ATL99s Role in Plant Defense Figure 4. The basal expression of ATL9 and its induction by chitin depends on NADPH oxidases. Plants were treated with chitin for 30 minutes and harvested for analysis. Expression levels of ATL9 were measured by qRT-PCR. The inset shows the relative expression of ATL9 transcript compared to actin (RE/Actin) in control plants. Data represent the ratio of expression between mock and treated plants of three independent biological samples. doi:10.1371/journal.pone.0014426.g004 taken at four early time points: 1 hr., 1.5 hr., 2 hr. and 4 hr. post- infection and at two late infection time points; 24 hr. and 48 hours post-infection (hpi). Quantitative analysis of the gene expression revealed that ATL9 is induced very early in the infection process (1 hr.; Figure 5) and then expression quickly drops off to negligible levels at 4 hpi in a pattern similar to that of PR1 expression (Figure 5). ATL9 expression again rose in the later time points (24 and 48 hpi). By 24 hpi, ATL9 expression was again induced to levels similar to those observed in the 1 hour time point (Figure 5). AtrbohD, AtrbohF, and PDF1.2 were not significantly induced early in the infection process but were present at higher levels in the later stages of the infection beginning at 24 hpi (Figure 5). These data may indicate that an early step in pathogen recognition or the infection process is critical for activation of AtrbohD, AtrbohF, and PDF1.2. ATL9 has ubiquitin ligase activity The RING zinc-finger domain is known to be essential for the function of ubiquitin E3 ligases [39] and two other members of the ATL family, ATL2 and ATL6, have been identified as E3 ligases in Arabidopsis [18,21]. In order to determine whether ATL9 might function as an E3 ligase, we analyzed ATL9 activity using an in vitro ubiquitination assay (Figure 6). A GST:ATL9 fusion protein was affinity purified and its E3 ligase activity was assayed in vitro. Multiple forms of ubiquitinated proteins were detected in the complete assay by the anti-ubiquitin antibodies (Figure 6, lane 1). The omission of AtUBC8 (Figure 6, lane 2), GST-ATL9 (Figure 6, lane 3) or ubiquitin (Figure 6, lane 4) from the assay resulted in a loss of protein polyubiquitination (Figure 6, lane 1) confirming that ATL9 has ubiquitin ligase activity. The ATL9 protein is localized to the Endoplasmic Reticulum (ER) Defense-related proteins can be found in a diverse array of cellular compartments. To determine the localization of ATL9, a construct was made containing a C-terminal fusion of the green fluorescent protein (GFP) to the ATL9 gene under the control of its native promoter (ATL9p:ATL9:GFP). Stable transgenic lines were created by transforming Col-0 plants with Agrobacterium tumefaciens containing the ATL9p:ATL9:GFP construct. Upon visualization, fluorescent signal was detected in the endoplasmic reticulum (ER) of leaf epidermal cells (Figure 7A–C). Localization of ATL9 was observed in the ER of all tissues tested and was particularly high in roots (Figure 7A–C; data not shown). For more precise localization a second construct containing a fusion of GFP to the C-terminus of ATL9 driven by the CaMV 35S promoter (35S:ATL9:GFP) was Figure 5. Relative expression levels of selected genes at various time points after inoculation with Golovinomyces cichoracearum. Col-0 plants were inoculated with G. cichoracearum at high concentration. qRT-PCR data represent the ratio between mock and treated plants of three independent biological samples. doi:10.1371/journal.pone.0014426.g005 PLoS ONE | www.plosone.org 6 December 2010 | Volume 5 | Issue 12 | e14426