Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis C W OPEN Thomas Hackenberg, a Trine Juul, a Aija Auzina, a Sonia Gwiżdż, a Anna Małolepszy, a Katrien Van Der Kelen, b,c Svend Dam, a Simon Bressendorff, d Andrea Lorentzen, e Peter Roepstorff, e Kåre Lehmann Nielsen, f Jan-Elo Jørgensen, a Daniel Hofius, d,g Frank Van Breusegem, b,c Morten Petersen, d and Stig Uggerhøj Andersen a,1 a Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark b VIB Department of Plant Systems Biology, Ghent University, B-9052 Ghent, Belgium c Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium d Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark e Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark f Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark g Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala BioCenter, 75007 Uppsala, Sweden Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea- induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death. INTRODUCTION Programmed cell death (PCD) is a necessary part of the life of multicellular organisms. It is associated with normal development and with immune responses aimed at pathogen clearance and destruction of otherwise harmful cells. Plant and animal innate immune systems detect conserved microbe-associated molec- ular patterns, such as flagellin-derived peptides, using pattern recognition receptors (Ausubel, 2005). To circumvent this de- tection system, adapted plant pathogens inject effectors directly into plant cells. In resistant plants, plant immune receptors known as R proteins detect the presence of effectors. This recognition leads to effector-triggered immunity, which often culminates in a hypersensitive response (HR) leading to accumulation of reactive oxygen species (ROS) and PCD (Jones and Dangl, 2006). Since the discovery more than two decades ago of HR- associated ROS accumulation (Doke, 1983), also known as the oxidative burst, its causal relationship with PCD has been an active area of research. Over the years, evidence has accumu- lated that ROS trigger PCD and are not merely cell death by- products. For instance, pharmacological treatments with ROS antagonists block induction of HR-associated PCD (Lamb and Dixon, 1997), transgenic plants with lower levels of antioxidative enzymes display aggravated PCD responses (Van Breusegem and Dat, 2006), and superoxide induces runaway cell death in Arabidopsis thaliana lesions simulating disease resistance1 mutants (Jabs et al., 1996). These observations highlight the importance of understanding how the plant interprets ROS sig- naling to induce PCD. Modulation of ROS levels during a HR could result in oxidative modification of countless proteins, emphasizing the complex nature of this topic and the potential difficulties in characterizing the nature of the ROS signal that induces PCD. Since ROS signaling events resulting in PCD occur down- stream of pathogen effector recognition by plant R proteins, genetic identification of signaling components required for R protein–dependent PCD is one possible avenue of investigation. It was revealed that autophagy components are involved in PCD execution downstream of the R protein RPM1 using an avrRpm1- expressing strain of the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst) in a reverse genetics approach (Hofius et al., 2009). Furthermore, accumulating evidence suggests the existence of autophagy-dependent cell death pathways across the eukaryotic kingdom (Hofius et al., 2011). Autophagy facilitates 1 Address correspondence to sua@mb.au.dk. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Stig Uggerhøj Andersen (sua@mb.au.dk). C Some figures in this article are displayed in color online but in black and white in the print edition. W Online version contains Web-only data. OPEN Articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.113.117192 This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online reduces the time to publication by several weeks. The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved. 1 of 11