ATL99s Role in Plant Defense Figure 9. Proposed role of ATL9 in chitin-NADPH mediated innate immunity. 1. Recognition of chitin released from fungal cell wall by LysM-RLK1 receptor. 2. Increase of intra-cellular calcium leading to the activation of the NADPH oxidases AtrbohD and F at the membrane initiating the production of ROS and activation of a MAPK cascade(s). 3. Induction of ATL9 and insertion into the ER membrane. We propose that ATL9 is involved tagging an inhibitor of plant defense for degradation by the proteosome 4. Defense response to fungal pathogen is activated after inhibitor degradation. 5. The ATL9 protein is rapidly degraded in the cell by either the proteosome pathway or a calcium-mediated pathway (similar to the mammalian protein calpain). doi:10.1371/journal.pone.0014426.g009 defense responses would be activated. After ubiquitylation of its target protein, ATL9 is rapidly degraded via its PEST domain (step 5, Figure 9). A similar mechanism to the one we propose has been described during recognition of human PAMPs [64]. We predict that the target of ATL9 is a negative regulator of defense responses since atl9 mutants are more susceptible to fungal pathogens. In these mutant plants, we suspect that the negative regulator cannot be ubiquitinated upon receipt of appropriate signals and thus defense responses cannot be activated (Figure 9). Our data clearly indicate that ATL9 is an E3 ligase that is essential for defense against powdery mildew and requires the expression of NADPH oxidases for its activity. This study is the first to directly implicate an ATL family member in chitin responses mediated by NADPH oxidase activity. Future studies identifying ATL99s targets and its possible interacting partners will be integral in order to better assess its role in innate immunity and defense against fungal pathogens. Materials and Methods Biological Materials Arabidopsis thaliana ecotype Columbia (Col-0) was used as the control in all experiments. All mutants and transgenic plants were in the Col-0 background. Mutant ein-2-5 [65] and the transgenic line 35S:ERF1.14 [66] was provided by Antonio Molina (E.T.S.I. Agronomos, Madrid, Spain. The sid2-1 mutant [67], Arabidopsis ecotype Kashmir 1 (Kas-1) [63] and Arabidopsis lines expressing GFP in the plasma membrane (line 29-1 encoding LTI6b) [68] were obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University, USA). T-DNA insertional lines used in this work were: SALK_066755 (atl9-1), SALK_036065 (atl9-2), and SALK_036066 (atl9-3). All T-DNA insertional lines were generated by SIGnAL at the SALK Institute [69,70]. The following gene-specific primers were used to screen for the T-DNA insertion in ATL9: (1) SALK_066755: 59- TTGGCATGTA- PLoS ONE | www.plosone.org 11 December 2010 | Volume 5 | Issue 12 | e14426