ATL99s Role in Plant Defense Figure 7. The ATL9 protein localizes to the ER. A–C) Confocal images of leaf epidermal cells in transgenic Arabidopsis plants expressing ATL9p:ATL9:GFP showing protein localization to the endoplasmic reticulum. B) Confocal image of a leaf trichome showing ATL9p:ATL9:GFP localizing to the ER. D–F) Co-localization of the 35S:ATL9:GFP fusion and ER-rk marker. Onion epidermal cells were co-bombarded with 35S:ATL9:GFP and ER-rk and visualized using fluorescence microscopy. D) 35S:ATL9:GFP. E) ER-rk (mCherry). F) GFP/mCherry overlay. Bars: 3 mm (A,B), 1 mm (C), 10 mm (D-F). doi:10.1371/journal.pone.0014426.g007 the responses of the known elicitor receptors, CERK1 (LysM RLK1), FLS2, BAK1 and EFR in the atl9 mutant. The induction of the gene encoding the chitin receptor LysM RLK1 was essentially unchanged in the atl9 mutant and wild-type plants. Surprisingly, expression levels of the flagellin receptor gene, FLS2 were strongly induced by chitin in Col-0 plants but not in the mutant (Table S1), suggesting that induction of FLS2 may be dependent on ATL9 expression. Expression of BAK1 which acts as a co-receptor of flagellin with FLS2 [9,42] and the bacterial EF-Tu receptor EFR [7] were both induced by chitin treatment but at similar levels in both the atl9 mutant and in wild-type plants. In addition, ACRE1b, a gene highly induced by treatment with flg22 [5] was also strongly induced by chitin treatment in wild-type plants. Taken together these results indicate that activation of elicitor-mediated signaling by chitin and flagellin are closely intertwined in plants. To better understand the interactions between chitin-mediated defense signaling and signaling mediated through SA, JA and ET the expression levels of key genes in each of the signal transduction pathways were monitored (Table S1). The induction of MAPK3 and MAPK5, both known to be important in signaling pathways initiated by flagellin [43] were very similar in atl9 and Col-0 plants indicating that these MAP kinases are upstream of ATL9 activity or are working in an independent signal transduction network. A similar induction by chitin in both the atl9 mutant and wild-type was also observed for MAPKK4. Other genes involved in SA-, JA- and ET-mediated signaling (RAR1, SGT1, HSP90, COI1, ETR1, EIN3, PAD2, JAR1 or SAG101) showed no differences in expression levels in either chitin-treated control or atl9 plants or in mock- treated plants pointing to the fact that these genes are not influenced by ATL9 expression. However, the expression of the lipoxygenase-3 (LOX3) gene, which is involved in octadecanoid biosynthesis leading to the production of JA in plants, was highly induced in both atl9 and Col-0 plants by chitin. Discussion ATL9 is an E3 ubiquitin ligase that is integral to defense against fungal pathogens Recent work has highlighted the ubiquitin-proteasome system (UPS) and its associated E3 ubiquitin ligases as regulators of the plant defense response and it is clear that these proteins play an important part in disease resistance [12,44]. In the current study we have shown that ATL9 is a RING-type E3 ubiquitin ligase strongly induced by chitin. The ATL gene family encodes a group of proteins that share three specific characteristics: 1) rapid induction (,1 hour) after elicitor treatment, 2) a highly conserved RING-H2 zinc-finger domain with at least six cysteines and three histidines conserved and 3) at least one amino-terminal trans- membrane domain. Most members of the ATL gene family are predicted to function as single subunit E3 ubiquitin ligases with seventeen members of the ATL family known to be expressed in Arabidopsis [25]. Although determination of a common function for them is still in progress, mutations in members of the ATL family have been shown to have an altered defense response to pathogens [25]. The ATL2 gene was shown to be specifically induced by chitin but not by other elicitors of classic defense pathways [18]. Constitutive over-expression mutants of ATL2 induced high levels of pathogen-related genes such as NPR1-1 and the phenylpropanoid biosynthetic enzymes phenylalanine ammo- nia lyase and chalcone synthase [21,45]. The EL5 gene in rice is also a member of the ATL family and is rapidly and transiently PLoS ONE | www.plosone.org 8 December 2010 | Volume 5 | Issue 12 | e14426