed to IAA (Figure 4C). These results suggest that the cyanide produced by different strains of P. aeruginosa cause the inhibition of primary root growth of A. thaliana Col-0 through a complete suppression of the auxin biosynthesis/perception at the root tip. Indirect exposure of the pseudomonad strains and cyanide suppress Bacillus subtilis colonization on A. thaliana Col-0 roots Since the pseudomonads are common soil bacteria, we suspected that apart from their effect on plant roots, they might also influence other rhizospheric processes such as beneficial bacteria-root associations. Therefore, we studied the possible effects of pseudomonad cyanogenesis on B. subtilis colonization and biofilm formation on Arabidopsis. B. subtilis, a biocontrol PGPR (plant growth-promoting rhizobacteria), forms biofilms on A. thaliana roots and protects the plant from pathogenic infections [37,39]. The experiment was conducted in vitro; in six well plates using 10-day-old A. thaliana seedlings grown in MS liquid medium with 1% sucrose (see materials and methods). The results presented in Figure 5 show a complete suppression of B. subtilis FB17 biofilm formation on the roots subjected to indirect exposure with P. aeruginosa strains PAO1/PA14, P. fluorescens strain CHAO and HCN (KCN+HCL) (Figure 5A). The control which did not receive any bacterial culture, and the treatments, with bacterial cultures of cyanide impaired mutants PAO6344, and CHAO77 formed extensive biofilms. Although indirect exposure of the pseudomonad strains and cyanide caused complete suppression of B. subtilis biofilm formation on Arabidopsis roots, they did not however, affect the single cell growth of B. subtilis in the Arabidopsis culture medium as indicated by FB17 colony forming units (CFU) PLoS ONE | www.plosone.org 5 April 2008 | Volume 3 | Issue 4 | e2073