Cyanogenic pseudomonads influence multitrophic interactions in the rhizosphere

Abstract

In the rhizosphere, plant roots cope with both pathogenic and beneficial bacterial interactions. The exometabolite production in certain bacterial species may regulate root growth and other root-microbe interactions in the rhizosphere. Here, we elucidated the role of cyanide production in pseudomonad virulence affecting plant root growth and other rhizospheric processes. Exposure of Arabidopsis thaliana Col-0 seedlings to both direct (with KCN) and indirect forms of cyanide from different pseudomonad strains caused significant inhibition of primary root growth. Further, we report that this growth inhibition was caused by the suppression of an auxin responsive gene, specifically at the root tip region by pseudomonad cyanogenesis. Additionally, pseudomonad cyanogenesis also affected other beneficial rhizospheric processes such as Bacillus subtilis colonization by biofilm formation on A. thaliana Col-0 roots. The effect of cyanogenesis on B. subtilis biofilm formation was further established by the down regulation of important B. subtilis biofilm operons epsA and yqxM. Our results show, the functional significance of pseudomonad cyanogenesis in regulating multitrophic rhizospheric interactions.

Figure 1. Direct and indirect effect of pseudomonad strains on the growth of A. thaliana Col-0 roots (A&B).

Severe reduction of primary root length (A&B) followed by the death of the seedlings was observed 5 days post-inoculation in the case of PAO1, PA14 and CHAO cultured at a distance of 1.5 cm from the primary root tip. The data also shows less reduction in primary root length in the case of PAO6344, CHAO77, when compared to PA01, PA14 and CHAO, Different letters indicate a statistically significant difference between treatments (Fisher's LSD, P<0.05). Data is the mean±SD of 12 replicates and the experiment was repeated on two independent occasions.

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 mean±SD of 3 replicates and the experiment was repeated on two independent occasions.

Figure 3. Direct and indirect effect of different doses (0–700 µM) 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; r ² = 0.8581) and HCN (D; r ² = 0.779). Data is the mean±SD of 12 replicates and the experiment was repeated on two independent occasions.

Figure 4. Suppression of DR5::GUS expression in A. thaliana by the indirect exposure of the pseudomonad strains (A) and cyanide (B) and the effect of exogenous IAA on cyanide mediated down regulation of DR5::GUS expression (C).

The images show complete suppression of DR5::GUS expression in the Col-0 DR5::GUS transgenic seedling roots when indirectly exposed to P. aeruginosa strains PAO1 and PA14, CHAO and cyanide. The images were representative of ten independent plants imaged (A) (bar = 100 µm). The figure also shows the effect of different doses (0–700 µM) of cyanide on DR5::GUS expression. The arrows in the panel show the localized DR5 expression.

Figure 5. Suppression of B. subtilis biofilm formation on A. thaliana Col-0 roots by indirect exposure of the pseudomonad strains and cyanide (A) and the effect of indirect exposure of the pseudomonad strains and cyanide on single cell growth of B. subtilis (B).

The images show complete suppression of B. subtilis biofilm formation when exposed to the indirect exposure of the strains PAO1, PA14, CHAO and HCN when compared to control plants not exposed to either bacterial culture or HCN and ΔhcnB mutants PAO6344 and CHAO77. The data also shows extensive colonization and biofilm formation by B. subtilis with indirect bacterial exposure from the hcnB mutants PAO6344 and CHAO77. The images were representative of the roots from six independent plants imaged.

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 37°C with or without exposure to pseudomonad strains and HCN. Growth (A) and β-galactosidase activity (B) were measured at regular intervals and plotted as a function of time. Data is the mean±SD 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.

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 37°C with or without exposure to pseudomonad strains and HCN. Growth (A) and β-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 mean±SD of 12 replicates and the experiment was repeated on two independent occasions.