Cyanogenic Pseudomonads Influence Multitrophic Interactions in the Rhizosphere Thimmaraju Rudrappa1,2, Robert E. Splaine1,2, Meredith L. Biedrzycki1,2, Harsh P. Bais1,2* 1 Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, United States of America, 2 Delaware Biotechnology Institute, Newark, Delaware, United States of America 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. Citation: Rudrappa T, Splaine RE, Biedrzycki ML, Bais HP (2008) Cyanogenic Pseudomonads Influence Multitrophic Interactions in the Rhizosphere. PLoS ONE 3(4): e2073. doi:10.1371/journal.pone.0002073 Editor: Hany A. El-Shemy, Cairo University, Egypt Received January 16, 2008; Accepted March 21, 2008; Published April 30, 2008 Copyright: 2008 Rudrappa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: HPB acknowledges the University of Delaware and EPSCoR for faculty start-up grant. RES acknowledges the Science and Engineering program at University of Delaware for the undergraduate research funding support. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hbais@udel.edu Introduction Cyanogenesis, a process by which cyanide (CN2 or HCN) 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 has often been reported to occur in the 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 sorption and mobility of cyanide produced from such organisms is mainly through soil surfaces and solutions. It is known that cyanide produced in the soil is usually associated with various metal ions, allowing for its rapid mobility in the ground water and subsequent diffusion in the atmosphere [8]. The two most extensively studied bacteria for cyanogenesis, as stated earlier P. aeruginosa and P. fluorescens, are both commonly found in the soil. Pseudomonas aeruginosa, a Gram-negative bacterium, has been reported to cause opportunistic infections in humans, animals and plants [9–13]. The ability of P. aeruginosa to infect humans and plants has been ascribed to its production of various secreted and surface associated virulence factors [11]. Apart from various protein toxins, P. aeruginosa also produces small molecular toxins such as cyanide that facilitate the overall virulence of this opportunistic bacterium against multiple hosts [14–21]. Interest- ingly, certain pseudomonads have been characterized as root colonizers of several food crops that evade pathogenesis against multiple pathogens [22]. Siderophores and cyanide production ability in various pseudomonads are linked to antagonistic and disease suppressing activity against various plant pathogens [23]. The discovery that P. aeruginosa has the ability to infect plants allowed for the use of the A. thaliana - P. aeruginosa infection model by various research groups [11,20]. The two contrasting ecological roles of pseudomonads as a biocontrol agent and opportunist plant pathogen suggests that both root colonization and pathogenesis inflicted by this bacterium are highly species specific interactions. Apart from bacteria, plants have also been reported to produce cyanide to defend themselves against herbivores [24]. In plants, cyanide is produced as cyanogenic glycosides and stored in the vacuole of the cell. Storage in the vacuole allows for separation from the enzymes, which act on the glycosides, and prevents the cells from auto-toxicity [24]. 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 [24]. The potential of cyanogenic glycosides in plants as chemical defense has been demonstrated in Sorghum bicolor [25]. Additionally, cyanogenesis plays an important role in the fitness of Trifolium repens against herbivores [26]. The quantitative analysis of the amount of cyanide containing compounds stored in a given tissue and their capacity to release HCN per unit time as a reaction to herbivory has been studied in lima bean (Phaseolus lunatus L.) [27]. Despite PLoS ONE | www.plosone.org 1 April 2008 | Volume 3 | Issue 4 | e2073