sites of infection or penetration. Transgenic plants expressing an ATL9p:ATL9:GFP fusion construct showed that ATL9 was localized to the ER membrane (Figure 7A–C). Localization of the ATL9 protein at the ER membrane not only allows the protein to remain close to the site of infection/penetration but also places the protein where it can be quickly degraded after carrying out its E3 activity. We hypothesize that ATL9 may attach ubiquitin to either plant- or fungal-derived proteins that are active near sites of infection. Several important genes in plant defense have been shown to localize to the ER. The Cf-9 resistance gene, which confers resistance to Cladosporium fulvum races expressing the Avr-9 avirulence gene, contains a C-terminal di-lysine motif (KKXX) targeting Cf-9 to the ER [53]. ACRE- 132, also a member of the ATL family, has been shown to be induced during Avr-9/Cf-9 interaction and may be an active player in that response [46]. The barley HSP90 protein is localized to the ER plasma membrane like ATL9 [54] and has been shown to interact with RAR1 and SGT1, two genes involved in R-gene mediated resistance to fungi, in yeast two- hybrid experiments. HSP90, RAR1 and SGT1 are hypothesized to form a chaperone complex that mediates the folding of R- proteins and their incorporation into functional complexes. Data supporting the role of an HSP90-RAR1-SGT1 complex as a chaperone active in R-gene mediated defenses have been shown in both barley and Arabidopsis [44,55,56]. Though our understanding of the role of ATL family members in defense is incomplete, it is intriguing to speculate that ATL9 or other family members may form complexes similar to HSP90-RAR1-SGT1 that are necessary for defense against G. cichoracearum. E3 ligases in the ER are often associated with endoplasmic reticulum associated degradation (ERAD) a process involved in targeting misfolded proteins for ubiquitination and subsequent degradation by the proteasome [57,58]. In the ER, the ATL9 protein would have its RING domain and PEST domain exposed to the ER lumen. We hypothesize that ATL9 acts to ubiquitinate a negative regulator of plant defense responses in the ER lumen or some unknown luminal protein that is not properly folded during stress-induced ERAD. The ATL9 protein shares homology with the well-characterized ERAD E39s Hrd1 and gp78 [59]. Both Hrd1 and gp78 are known to be induced during cellular stress and participate in the unfolded protein response. The yeast Hrd1 protein is at the center of a large protein complex involved in the ubiquitylation of ER luminal and membrane proteins. Since ATL9 is an ER membrane resident protein whose RING domain projects into the ER lumen, it is interesting to speculate that ATL9 may also function in a group of proteins similar to the Hrd1 complex that is involved in ERAD induced by biotic stresses. Further experiments directed towards identifying both ATL9 interacting partners and its’ protein target will be helpful in understanding its precise role. Expression and chitin ATL9 gene response depends on NADPH oxidases The oxidative burst and the production of ROS is an essential component of plant defense. This pathway is mediated by the activation of the NADPH complex which is necessary for the production of H2O2. Following H2O2 production, several downstream signaling events are initiated including calcium mobilization to the cytoplasm, protein phosphorylation, MAP kinase activation and defense gene expression [53,60]. In Arabidopsis, the NADPH family of oxidases consists of ten members (AtrbohA –AtrbohJ) which are homologs of gp9phox, a subunit of the mammalian NADPH oxidase complex [33]. Infection studies of Arabidopsis mutants in AtrbohD and AtrbohF ATL99s Role in Plant Defense have shown that AtrbohD is responsible for reactive oxygen species produced after infection with avirulent bacteria or oomycete pathogens whereas AtrbohF is integral to the regulation of the hypersensitive response [61]. In the current study, we demonstrate that the induction of ATL9 by chitin is dependent on both AtrbohD and AtrbohF and that MAPK3 activation is also dependent on Atrboh expression. These results suggest that the early steps in chitin recognition are reliant on Atrboh functionality (Figure 3). Our data also demonstrate that both AtrbohD and AtrbohF mutants are more susceptible to G. cichoracearum than wild type plants (Figure 3A–B), indicating that Atrboh activity is necessary for defense against powdery mildew. Recent work [62] has demonstrated that Atrboh genes can modulate and antagonize SA-dependent cell death signals. ROS produced by AtrbohD limited the spread of the cell death induced by the bacterial pathogen P. syringae DC3000 and the necrotroph B. cinerea. Our current study clearly shows that both ArbohD, AtrbohF mutants were more susceptible than wild type to G. cichoracearum and those quantitative results are directly correlated with the low levels of hydrogen peroxide accumulation at the point(s) of penetration detected in these mutants (Figure 3B–C). Hydrogen peroxide production in AtrbohD, AtrbohF, and AtrbohD/ F as well as in the atl9 mutants was essentially half of that in wild- type plants. Interestingly the Kas-1 accession [63] that is resistant to G. cichoracearum had increased levels of peroxide (Figure 3C) at sites of attempted penetration (Figure 3B) although no cell death was observed in these cells and plants were phenotypically resistant. These data imply that careful regulation of hydrogen peroxide production in Kas-1 plants is sufficient to inhibit fungal penetration without cell death [62] suggesting that salicylic acid may activate defense signaling in cells that are spatially removed from infection sites without activating cell death. Torres et al. [62] further hypothesize that Atrbohs are necessary for the suppression of unwanted cell death in cells where increased levels of SA are required to activate defense responses. G. cichoracearum resistance in Kas-1 plants may be dependent on Atrboh’s ability to induce higher levels of H2O2 production at sites where the fungus is attempting to penetrate epidermal cells while simultaneously repressing cell death in those same cells. This hypothesis is supported by the fact that Atrboh mutants had virtually no peroxidase activity at penetration sites and were much more susceptible to G. cichoracearum (Figure 3A–B). Although a connection between the Atrboh genes, chitin induction of ATL9 and resistance to G. cichoracearum remains to be definitively established, our results clearly show that NADPH oxidase activity is required for ATL9 induction after chitin recognition. This is in line with our results showing that the expression levels of ATL9 are impaired in both mutants compared with the wild type plants (inset Figure 4B),as in root tissues of AtrbohC. The activity of AtrbohD, AtrbohF and ATL9 are needed for effective defense against powdery mildew. This study provides evidence for the first time that AtrbohD and AtrbohF activity, in plant leaves, are required for G. cichoracearum resistance and for some chitin-elicited defense responses. Possible mode of action of ATL9 in chitin recognition and chitin-mediated innate immunity We hypothesize that upon chitin recognition/fungal infection intracellular calcium levels increase leading to the activation of Arabidopsis NADPH oxidases (Figure 9). The subsequent production of ROS and activation of MAPK signaling cascades induce ATL9 expression and its transport to the ER membrane. Here ATL9, either alone or working in a complex would ubiquitinate its target protein(s) for degradation and appropriate PLoS ONE | www.plosone.org 10 December 2010 | Volume 5 | Issue 12 | e14426