NPR1 Protein Regulates Pathogenic and Symbiotic Interactions between Rhizobium and Legumes and Non- Legumes Smadar Peleg-Grossman, Yael Golani, Yuval Kaye, Naomi Melamed-Book, Alex Levine* Department of Plant and Environmental Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Abstract Background: Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs) produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non- legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA). SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1. Methodology/Principal Findings: We used Arabidopsis mutants in SA defense pathway to test the role of NPR1 in symbiotic interactions. Inoculation of Sinorhizobium meliloti or purified NF on Medicago truncatula or nim1/npr1 A. thaliana mutants induced root hair deformation and transcription of early and late nodulins. Application of S. meliloti or NF on M. truncatula or A. thaliana roots also induced a strong oxidative burst that lasted much longer than in plants inoculated with pathogenic or mutualistic bacteria. Transient overexpression of NPR1 in M. truncatula suppressed root hair curling, while inhibition of NPR1 expression by RNAi accelerated curling. Conclusions/Significance: We show that, while NPR1 has a positive effect on pathogen resistance, it has a negative effect on symbiotic interactions, by inhibiting root hair deformation and nodulin expression. Our results also show that basic plant responses to Rhizobium inoculation are conserved in legumes and non-legumes. Citation: Peleg-Grossman S, Golani Y, Kaye Y, Melamed-Book N, Levine A (2009) NPR1 Protein Regulates Pathogenic and Symbiotic Interactions between Rhizobium and Legumes and Non-Legumes. PLoS ONE 4(12): e8399. doi:10.1371/journal.pone.0008399 Editor: Karin Schumacher, University of Heidelberg, Germany Received September 15, 2009; Accepted November 24, 2009; Published December 21, 2009 Copyright: 2009 Peleg-Grossman 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: This work was supported in part by the Israel Science Foundation (ISF). YK and YG were supported by the Canadian Friends of the Hebrew University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alexl@cc.huji.ac.il Introduction Plants continually interact with soil micro-organisms that are broadly divided into pathogenic, saprophytic or symbiotic. While the pathogenic and saprophytic interactions are common to all plant species, symbiosis with the nitrogen-fixing rhizobacteria is a relatively recent evolutionary development that is restricted to plants of the legumes family. Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from the Rhizo- bium genus, which provide plants with a source of available nitrogen. Symbiosis is regulated by complex mutual interactions between the organisms. Symbiosis between legumes and Rhizobium is initiated by specific nodulation (Nod) factors (NFs) that are secreted into the soil by the bacteria. In response to compatible NFs the legume root hairs begin to curl, entrapping the bacteria. The cell wall within the curl undergoes a local hydrolysis, allowing bacteria to enter the root hair and form an intracellular infection thread from the curled region to the root cortex [1]. The root cells in the cortex undergo reprogramming and begin to divide rapidly, giving rise to a nodule primordium, a specific plant organ that provides favorable environment for nitrogen fixation by Rhizobium [2]. In contrast to symbiotic interaction with rhizobacteria, plants mount a defense response when challenged with pathogenic bacteria. Several signaling pathways that mediate local and systemic plant responses to pathogens have been identified. One of the major signaling pathways induced during pathogenic interactions, including local and systemic defense responses to pathogens is mediated by salicylic acid (SA); for reviews: [3,4]. SA is sensed and transduced by NPR1 protein, which is a redox- sensitive protein that contains several ankyrin repeats and has limited homology to IkBa [5]. During pathogenesis response, the challenged cells undergo an oxidative burst followed by reduction of two conserved cysteines in the NPR1, leading to its monomerization and nuclear localization. In its reduced form, the NPR1 protein interacts with bZIP transcription factors of the TGA/OBF family, and activates the SA-responsive element in the promoters of defense genes, such as pathogenesis related protein, PR1 [6]. Increased production of SA, or NPR1 overexpression cause enhanced disease resistance (edr1) phenotype in heterologous plant species, suggesting an evolutionary conserved SA-mediated signaling pathway in different plants [7]. Moreover, mutations that block SA perception and signaling, such as nim1/npr1, as well as PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8399