the rice resistance (R) protein Xa21 has been shown to interact with an E3 ubiquitin ligase XB3 [15]. Interaction between XB3 and Xa21 is required for the accumulation of the XA21 protein and is necessary for Xa21-mediated resistance to Xanthomonas oryzae pv. oryza in rice [15,16]. A RING-finger type protein from pepper CaRFP1 was shown to physically interact with PR-1 (pathogenesis related-1) protein in leaves of plants after infection with both bacterial and fungal pathogens [17]. Over-expression of CaRFP1 in transgenic Arabidopsis conferred disease susceptibility to Pseudomonas syringae pv. tomato and reduced PR-2 and PR-5 expression suggesting that CaRFP1 is an E3 ligase that targets PR proteins [17]. E3 ligases also appear to play a prominent role in elicitor- mediated defense responses. In particular, members of the ATL (Arabidopsis to´xicos en levadura) [18,19] gene family have been shown to be activated by elicitors and to play important roles in defense pathways. The Arabidopsis ATL gene family contains 80 members and is a conserved group of RING zinc-finger proteins that encode putative E3 ubiquitin ligases [20]. ATL2 and ATL6 in Arabidopsis and EL5 in rice, all encoding RING-finger type E3 ligases, have been shown to be rapidly induced in response to the elicitor chitin [18,21,22,23]. Recent work by Hondo et al. [24] demonstrated that the tomato ortholog of Arabidopsis ATL2, LeATL6, responded to cell wall protein fraction elicitor from the biocontrol agent Pythium oligandrum and appeared to regulate the jasmonic acid- dependent defense gene expression. In a screen for chitin- responsive genes in Arabidopsis, we identified an ATL family member, ATL9 (At2g35000; ATL2G), that responded strongly to chitin treatment [6]. Loss-of-function mutations in this gene resulted in increased susceptibility to the powdery mildew pathogen, Golovinomyces cichoracearum ( = Erysiphe cichoracearum) [6]. Our results here confirm that ATL9 is an E3 ubiquitin ligase and show that it is localized to the endoplasmic reticulum. ATL9 expression is induced by infection with G. cichoracearum and ATL9 function is required for basal defense against this biotrophic pathogen. Interestingly, ATL9 expression appears to be dependent on NADPH oxidases and mutations in ATL9 lead to an impairment in the ability of plants to produce reactive oxygen species (ROS) after infection. Expression profiling of atl9 revealed a complex interplay between chitin-mediated signaling and other defense pathways. Results ATL9 (Arabidopsis to´ xicos en levadura 9) encodes an E3 ubiquitin ligase with homology to a family of genes induced by wounding and abiotic stress Previous studies by our group have shown that mutants in the gene At2g35000 were more susceptible to fungal infection than wild-type plants [6]. At2g35000 belongs to the ATL family [18,19] of RING (really interesting new gene) zinc-finger proteins and was designated as ATL9 in a previous review [12]. The ATL9 protein consists of 378 amino acids and contains an N-terminal signal peptide; two predicted transmembrane domains, a C3HC4 RING zinc-finger domain, a PEST domain and a C-terminal coiled coil region (Figure 1A). Three members of the Arabidopsis ATL gene family, ATL2, ATL6 and ATL17, are presumed to play a role in defense although their precise functions are unknown at present [21,25]. Using database searches we identified a total of eight proteins with a high percentage of homology to ATL9, including several ATLs in other plant species such as Oryza sativa (EL5) [26], Nicotiana tabacum (Avr9) [27] or Poplar (PtaRHE1) [28]. An alignment of the eight protein sequences along with their putative function in defense is shown in Figure 1B. Of the eight proteins, ATL99s Role in Plant Defense seven are known to be specifically induced by elicitors and four of the proteins (ATL9, ATL2/ACRE132, ATL6 and ATL17) are induced specifically by flagellin or chitin (Figure 1B). All eight proteins contain a RING zinc-finger motif with six highly conserved cysteine residues that match the consensus sequence for the C3HC4 type (RING zinc-finger) domain group (http:// www.sanger.ac.uk/Software/Pfam/). Of the genes found only three, ATL9 in Arabidopsis, NtACRE132 in tobacco and OsBIRF1 in rice have been tested for their putative role in response to pathogens (Figure 1B). The induction of ATL9 is independent of the classical defense pathways Studies have shown that chitin-induced defense responses act through an independent signaling pathway and are not dependent on SA-, JA- or ET-mediated responses [29]. In some cases, however, expression levels of chitin-induced genes were found to be slightly compromised in mutants defective in the SA- and JA- dependent signaling pathways suggesting some level of cross-talk [29]. In order to determine whether the induction of ATL9 was mediated solely by chitin or might also be regulated by the SA-, JA- or ET-dependent signaling pathways, Col-0 plants and mutants impaired in each signaling pathway (SA: sid2-1, npr1-1; JA: jar1; ET: ein2-5) were treated with chitin for 30 minutes and ATL9 expression was monitored. All plants tested showed induction of ATL9 when treated with chitin compared to untreated controls, although in all cases levels of induction in the mutant lines were lower than in wild type. Induction of ATL9 by chitin treatment was higher in Col-0 plants than in any other line tested (Figure 2A). Among the mutants, the highest levels of ATL9 induction were detected in the ein2-5 and npr1-1 mutants, while ATL9 levels in sid2-1 and jar-1 were the lowest (Figure 2A). The basal levels of ATL9 in each of the mutants are slightly lower or higher than in Col-0 (inset Figure 2A). From these data it appears that the induction of ATL9 expression by chitin is not dependent on the SA and JA signaling pathways. To resolve the contributions of each signaling pathway to overall levels of ATL9 expression, we treated wild type plants with chitin, SA, 1-aminocyclopropane-1-carboxylic acid (ACC) and JA [30,31]. Expression of ATL9 and marker genes associated with each pathway (SA: PR1; JA- and Ethylene: PDF1.2) were quantified 30 minutes after treatment. Only treatment with crab-shell chitin (CSC) induced expression of ATL9 (Figure 2B). Exposure of plants to ACC, JA and SA resulted in no appreciable expression of the gene (Figure 2B) providing evidence that ATL9 induction is uniquely associated with chitin-mediated defense responses. As expected, marker genes associated with each of the classical defense pathways were induced by their corresponding signaling molecule (Figure 2B). These results suggest that the SA-, ethylene- and JA-mediated defense pathways do not directly regulate ATL9 expression but might feedback on the chitin- induced expression of ATL9. ATL9 is involved in plant defense against G. cichoracearum Mutations in ATL9 rendered plants more susceptible to infection by the biotrophic fungus G. cichoracearum compared to the wild type plants. To more precisely quantify the susceptibility of atl9 mutants to G. cichoracearum, inoculations were performed using three independent atl9 T-DNA insertional lines, 35S:ATL9 plants, Col-0, the Kas-1 Arabidopsis accession and sid2-1 plants. Counts of mature conidiophores 6 dpi showed that all three atl9 mutants (atl9-1, atl9-2, atl9-3) supported more than two times the PLoS ONE | www.plosone.org 2 December 2010 | Volume 5 | Issue 12 | e14426