(Invitrogen) and the DNA sequence analyzed. ORF’s determined to be correct were introduced into the pDEST15 (Invitrogen, Carlsbad, CA) protein expression vector to produce in-frame fusions with the GST tag. AtUBC8 was cloned in a similar manner and introduced into the pDEST17 (Invitrogen, Carlsbad, CA) vector to produce an in-frame fusion with the 6X HIS tag. The expression clone containing yeast E1 was provided by M. Wogulis (University of California-Davis). Ubiquitination assays were carried out as described previously [80]. Reactions (30 mL) containing 50 mM Tris-HCl, pH 7.5; 10 mMMgCl2; 0.05 mM ZnCl2; 1 mM ATP; 0.2 mM dithiothreitol; 10 mM phosphocre- atine;0.1 unit of creatine kinase (Sigma); 50 ng of yeast E1 (Boston Biochem, Cambridge, MA); 250 ng of purified E2 AtUBC8; 250 ng of eluted/bead-bound GST-RING, GST-mutated RING protein, or GST-CIP8 (positive control); and 2 mg ubiquitin (Sigma) were incubated at 30uC for 2 h. Reactions were stopped by adding 6 mL of 53 SDS-PAGE sample buffer (125 mM Tris- HCl, pH 6.8, 20% [v/v] glycerin, 4% [w/v] SDS, and 10% [v/v] b-mercaptoethanol) and analyzed by SDS-PAGE electrophoresis followed by western blotting using ubiquitin antibodies. For zinc- chelating experiments, bead-bound GST-RING protein was either incubated in 50 mM Tris-HCl, pH 7.4, containing 5 mM TPEN or 0.5% (v/v) ethanol (vehicle; mock treatment) for 16 h at 4uC with at least three solution changes. Beads were then washed three times in 50 mM Tris-HCl, pH 7.4. An aliquot of TPEN-treated bead-bound GST-RING protein was incubated with 1 mM ZnCl2 for 4 h at 4uC with at least three solution changes, followed by three washes in 50 mM Tris-HCl, pH 7.4. Mock-, TPEN-, and TPEN plus ZnCl2-treated bead-bound GST-RING protein were then used in ubiquitination assays. Protein expression and purification GST:ATL9 fusions were expressed in E. coli strain BL21 (DE3) pLysS in 50 ml cultures. Transformed cells were grown at 37uC for 2 to 3 hours or to an OD600 of 0.4–0.6 before induction with 0.5 mM IPTG for 3 to 4 hours at 37uC. Cells were harvested by centrifugation and lysed in 2 ml of lysis buffer containing 25 mM Tris-HCl pH 7.5, 500 mM NaCl, and 0.01% Triton X-100. For purification, 100 ml of glutathione sepharose beads (Sigma- Aldrich, St. Louis, MO) was added to cleared lysates and incubated for two hours at 4uC. Beads were then washed four times with 1 ml of wash buffer containing 25 mM Tris-HCl pH 7.5, 300 mM NaCl, and 0.01% (v/v) Triton X-100. GST fusion proteins were eluted with 100 ml of elution buffer containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.01% (v/v) Triton X-100 supplemented with 15 mM reduced glutathione or the GST fusion proteins were left bound to the beads. 40 ml of glycerol was added to the eluted protein. Bead bound GST proteins were stored in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% (v/v) Triton X-100 and 40% (v/v) glycerol. Proteins were stored at 280uC. 6X HIS tagged AtUBC8 was expressed and purified in a similar manner. The 6X HIS fusion was expressed in E. coli strain BL21 AI and induced with 0.2% (w/v) arabinose. Lysis and wash buffer were supplemented with 5 mM imidazole and elution buffer supplemented with 300 mM imidazole. Yeast E1 was expressed as an Intein fusion and purified using the IMPACT (Intein mediated purification with an affinity chitin-binding tag) system as per the manufacturer’s instructions (New England Biolabs, Beverly, Massachusetts, USA). SDS-page electrophoresis followed by Coomassie blue staining and Bradford assays (Biorad, Philadel- phia, USA) were used to quantify purified proteins. Western blot analysis using GST antibodies or HIS antibodies (Amersham, ATL99s Role in Plant Defense Buckinghamshire, UK) was also used to confirm the presence and integrity of the fusion protein. Bioinformatics Analysis Additional data analysis and information about gene expression of ATL9 was obtained from the following web pages: http:// affymetrix.arabidopsis.info/narrays/, https://www.genevestigator. ethz.ch/. Web analysis was used to obtain microarray data regarding ATL9 gene expression in different tissues, conditions, and treatments and to examine co-expression patterns of ATL9 and other genes. The Aramemnon program (http://aramemnon. botanik.uni-koeln.de/) was used to analyze the structure of ATL9 and to predict its cellular localization. Tools used for general biomolecular analysis, BLAST, sequence alignment and determi- nation of protein-specific domains were: http://www.us.expasy. com/tools/, http://www.ncbi.nlm.nih.gov/, and http://www.ebi. ac.uk/Tools/. Microarray Expression Analysis Seedlings of ATL9-1 and Col-0 were treated with hydrolyzed crab shell chitin or with water as a mock control in liquid culture for 30 min as described previously [29]. Four independent biological replicates were performed for each experiment. RNA extractions, labeling and hybridization to ATH1 Affymetrix GeneChipsH (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix, Inc., Santa Clara, CA) were performed as described previously [81] with the exception that 15 mg of RNA were biotinylated to obtain cRNA. Data were extracted from the GeneChipH images using MAS5.0 (Affymetrix) software and imported into GeneSpring 6.0 software (Silicon Genetics, Redwood City, CA) for normalization and further analysis. Fold change threshold is shown in Table1. This data set has been made available via the Gene Expression Omnibus (GSE2169) (http:// www.ncbi.nlm.nih.gov/geo/). Supporting Information Figure S1 Transient Expression of ATL9 in Tobacco Epidermal Cells. A) Expression of 35S:GFP:ATL9 construct in tobacco epidermal cells shows localization to the ER with no nuclear localization. B) Expression of 35S:GFP negative control in tobacco epidermal cells shows GFP localizing to the nucleus when it is not fused to ATL9. C) Western blot confirming sizes of ATL9 and GFP fusion proteins from A and B. Blot was probed with a monoclonal anti-GFP antibody. Bars: 5 mm. Found at: doi:10.1371/journal.pone.0014426.s001 (1.42 MB TIF) Table S1 Microarray data for selected chitin-responsive genes. Found at: doi:10.1371/journal.pone.0014426.s002 (0.18 MB DOC) Acknowledgments We thank Jonathan Jones for seeds of the Atrboh mutants, Rachel Huntley for help with qRT-PCR experiments, Plinio Guzman for atl2 seeds, P. Gonza´lez Melendi, John Yoder and S. Koh for confocal microscopy support, Mary Beth Mudgett’s laboratory for technical help and Antonio Molina for scientific discussions. Author Contributions Conceived and designed the experiments: MBL JC KR SS. Performed the experiments: MBL SS XY JA. Analyzed the data: MBL SS KR. Contributed reagents/materials/analysis tools: JC KR SS. Wrote the paper: KR. Laboratory support: SS. PLoS ONE | www.plosone.org 14 December 2010 | Volume 5 | Issue 12 | e14426