seedlings treated with 1-mM DEX for 12 hr were processed as previously described [55]. Briefly, chromatin was isolated from 0.8 g of frozen tissue and sonicated with a Bioruptor sonicator (15 s on and 15 s off cycles, medium-energy settings) for 6 min. Immunoprecipitation was performed by incubating chromatin with 2 mg of anti-myc antibody (Millipore) or mouse IgG (negative control) for 1 hr at 4uC. The protein-chromatin immunocomplex was captured using Protein G-Dynal magnetic beads (Invitrogen). After Proteinase K digestion, the immunoprecipitated DNA was purified using a ChIP DNA Clean and Concentrator kit (Zymo Research Corporation). Immunoprecipitated DNA and input DNA were analyzed by qPCR using primers specific for the promoter regions of PAD3 and WRKY33. The primer pairs (forward and backward) used for ChIP-qPCR were ACS2 (59- AGGCCATAAGCCCATTCAAA-39 and 59-GCCTACAGTG- CACGACTTCA-39) and ACS6 (59-AAAGTCGTTGAGAT TGTGTTGG-39 and 59-TGGCAGCCTTAAAGACCAGT-39), which are in proximity of the W-boxes in the promoters. ChIP results are presented as percentage of input DNA. Accession numbers Sequence data for this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: MPK3 (At3g45640), MPK6 (At2g 43790), EF1a (At5g60390), ACS1 (At3g61510), ACS2 (At1g01480), ACS4 (At2g22810), ACS5 (At5g65800), ACS6 (At4g11280), ACS7 (At4g26200), ACS8 (At4g37770), ACS9 (At3g49700), ACS11 (At4g08040), and WRKY33 (At2g38470). Supporting Information Figure S1 Mutation in ACS7, a Type III ACS isoform, slightly reduced B. cinerea-induced ethylene production. Two-week-old acs7-1 and acs7-2 as well as their respective wild-type controls, Ws- 0 and Ler-0, grown in GC vials were inoculated with B. cinerea spores. Ethylene accumulation in the headspace was determined at the indicated times. Error bars indicate standard deviations (n = 3). (TIF) Figure S2 Comparison of basal-level and B. cinerea-induced ethylene production in acs2-1/acs6-2 and acs2-1/acs6-1 double mutants. (A) B. cinerea-induced ethylene production in wild type, acs2-1/acs6-2, and acs2-1/acs6-1 plants. Twelve-day-old seedlings grown in GC vials were inoculated with B. cinerea spores. Ethylene accumulation in GC vials was monitored at indicated times. Error bars indicate standard deviations (n = 3). (B) Basal level ethylene production in wild type, acs2-1/acs6-2, and acs2-1/acs6-1 seedlings. Twelve-day-old seedlings grown in GC vials were mock inoculat- References 1. Wang KL-C, Li H, Ecker JR (2002) Ethylene biosynthesis and signaling networks. Plant Cell 14: S131–S151. 2. Schaller GE, Kieber JJ (2002) Ethylene. In: Somerville CR, Meyerowitz EM, editors. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists. pp. 1–19, doi/10.1199/tab.0071, http://www.aspb.org.publications/ arabidopsis/. 3. Klee HJ (2004) Ethylene Signal Transduction. Moving beyond Arabidopsis. Plant Physiol 135: 660–667. 4. Zarembinski TI, Theologis A (1994) Ethylene biosynthesis and action: a case of conservation. Plant Mol Biol 26: 1579–1597. 5. Kendrick MD, Chang C (2008) Ethylene signaling: new levels of complexity and regulation. Curr Opin Plant Biol 11: 479–485. 6. Broekaert WF, Delaure SL, De Bolle MFC, Cammue BPA (2006) The role of ethylene in host-pathogen interactions. Ann Rev Phytopathol 44: 393–416. 7. van Loon LC, Geraats BPJ, Linthorst HJM (2006) Ethylene as a modulator of disease resistance in plants. Trends Plant Sci 11: 184–191. 8. Chang C, Bleecker AB (2004) Ethylene Biology. More Than a Gas. Plant Physiol 136: 2895–2899. Dual-Level Regulation of Ethylene Biosynthesis ed. Ethylene accumulation in GC vials was measured after 24 hours. Error bars indicate standard deviations (n = 3). (TIF) Figure S3 Basal level ethylene production in various acs mutants. (A) Basal level ethylene production in wild type (Col-0, acs2-1/acs6-2 double and acs2-1/6-2/7-1 triple mutant. Twelve-day-old seedlings grown in GC vials were mock inoculated. Ethylene accumulation in GC vials was measured 24 hours later. Error bars indicate standard deviations (n = 3). (B) Basal level ethylene production in the high- order acs mutants generated in Dr. Athanasios Theologis’ lab. Twelve-day-old seedlings grown in GC vials were mock inoculated. Ethylene accumulation in GC vials was measured 24 hours later. Error bars indicate standard deviations (n = 3). The allele numbers are omitted for easy labeling. They are acs1-1, acs2-1, acs4-1, acs5-2, acs6-1, acs7-1, acs9-1, and acs11-1. (TIF) Figure S4 Activation of ACS7, ACS8, and ACS11 in the wrky33 mutant after B. cinerea inoculation. B. cinerea-induced ACS7, ACS8, and ACS11 expression is not compromised in wrky33 mutant. Total RNA from the experiment shown in Figure 8 was reverse transcribed. Expressions of ACS7 (A), ACS8 (B), and ACS11 (C) genes were quantified by real-time PCR. ACS transcript levels were calculated as percentage of EF1a transcript. Error bars indicate standard deviations (n = 3). (TIF) Figure S5 Ethylene production in acs2-1/acs6-2/acs7-1/GVG- ACS6 transgenic seedlings after DEX treatment. Twelve-day-old wild-type (Col-0), acs2-1/acs6-2/acs7-1, and acs2-1/acs6-2/acs7-1/ GVG-ACS6 transgenic seedlings (line #5 and #12) grown in GC vials were treated with DEX (+DEX, final concentration of 1 mM) or ethanol solvent control (2DEX), but without B. cinerea inoculation. Ethylene accumulation in GC vials was measured after 24 hours. Error bars indicate standard deviations (n = 3). 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