Rhizospheric pseudomonads

Abstract

Of the different groups of soil microorganisms, pseudomonads are one of the important class, playing various roles in the plants growth and development. Although they have been reported to inflict both beneficial and harmful effect on plants, they act through various mechanisms. Among the different mechanisms, cyanogenesis is one of the important factors used by pseudomonads to cause positive and less studied negative effects in the rhizosphere. By employing a bioassay driven approach, we dissected the direct effect of pseudomonad cyanogenesis on host plants and also its indirect effect through the inhibition of beneficial biofilm formation by B. subtilis. This study may further our understanding on the multi-tropic rhizospheric interactions mediated by rhizospheric pseudomonads.

Cyanogenesis, a process by which cyanide (HCN or CN^-) is produced, has been demonstrated to occur in both bacteria and plants. Among bacteria, it has been studied extensively in fluorescent pseudomonads, especially Pseudomonas fluorescens and Pseudomonas aeruginosa.¹ In addition to its activity in Pseudomonas, cyanogenesis has also been reported in Chromobacterium violaceum and in case of cyanobacteria such as Anacystis nidulans, Nostoc muscorum and Plectonema boryanum.^2–5 Additionally some strains of Rhizobium leguminosarum have also been reported to produce cyanide as free-living bacteria.⁶ In most of these cases, cyanide has been reported to be synthesized from the amino acid glycine.⁷ The cyanide produced from such organisms is readily converted to hydrogen cyanide (HCN) and diffused into the air under physiological conditions (pH 7.0). The two most extensively studied bacteria for cyanogenesis, as stated earlier P. aeruginosa and P. fluorescens, are both commonly found in the soil.

Apart from bacteria, plants have also been reported to produce cyanide to defend them against herbivores.⁸ In plants, cyanide is produced as cyanogenic glycosides and stored in the vacuole of the cell. Compartmentalization of the glycoside acting enzymes prevents the cells from auto-toxicity⁸. When cells are damaged by herbivores, the cyanogenic glycosides and the enzymes are released from their separate compartments and react to produce HCN, which is toxic to herbivores.⁸ The potential of cyanogenic glycosides in plants as chemical defense has been demonstrated in Sorghum bicolor, Trifolium repens and Phaseolus lunatus L.^9–11 Despite these foliar examples, there are no reports of secretion of cyanide into the rhizosphere through plant roots. Although there were reports of rhizosphere fluorescent bacteria producing toxic levels of cyanide resulting in inhibition of root growth,^12–15 no study has attempted to test its effect on other rhizospheric interactions such plant growth promoting rhizobacteria (PGPR). We used a bioassay drive approach to test the phytotoxicity of pseudomonad cyanogenesis on A. thaliana. In addition we also tested the effect of cyanogenesis on A. thaliana-B. subtilis (a PGPR) interactions. We studied the direct and indirect effects of different strains of both P. aeruginosa and P. fluorescens including cyanogenic mutants. All the wild type strains tested such as PAO1, PA14 (P. aeruginosa) and CHAO (P. fluorescens) showed a significant primary root growth inhibition. Where as the reduced primary root growth inhibition by cyanide mutants PAO6344 and CHAO77 confirmed the root growth inhibition was in fact because of production and release of cyanide.¹⁶ In order to support the data from indirect and direct assay on the effect of pseudomonads on the growth of Arabidopsis primary root and also establish that pseudomonads produced cyanide, we estimated the cyanide ions released in pseudomonad cultures. Further we observed a significantly higher cyanide production in the culture of P. fluorescens strain CHAO followed by P. aeruginosa strains PAO1 and PA14.¹⁶ However, significantly less cyanide production was observed in case of cyanide mutants PAO6344, CHAO77 compared to the respective parental (PAO1 and CHAO) strains. This data was consistent and directly correlated with the root inhibition phenotype observed with pseudomonad strains. These observations further lead us to the hypothesis that if, pseudomonad cultures are accumulating cyanide ions, exogenously supplied cyanide also should inhibit the root growth. Accordingly, the A. thaliana plants exposed to both direct (KCN) and indirect (HCN) cyanide showed a significantly higher inhibition of primary root growth.¹⁶ This result along with the earlier observation with the cyanide mutants conclusively proved the involvement of cyanogenesis mediated growth response. Since, we observed the inhibition of primary root growth consistently throughout by both cyanide and cyanide generating pseudomonads, we speculated for its effect on biosynthesis/perception of auxin at the root tip. Auxin is an important plant hormone which controls primary root growth by regulating cell proliferation and enlargement.¹⁷ Our experiments with a transgenic line carrying an auxin responsive promoter element DR5, fused with GUS (DR5::GUS) revealed a complete suppression of DR5::GUS expression in compartment plate assays carried out with P. aeruginosa strains PAO1/PA14, P. fluorescens strain CHAO and cyanide (KCN) treatment. However, the cyanide mutants PAO6344 and CHAO77, showed stable expression of DR5::GUS similar to the control untreated plants. A dose response experiment with HCN (0–700 µM KCN + 0.1 M HCl) also resulted in the complete suppression of DR5::GUS expression at ≥100 µM.

Since we observed a severe root inhibitory phenotype in A. thaliana in response to Pseudomonad cyanogenesis, we further questioned its effect on the root-microbe interactions in the rhizosphere, especially the beneficial associations. We verified this question by using B. subtilis, which is a soil bacterium like pseudomonads widely used as a biocontrol agent is known to form protective biofilms on the root surface and trigger induced systemic resistance (ISR) in plants.^18–20 The experiments, carried out to test the effect of pseudomonad cyanogenesis on the colonization and biofilm formation by B. subtilis revealed 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). The treatments, with bacterial cultures of cyanide mutants PAO6344, and CHAO77 formed extensive biofilms similar to controls which did not receive any bacterial cultures. Although the cyanide exposure and treatment with wild type pseudomonad strains caused complete suppression of colonization and biofilm formation, they did not affect the planktonic growth of B. subtilis in the Arabidopsis culture medium.¹⁶ These data clearly indicated that pseudomonad cyanogenesis influence other rhizospheric processes such as protective biofilm formation by a beneficial biocontrol PGPR such as B. subtilis in addition to root growth inhibition. As a mechanistic insight in to such an effect we observed the suppression of the transcription of two key operons involved in B. subtilis biofilm formation epsA and yqxM.^21–26 Treatment with PAO1, cyanide and CHAO suppressed the induction of epsA by more than 40% and yqxM by about 50% compared to the untreated control and cyanide biosynthetic mutants.

In sum our studies conclusively showed that pseudomonad cyanogenesis affects A. thaliana Col-0 primary root growth by inhibiting auxin synthesis/perception. In addition, we also found that cyanogenesis affected one of the multitrophic rhizospheric processes; in particular, B. subtilis biofilm formation on Arabidopsis roots. Therefore, our findings highlighted multiple roles of a bacterial virulence factor, inflicting damage to a host through different mechanisms. Further, the study also implicated cyanogenesis as a multi-host virulence factor. Similarly, the pseudomonad cyanogenesis also been recently reported to have a strong host plant selectivity.²⁷ Further studies involving the effect of pseudomonad cyanogenesis on other beneficial interactions and root exudation profile as a marker for perturbation in plant defense are needed for broader understanding of such processes.

Addendum to: Rudrappa T, Splaine RE, Biedrzycki ML, Bais HP. Cyanogenic pseudomonads influence multitrophic interactions in the rhizosphere. PLoS ONE. 2008;3:2073. doi: 10.1371/journal.pone.0002073.