Real Time Quantitative RT-PCR and Other PCR assays Total RNA was isolated from frozen tissues using TRizol Reagent (InvitrogenH, Carlsbad, CA) according to the manufac- turer’s protocol. RNA samples were treated with RQ1 DNase (Promega, Madison, WI). Trace amounts of genomic DNA were removed by digestion with Turbo DNA-freeTM (Ambion, Austin, TX). First-strand cDNA synthesis was primed with an oligo (dT)15 anchor primer and cDNA was synthesized using the First-Strand Synthesis Kit (Amersham-Pharmacia, Rainham, UK) according to the manufacturer’s protocol. An aliquot of 1.5 ml of the first-strand synthesis reaction was used as template for PCR amplification. To ensure that the sequence amplified was specific, a nested PCR was performed using 1 ml of a 1:50 dilution of the products synthesized in the first PCR reaction as a template. The RT-PCR, PCR and nested PCR program consisted of: 3 min at 96uC, 40 cycles of 30 s at 94uC, 30 s at 65uC, and 1 min at 72uC. The final extension step consisted of 7 min at 72uC. Amplified PCR fragments were visualized using 1.5% agarose gels. Quantitative RT-PCR experiments were performed using a SYBRH Green qPCR kit (Finnzymes, Espoo, Finland) with reactions at a final volume of 20 ml per well and using the cycle protocol recommended by the manufacturer. Samples were run in a DNA Engine OpticonH 2 System instrument with PTC-200 DNA Engine Cycler and CFD-3220 OpticonTM 2 Detector (BioRad, Hercules, CA). Gene-specific primers were designed using the Primer Express 2.0 program (Applied Biosystems, Foster City, CA) and minimal self-hybridization and dimer formation of primers was determined using the Oligo 6.0 program (Molecular Biology Insights, West Cascade, CO). Primers with annealing temperatures of 62uC to 65uC that amplified products with lengths of about 300 bp were selected and then verified for specificity by BLAST searches. The efficiency of amplification for each gene was calculated as recommended by the manufacturer (BioRad, Hercules, CA) [75]. The following gene specific primers were used for RT-PCR and quantitative RT-PCR; ATL9 (At2g35000): 59-GTCGGAAGATTCTTCGGCGCATCTCC-39 (forward) and 59-CGACCGGACATTCGTTAATTCAAC-39 (reverse); PR1 (At2g 14610): 59-GATAGCCCACAAGATTATCGG-39 (forward) and 59-CTCGTTCACATAATTCCCACG-39 (reverse); ATRBOHD (At5g47910): 59-ATGAAAATGAGACGAGGCAATTC-39 (for- ward) and 59-GGATACTGATCATAGGCGTGGCTCC-39 (re- verse); ATRBOHF (At1g64060): 59-CTTCCGATATCCTTCAA- CAACTC-39 (forward) and 59-GAGATTGCCTTTATACTAT- AAGTG-39 (reverse); MAPK3 (At3g45640): 59-ATGAACACCGG- CGGTGGCC-39 (forward) and 59-GGCATTCACGGGGCTGC- TG-39 (reverse); b-ACTIN (At3g18780): 59-GTTGGTGATGAAG- CACAATCCAAG-39 (forward) and 59-CTGGAACAAGACTT- CTGGGCATCT-39 (reverse). Data acquisition was performed using the Opticon Monitor Analysis software (version 2.01) and changes in transcript levels were determined by the 22DDCT method in Microsoft EXCEL [76]. Data points were compared using a T- test. Three independent biological replicates were used in each experiment. Transient Expression in Tobacco and Immunoblot Analysis Agrobacterium-mediated transient expression in tobacco was performed as described previously [77], with the exception that Agrobacterium tumefaciens GV3101 (at O.D.600 of 2) was used. Agrobacterium containing the transformation constructs described above, 35S:ATL9:GFP and 35S:GFP (negative control) [68] were introduced into tobacco leaves by infiltration. After two days, infiltrated tissues were harvested for western blot analysis using the monoclonal anti-GFP antibody, JL-8 (BD Biosciences, Palo Alto, ATL99s Role in Plant Defense USA) at a 1:1000 dilution [78]. Experiments were performed three times with three replicates in each experiment. Microparticle Bombardment of Onion Epidermal Cells Tungsten microcarriers (1.1 mm) were prepared and coated with an equal molar ratio of 35S:ATL9:GFP to ER luminal marker, ER-rk [40] plasmid DNA (1 mg/mL) according to manufacturer’s instructions (Bio-Rad). Fresh onion epidermal peels were trans- ferred to MS medium with vitamins (0.5% MS Salts with vitamins, 0.7% Agar, pH 5.7) less than one hour before bombardment. Microcarriers were bombarded into onion epidermal cells using Bio-Rad PDS-1000/He Particle Delivery System (Bio-Rad). Bombardment was executed following the manufacturer’s protocol under the following conditions: 900 PSI rupture disk, helium vacuum of 27 in. Hg, and a distance of 6 cm from the microcarriers to the sample. Immediately after bombardment, onion epidermal cells were incubated in the dark at room temperature (22–23uC) for 12–24 hours before observation using fluorescent microscopy. Microscopy and Photography Techniques Confocal fluorescence images of Arabidopsis and tobacco tissues expressing GFP fusion constructs were observed using a Nikon Diaphot 200 inverted fluorescence microscope equipped with Nikon 60X 1.2 numerical aperture water immersion objective (Nikon, Japan) and a BioRad MRC 1024 confocal head with inverted fluorescence microscope (BioRad, Philadelphia, USA). Samples were prepared in water as previously described [68]. Confocal images were processed using Image J (v. 1.30, N.I.H. USA), Cas40 (Confocal Assistant v. 4.02, USA) and Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA) programs. For subcellular localization in onion cells using fluorescent microscopy, onion epidermal cells were mounted in distilled H2O ,12 hours after bombardment. GFP and mCherry fluorophores were visualized concurrently with a Nikon Eclipse 90i epi- fluorescent microscope (Nikon, Melville, NY) equipped with an OptiGrid imaging system (Qioptiq, Paris, France) using the FITC HYQ (Excitation: 460–500 nm; Emission: 510–560 nm) and TRITC HYQ (Excitation: 530–560 nm; Emssion: 590–650 nm) filters. Images were generated and/or merged using NIS-Elements software (Version 3.2, Nikon). For light microscopy experiments, a Nikon Eclipse E600 light microscope was used and images were recorded with a Nikon Spot Advance 32 camera (Nikon, Japan). To determine the number of conidiophores per fungal colony, leaves were inoculated at low density with G. cichoracearum and stained with trypan blue either 6 or 7 dpi [37,71]. To assess cell death, leaves were stained with trypan blue in phenol [79]. Photographs of Arabidopsis leaves inoculated with G. cichoracearum were taken using a Nikon Coolpix E995. Tissues that were double stained with Trypan blue (TB) and Diamino-benzidine (DAB) were first stained with DAB and subsequently with TB. Osmotic shock was induced by immersion of Arabidopsis leaves in sodium chloride 0.5 M for 10 minutes. Ubiquitination Assays and Cloning of ATL9 for expression assays Total RNA isolated from leaves of 2–4 week old Arabidopsis thaliana ecotype Col-0 plants was used in reverse transcription reactions followed by PCR to amplify the predicted open reading frame (ORF) of ATL9 (At2g35000). The Qiagen RNeasy plant RNA extraction kit (Qiagen, Valencia, CA) was used to isolate total RNA as per manufacturer instructions. The amplified cDNA was first introduced into the Gateway entry vector, pDONR PLoS ONE | www.plosone.org 13 December 2010 | Volume 5 | Issue 12 | e14426