Figure 6. ATL9 is capable of mediating protein ubiquitination in a E2-dependent manner. The complete in vitro ubiquitination assay (lane 1) contained recombinant yeast E1 enzyme, recombinant 6x histidine-tagged Arabidopsis E2 enzyme UBC8, GST-tagged ATL9 and ubiquitin. Omission of AtUBC8 (lane 2), GST-ATL9 (lane 3) or ubiquitin (lane 4) from the assay resulted in a loss of protein polyubiquitination as indicated by a lack of the ubiquitinated proteins compared to the complete assay (lane 1). Ubiquitinated proteins were visualized by western blot analysis using anti-ubiquitin antibodies. * Indicate non- specific cross-reactive proteins. ¤ Represent AtUBC8-Ub(n). Molecular weight markers (kDa) are shown to the left of the blot. doi:10.1371/journal.pone.0014426.g006 co-bombarded into onion epidermal cells with a known ER luminal marker ER-rk (signal peptide of AtWAK2:mCherry:H- DEL) [40]. When visualized with fluorescent microscopy, 35S:ATL9:GFP co-localized with the ER-rk marker in the onion epidermal cells confirming its localization to the ER (Figure 7D– F). In parallel we also transiently expressed an N-terminal construct of ATL9, 35S:GFP:ATL9, in tobacco with a 35S:GFP construct as an internal negative control for GFP localization to the nucleus. Again, ATL9 was expressed transiently in the ER of tobacco epidermal cells confirming our results in Arabidopsis and onion (Figure S1A). Fluorescence was detectable within the nucleus in the tobacco cells expressing the 35S:GFP construct and the GFP signal could be seen in the ER surrounding the nuclear membrane (Figure S1B). A western blot with the protein extract from the transiently transformed tobacco tissue was performed and hybridized with anti-GFP antibody to confirm the size of the ATL9 and GFP fusion proteins (Figure S1C). Since some PAMP response related proteins are transported to the vesicle after stress responses [41], we wanted to determine if ATL9 responded in a similar manner. To determine if ATL9 localization changed after elicitor treatment, transgenic plants expressing ATL9p:ATL9:GFP were treated with chitin (100 mg/L) by direct infiltration of the leaves and observed at five minute intervals for one hour. No change in ATL9 localization was ATL99s Role in Plant Defense detected regardless of the time after treatment with chitin (data not shown). Microarray expression analysis of atl9 To identify genes whose expression is dependent on ATL9 function, Col-0 plants and atl9 mutants were treated with chitin for 30 minutes and their expression profiles were examined using Affymetrix ATH1 arrays. Of the 22,677 genes represented, 16,530 were defined as present and of these 4,375 genes showed altered expression in the atl9 mutant compared to wild-type plants (Figure 8A and 8B). Genes were divided into (1) those that exhibited genotype-specific differences in expression between wild type and atl9 and (2) those that responded to treatment with chitin. Figure 8B shows that the majority of genes that exhibited altered expression responded to chitin treatment regardless of plant genotype (3510), while a smaller number (525) fluctuated between wild-type plants and the atl9 mutant irrespective of treatment. An intermediate number of genes (340) were changed both by the loss of ATL9 expression and by chitin treatment (Figure 8B). Data on selected genes within each category are given in Table S1. Several genes with known roles in plant defense were identified that were specifically altered between wild-type plants and atl9 mutants (Table S1). Two genes associated with SA-mediated defense responses, PR-1 and SID2/ICS1 were repressed in plants lacking ATL9 (Table S1). PR-1 levels (At2g14580) were decreased 3-fold in the atl9 mutant compared with Col-0 plants. This finding is surprising since we have shown that PR-1 gene expression is induced by infection with G. cichoracearum but not by chitin-treatment alone (Figure 5, Table S1). Expression of SID2/ICS1 (isochorismate synthase; At1g74710) was slightly down-regulated in the atl9 mutant (0.77-fold) compared to Col-0 though in both cases the gene was induced by chitin. PEN3 expression was reduced by 50% in the atl9 mutants in contrast to PEN1 and PEN2 whose expression was unchanged (Table S1). Expression of PDF2.5, an antimicrobial peptide, was slightly lower in atl9 plants compared to Col-0. However, when treated with chitin PDF2.5 was strongly induced in wild-type plants but its expression remained unchanged in the atl9 mutant (Table S1). Similarly the PCC1 gene (pathogen and circadian controlled 1) was down-regulated in response to the loss of ATL9. As in the case of PDF2.5, PCC1 was strongly up-regulated in Col-0 plants upon chitin treatment and was down-regulated in the atl9 mutant (Table S1). Most of the defense genes surveyed were not significantly affected by the loss of atl9 expression and exhibited similar responses to chitin-treated control plants (Table S1). However, several defense genes had enhanced expression levels after chitin treatment in the atl9 mutant including PEN1, PAD4, two disease resistance-like proteins, ACRE1b, ATL17 and protein phosphatase 2C (Table S1). Interestingly, AtrbohD expression was induced to higher levels in chitin-treated atl9 compared with wild-type controls. Similar increases were not observed in the expression of AtrbohC or AtrbohF. Defense genes associated with elicitor-mediated responses To identify defense-related genes that were specifically linked to the chitin signaling pathway, we compared the expression of genes known to be induced by both abiotic and biotic stresses in chitin- treated Col-0 wild type and atl9 plants (Table S1). We first analyzed the expression levels of the ATL9 family members ATL2- ACRE132, ATL6 and ATL17 (Table S1). In all cases, the ATL family members were induced at significantly higher levels in wild- type plants, compared to atl9 mutants. This strongly suggests that ATL9 is important in initiating the expression of genes involved in chitin-mediated responses. We were also interested in determining PLoS ONE | www.plosone.org 7 December 2010 | Volume 5 | Issue 12 | e14426