,30 mg total RNA, 16 SYBR Green PCR Master Mix (Applied Biosystems) and 500 nM forward and reverse primers. Real-time PCR was performed in an ABI PRISM 7500 sequence detection system (Applied Biosystems) under the following thermal cycling conditions: 10 min at 95uC followed by a total of 40 cycles of 30 s at 95uC, 30 s at 60uC and 40 s at 72uC. For qRT-PCR, relative transcript abundance was calculated and normalized with respect to ACTIN2/7 to minimize variation in cDNA template levels, with the solvent-treated (control) and control Col-0 samples acting as calibrators (for microarrays validation assay for and JA responsive genes assay respectively). Data shown represent mean values obtained from at least three independent amplification reactions; the SE of the CTs averaged 0.1, demonstrating the high precision of the assays. All calculations and analyses were performed using 7500 Software v2.0.1 (Applied biosystems) and the 22DDCT method [83]. Amplification efficiency for the primer sets was determined by amplification of cDNA dilution series (1:5). The values obtained not change significantly between different cDNA smaples, and were always higher than 0.90. Specificity of the RT-PCR products was followed by a melting curve analysis with continual fluorescence data acquisition during the 65–95uC melt. Analysis of JA levels 250 mg of freshly harvested plant tissues were chilled in liquid nitrogen and JA extraction was performed as in [84] using dihydrojasmonate as internal standar, derivatized with chloro- form/N,N-diisoprpyl-etylamine 1:1. In order to analyze the samples by GC/MS the extract was added with 10 ml of PFBr and 200 ml of cloroform: N,N-diisopropilethilamine (1:1) then incubated at 65uC for 1 h. When cooled, the solvent was evaporated to dryness and resuspended in 100 ml methanol. Samples were analyzed in a gas chromatograph (Agilent Technologies 7890A) equipped with a capillary column J&W DB-1 (60 m6250 mm60.25 mm) coupled to a mass selective detector (Agilent 5973 Series MSD). Using an autosampler 7683B Series. 2 ml of the sample was injected in a splitless way. Operating conditions were: injector temperature 250uC; the oven temperature was programmed as: initial temperature 150uC for 3 min then increasing at the rate of 4uC per min to a final temperature of 280uC maintained for 20 min. Helium was used as carrier gas with a constant flow of 1 ml/min. The MS was set to scan from 40 to 600 uam in Synchronous SIM/Scan mode for selectively monitor the following ions for jasmonic acid derivative: 141, 181, 390, and 392. MS temperatures were: Source 230uC, MS Quadrupole 150uC. Microscope Analyzes For histochemical analysis of transgenic lines LOX2:GUS and PR1:GUS, 7 d-old transgenic seedlings expressing these marker constructs were incubated at 37 uC in a GUS reaction buffer (0.5 mg/ml of 5-bromo-4-chloro-3-indolyl-B-D-glucuronide in 100 mM sodium phosphate, pH 7.0). The stained seedlings were cleared by the method of Malamy and Benfey [85]. For each treatment, at least 9 transgenic plants were analyzed. A representative plant was chosen for each treatment and photographed using the Nomarski optics on a Leica DMR microscope. H2O2 production was detected by the endogenous peroxidase- dependent staining procedure using 3,3-diaminobenzidine (DAB) uptake [86]. Control, 15 and 30 mM N-isobutyl decanamide- treated 7 d seedlings were placed in a solution of 1 mg mL21 DAB, pH 3.8, and incubated in dark for 2 h. Subsequently, were immersed in boiling 96% (v/v) ethanol for 10 min and then stored Alkamide-Regulated Transcriptional Networks in 96% (v/v) ethanol. For each treatment, at least 9 treated- seedlings were analyzed. A representative plant was chosen for each treatment. H2O2 production was visualized as a reddish- brown precipitated coloration and photographed using the Nomarski optics on a Leica DMR microscope. Nitric Oxide (NO) was monitored by incubating Arabidopsis seedlings with 10 mM of the fluorescent probe 4,5-diaminofluor- escein diacetate (DAF-2DA) [87] in 0.1 M Tris–HCl (pH 7.4). Treated seedlings were incubated for 2 h in the dark, and washed three times for 20 min with fresh buffer. Fluorescence signals from at least 9 treated and control leaves were detected using a confocal laser scanning microscope (model BX50, Olympus), and moni- tored with an argon blue laser with an excitation line from 488 to 568 nm and an emission window from 585 to 610 nm. Fungal growth and plant inoculation Pathogenesis assays were modified from [88]. Botrytis cinerea was grown on agar PDA medium (PhytoTechnology) for 7–12 days at 22u C in darkness. Spores were collected with distilled water. Col-0 superficially sterilized seeds were germinated and grown in MS- agar medium into 100 ml flasks with transparent lid. At 20 days after germination, rosette leaves were placed in Petri dishes with 60 mM of N-isobutyl decanamide containing medium or medium supplied with the solvent. Inoculation was performed by placing a 5 ml drop of a suspension of 56105 conidiospores/ml on the surface of leaves. The samples were incubated at 22uC and analyzed at a further 3, 4 and 5 d period after inoculation. Susceptibility was evaluated by microscopic observation of necrotic symptoms under a dissecting microscope (Leica MZ6) connected to a digital color camera (Samsung SCC-131A). The percent of necrotic leaves was scored for 30 independent inoculated leaves. The disease symptoms on inoculated leaves and fungal growth over leaves was estimated by trypan blue staining and further cleared with chloral hydrate and the extension of necrotic lesions (lesion diameter) measured at 4 d after inoculation. For mutant inoculation, leaves from soil grown adult plants were incubated in agar solution supplied with solvent (ethanol) or N-isobutyl decanamide during 24 h prior to inoculation by leaves immersion into solution of 56105 conidio- spores/ml. Supporting Information Figure S1 Experimental design for microarray analysis. 6 day-old Arabidopsis Col-0 seedlings were grown on N-isobutyl decanamide-free medium and then transferred to control medium (A) supplied with the solvent, or to 60 mM N-isobutyl decanamide- containing medium (B). Pictures were taken 14 days after transfer (d.a.t.). Modified loop design including 4 independent replicates evaluated at 1, 3, 7, and 14 d.a.t. (C). A total of 16 slides were employed. Each replicate was conformed by at least 120 transferred seedlings, which were harvested from four independent plates. (TIF) Figure S2 Validation of microarray results via qRT- PCR. Quantitative real-time PCR analysis was performed for 15 genes in Arabidopsis (Col-0) seedlings, under the same conditions used for microarray analysis (1, 3, 7 and 14 days of treatment with 60 mM N-isobutyl decanamide). Fold-change (control to N- isobutyl-decanamide) expression for the indicated selected genes in a log2 scale is shown. Expression ratios obtained by microarray experiments (A). Estimates of the differences of expression levels were calculated using the mixed model as described in methods. Expression ratios obtained by qRT-PCR (B). RQ (relative PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27251