Enhanced Disease Susceptibility 1 and Salicylic Acid Act Redundantly to Regulate Resistance Gene-Mediated Signaling Srivathsa C. Venugopal1¤a, Rae-Dong Jeong 1 , Mihir K. Mandal1, Shifeng Zhu1, A. C. Chandra-Shekara1¤b, Ye Xia1, Matthew Hersh2, Arnold J. Stromberg2, DuRoy Navarre3, Aardra Kachroo1, Pradeep Kachroo 1 * 1 Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, United States of America, 2 Department of Statistics, University of Kentucky, Lexington, Kentucky, United States of America, 3 United States Department of Agriculture–Agricultural Research Service, Washington State University, Prosser, Washington, United States of America Abstract Resistance (R) protein–associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non–race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA–synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N- terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense. Citation: Venugopal SC, Jeong R-D, Mandal MK, Zhu S, Chandra-Shekara AC, et al. (2009) Enhanced Disease Susceptibility 1 and Salicylic Acid Act Redundantly to Regulate Resistance Gene-Mediated Signaling. PLoS Genet 5(7): e1000545. doi:10.1371/journal.pgen.1000545 Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America Received March 26, 2009; Accepted June 1, 2009; Published July 3, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by grants from NSF (MCB#0421914), USDA-NRI (2004-03287), and KSEF (419-RDE-004, 555-RDE-005, 622-RDE-006). 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: pk62@uky.edu ¤a Current address: Metahelix Life Sciences Private Limited, Bangalore, India ¤b Current address: Dow AgroSciences, Indianapolis, Indiana, United States of America Introduction Plants have evolved highly specific mechanisms to resist pathogens. One of the common ways to counter pathogen growth involves the deployment of resistant (R) proteins, which confer protection against specific races of pathogens carrying correspond- ing avirulence (Avr) genes [1]. Following recognition of the pathogen, one or more signal transduction pathways are induced in the host plant and these lead to the prevention of colonization by the pathogen. Induction of defense responses is often accompanied by localized cell death at the site of pathogen entry. This phenomenon, termed the hypersensitive response (HR), is one of the earliest visible manifestations of induced defense reactions and resembles programmed cell death in animals [1–6]. Concurrent with HR development, defense reactions are triggered in both local and distant parts of the plant and accompanied by a local and systemic increase in endogenous salicylic acid (SA) levels and the upregulation of a large set of defense genes, including those encoding pathogenesis-related (PR) proteins [7–9]. The SA signal transduction pathway plays a key role in plant defense signaling (see reviews in [10–12]). Arabidopsis mutants that are impaired in SA responsiveness, such as npr1 (Nonexpressor of PR; [13–15]), or are defective in pathogen-induced SA accumulation, such as eds1 (Enhanced Disease Susceptibility 1; [16]), eds5 (Enhanced Disease Susceptibility 5; [17]), sid2 (isochorishmate synthase; [18]) and pad4 (Phytoalexin Deficient 4; [19]), exhibit enhanced susceptibility to pathogen infection and show impaired PR gene expression. The EDS1, EDS5, PAD4, NPR1 and SID2 proteins participate in both basal disease resistance to virulent pathogens as well as R protein-mediated resistance to avirulent pathogens [20]. Defense signaling mediated via a majority of R proteins, which contain Toll-interleukin1-like (TIR) domains at their N-terminal ends, is dependent on EDS1 [21]. Conversely, the NDR1 (Non-race-specific Disease Resis- tance) protein is required for many R proteins that contain coiled- coil (CC) domains at their N-terminal ends. However, several CC- nucleotide binding site (NBS)-leucine rich repeat (LRR) type of R proteins, including RPP8, RPP13-Nd, HRT, and RPP7, signal resistance via a pathway(s) that is independent of NDR1 [21,22– 24]. Strikingly, the CC-NBS-LRR gene HRT, which confers resistance to Turnip Crinkle Virus (TCV), is dependent on EDS1 [23]. Besides HRT, the only other CC domain-containing R protein that utilizes an EDS1-dependent pathway is RPW8, which confers broad-spectrum resistance to powdery mildew [25]. PLoS Genetics | www.plosgenetics.org 1 July 2009 | Volume 5 | Issue 7 | e1000545