ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase- Mediated Defense Responses Marta Berrocal-Lobo1¤b, Sophia Stone3, Xin Yang2, Jay Antico2, Judy Callis3, Katrina M. Ramonell2*, Shauna Somerville1¤a 1 Department of Plant Biology, Carnegie Institution, Stanford, California, United States of America, 2 Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama, United States of America, 3 Section of Molecular and Cellular Biology, University of California Davis, Davis, California, United States of America Abstract Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. Citation: Berrocal-Lobo M, Stone S, Yang X, Antico J, Callis J, et al. (2010) ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses. PLoS ONE 5(12): e14426. doi:10.1371/journal.pone.0014426 Editor: Daniel J. Kliebenstein, University of California, United States of America Received June 24, 2010; Accepted November 23, 2010; Published December 23, 2010 Copyright: 2010 Berrocal-Lobo 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: Marta Berrocal-Lobo was supported by the Carnegie Institution of Washington and a postdoctoral fellowship from the Secretaria de Estado de Educacion y Universidades and Fondo Social Europeo, MECD/Fullbright (Spain). Katrina M. Ramonell was supported by the University of Alabama and by grants from the National Institutes of Health (grant no. 1-R15-GM073630-01) and the U.S. Department of Agriculture (grant no. 2007-01652 to K.M.R.). This work was supported in part by the National Science Foundation and the Carnegie Institution. 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: kramonel@bama.ua.edu ¤a Current address: Energy Biosciences Institute, University of California, Berkeley, California, United States of America ¤b Current address: Centro de Biotecnolog´ıa y Gen o´mica de Plantas (CBGP), (UPM-INIA), Campus de Montegancedo, Universidad Polite´cnica de Madrid, Pozuelo de Alarco´ n, Madrid, Spain Introduction Plants defend against pathogens using an innate system of defense that has both constitutive and inducible components. Constitutive defense responses are independent of the physical presence of a pathogen and are typically chemical and physical barriers that protect the plant from pathogen invasion [1]. Inducible plant defenses depend on pathogen recognition and fall into two major classes; specific gene-for-gene interactions and more general Pathogen or Microbe-Associated Molecular Pattern (PAMP or MAMP)-associated responses. In gene-for-gene interactions, a plant resistance (R) gene recognizes and interacts with a specific race(s) of pathogen that expresses a corresponding avirulence (Avr) gene [2]. In the absence of gene-for-gene interactions, general elicitors or PAMPs, [3] are recognized by the host and activate broad-spectrum defense responses. Common PAMPs such as the oligosaccharide chitin (b-1, 4 linked N-acetylglucosamine) and the bacterial proteins flagellin and elongation factor Tu (EF-Tu) are known to activate strong defense responses [4,5,6,7,8]. Several receptors associated with these PAMPs have been characterized including: the FLS2 receptor that recognizes flagellin [9]; the EFR receptor, which perceives the first 18 amino acids of bacterial elongation factor Tu (EF-Tu) [7], chitin oligosaccharide elicitor binding protein (CeBiP) [10], a transmembrane protein with two extracellular Lysine motifs (LysM) that is involved in chitin recognition and the LysM-RLK CERK1 that is required for chitin-initiated responses and downstream signalling [10,11]. While our knowledge of how plants perceive pathogens and activate associated defense signaling pathways is increasing rapidly, less is known about how these processes are regulated during the infection. A predominant theme that is emerging is that of ubiquitination as a means of targeting components of defense signaling pathways for degradation to curtail the plant immune response [12,13]. Studies characterizing the roles of several ubiquitin E3 ligases in defense have begun to provide clues about the regulation of pathogen-induced signaling [12,14]. For instance PLoS ONE | www.plosone.org 1 December 2010 | Volume 5 | Issue 12 | e14426