GAAACATAATTAGCG -39(forward) and 59-GACGACGAC- GACAGCACTGAA-39 (reverse), (2) SALK_036065: 59 –CGTT- CATCTGGTCGGAGCCGTTCG-39 (forward) and 59-GCGT- TTGATGCCTCCTTGTTG-39 (reverse), and (3) SALK_036- 066: 59 CGTTCATCTGGTCGGAGCCGTTCG-39 (forward) and 59-GCGTTTGATGCCTCCTTGTTG-39 (reverse). Nested primers for confirming the T-DNA insertions in ATL9 were as follows: (1) SALK_066755: 59-TTGGCATGTAGAAACATAAT- TAGC-39 (forward) and 59-GACGACGACGACAGCACTGAA- 39 (reverse); (2) SALK_036065: 59-CGATGTCGGAAGATTCT- TCGG-39 (forward) and 59-GTCTGGCTCTCAGAACACTCC- 39 (reverse) and (3) SALK_036066: 59- CGATGTCGGAA- GATTCTTCGG-39 (forward) and the reverse primer was the same as in SALK_036065 (Oligo 6.0 Primer Analysis Software, LSR, Minnesota, MN, USA). Seeds of homozygous lines from the three atl9 mutants will be made available via the Arabidopsis Biological Resource Center (Ohio State University, USA). The fungal pathogens used in this work were Golovinomyces cichoracearum UCSC1 (Powdery mildew = Erysiphe cichoracearum UCSC1) [71]. Powdery mildew fungus was maintained as described previously [63]. Disease Assessments Powdery mildew inoculations and disease assessments were carried out as described in [37]. In brief, Arabidopsis seeds were planted in soil (Promix HP, Hummert International, St. Louis MO), placed in a cold room for three days and then transferred to a growth chamber (24uC day, 22uC night; 14 h day, 150 mE m22 sec21 of light; 60% relative humidity). After four weeks, plants were inoculated with powdery mildew and placed in a chamber under the same temperature and light conditions except at 80% relative humidity. Disease development was assessed in a qualitative manner by monitoring the appearance of powdery symptoms on inoculated leaves over a period of 10 days post inoculation [71]. At least 36 plants per genotype were inoculated in each experiment. For quantitative assessment, inoculated leaves were treated with ethanol 100% for two hours at 65uC and 90% relative humidity before staining with trypan blue solution (25 mg/ml trypan blue in a 1:1:1 solution of glycerol, lactic acid and water,) for 15 minutes. Leaves were harvested for staining 6 or 7 days after inoculation depending on the development of the fungal infection. The number of conidiophores per colony was then determined for at least 36 leaves per genotype. For diamino- benzidine (DAB)/trypan blue double staining, inoculated leaves were treated with 1mg/ml DAB for two minutes under vacuum and then covered with aluminum foil for six hours at room temperature. Leaves were then immersed in 100% ethanol for two hours at 65uC and 90% of humidity before performing the trypan blue staining as described. Chemical Treatments Seeds were surface sterilized and grown in liquid Murashige Skoog culture medium at a density of approximately 500 seeds (10 mg) per 125 ml flask. Flasks with seeds were incubated at 4uC for 6 days and then placed in a shaking incubator at 150 rpm for two weeks under constant illumination (125 mmol m22 s21) at 23uC. After fourteen days, seedlings were treated with either 100 mg ml21 hydrolyzed crab shell chitin (Sigma, St Louis, MO, USA) [29] or with salicylic acid (0.5mM; Sigma, St Louis, MO, USA), jasmonic acid (5 mM; Sigma, St Louis, MO, USA) or ACC 5 mg ml21 (50 mM; Sigma, St Louis, MO, USA) for thirty minutes. After 30 minutes of treatment seedlings were harvested, flash- frozen in liquid N2 and stored at 280uC until analysis. ATL99s Role in Plant Defense Generation of Transgenic Plants and Constructs The ATL9 sequence was PCR amplified using PlatinumH Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA). For GatewayH cloning, attB PCR primers were designed per manufac- turer’s recommendations (Invitrogen, Calsbad, CA). Primer se- quences were as follows: attB1 forward primer 59-GGGGA- CAAGTTTGTACAAAAAAGCAGGCTTC-39; attB2 reverse primer 59-GGGGACCAACTTTGTACAAGAAAGCTGGGTC- 39. Gene specific primers for ATL9 were: forward primer 59- CATACGTCGATTGGATTTTAATGG-39 and reverse primer 59- CCACTCGTTCATCTGGTCG-39. Final primers used for PCR amplification of the ATL9 gene were forward 59-GGGGA- CAAGTTTGTACAAAAAAGCAGGCTTCCATACGTCGATT- GGATTTTAATGG-39 and reverse 59-GGGGACCAACTTT- GTACAAGAAAGCTGGGTCCACTCGTTCATCTGGTCG- 39 using Col-0 wild type cDNA as the template. cDNA was prepared using an Amersham First-Strand cDNA Synthesis Kit (Amersham, Buckinghamshire, UK). Resulting PCR products were purified and entry clones were generated by recombination into the vector, pDONRTM207 using the BP Recombination Reaction (Invitrogen, Carlsbad, CA). For the ATL9p:ATL9:GFP construct the destination vector pGWB4 (attR1-CmR-ccdB-attR2-sGFP) was used. The generation of the constructs for the tobacco transformation were as follows: 35S:GFP:ATL9 utilized the destination vector pGWB6 (35S promoter- NsGFP-attR1-CmR-ccdB-attR2) and the destination vector pGWB5 (35S promoter-attR-CmR-ccdB-attR2-sGFP) was used to generate a 35S:GFP construct [68] for use as a control. All GatewayH binary vectors were kindly provided by Tsuyoshi Nakagawa at the Research Institute of Molecular Genetics, Shimane University, Japan. All plasmid inserts were sequenced prior to transformation and verified constructs were transformed into Agrobacterium tumefaciens strain C58C1 (pGV2260) via electro- poration. Both Col-0 plants and the atl9-1 mutant were transformed using the floral dip method [72,73]. Kanamycin resistant plants were then selected on plates and resistant progeny were transferred to soil and allowed to set seed. The T2 progeny from these transformants were used in the experiments described in the text. The transcript levels of ATL9 in all lines were verified using qRT- PCR as described below. Seeds of all transgenic lines will be made available via the Arabidopsis Biological Resource Center (ABRC, Ohio State University). Generation of Constructs for Microprojectile Bombardment ATL9 full length genomic DNA was amplified by attB primers containing ATL9 sequence. Forward Primer 59–GGGGACAAG- TTTGTACAAAAAAGCAGGCTTCATGGCGATCCTCGA- CACAAAG–39 and Reverse Primer 59– GGGGACCACTTTG- TACAAGAAAGCTGGGTCCACTCGTTCATCTGGTCGGA- GC –39. The resultant PCR product was cloned into pDONR221 (Invitrogen) using BP Clonase II enzyme mix (Invitrogen) to generate entry clones. The entry clones carrying ATL9 were subcloned into T-DNA binary vector pMDC83 [74] using LR Clonase enzyme mix (Invitrogen). The resultant 35S:ATL9:GFP plasmid was transformed into One-Shot TOP10 Chemically Competent E.coli (Invitrogen) and screened on LB agar plates containing kanamycin (50 mg/mL) and hygromycin (50 mg/mL). The plasmid was isolated from positive clones using QIAprep Spin miniprep kit (Qiagen). Plasmid DNA was concentrated to 1 mg/mL by ethanol precipitation for microparticle bombardment into onion epidermal cells. The plasmid for the ER localization, ER-rk, was acquired from the ABRC (stock number CD3-959). PLoS ONE | www.plosone.org 12 December 2010 | Volume 5 | Issue 12 | e14426