tions by affecting the level of other lipids or by modulating the levels of second messengers involved in signal transduction to these lipids such as Ca2+, NO, and/or ROS. In a developmental context, the relationship between NO and alkamides pathways in Arabidopsis was recently investigated, mitotic activation of pericycle cells from seedlings roots induced by N-isobutyl decanamide occurred in parallel and in a dependent way to NO synthesis [45]. However, whether NO mediates the defense responses to alkamides remains to be clarified. Our previous research revealed a genetic interaction of alkamides and senescence responses mediated by the DRR1 locus in Arabidopsis [32]. Leaf senescence is a metabolic active process controlled by a genetic program [72,73]. Interestingly, ultra- structural changes in senescing cells are accompanied by production of several metabolites that may influence interactions with other organisms. For example, antimicrobial compounds often accumulate in senescing tissues, preventing diseases [74]. In agreement with this, many senescence genes are transcriptionally up-regulated by N-isobutyl decanamide, i.e. PR genes, SAG genes (At2g29350, At4g17670, At5g47060), a member of TCP family (At5g40070), and JA-related genes. It is tempting to speculate that N-isobutyl decanamide can be recognized as a senescence-induced signal, thus influencing developmental and defense programs involving JA signaling and possibly other additional signaling/ metabolic pathways regulated by NO, ROS and/or MAPK messengers. N-isobutyl decanamide share structural similarity to N-decanoyl homoserine lactone (C:10 AHL) [25], a member of the bacterial quorum-sensing signals, which have been found to alter root development and activate defense responses in different plant species [30,29,25]. An interesting hypothesis is that small lipid signaling based on plant fatty acid amides and/or AHLs might be part of an ancestral inter-kingdom communication system between plants and their associated bacteria. Our data thus expand the repertoire of signaling lipid molecules known to trigger plant defenses and provide evidence that alkamides interact with the JA pathway. The use of alkamides and bacterially produced fatty amides in pathogen resistance by acting as defense elicitors in plants shows great potential towards application of these compounds to combat pathogen pests. Materials and Methods Plant material and growth conditions Arabidopsis thaliana ecotype Col-0 was used for all experiments unless indicated otherwise. Col-0, transgenic LOX2:GUS [75] and PR1:GUS, and mutants jar1 [16], mpk6 [76], coi1-1 [77] and eds16/ sid2-1 [78] seeds were surface sterilized with 95% (v/v) ethanol for 5 min and 20% (v/v) bleach for 7 min. After five washes in distilled water, seeds were germinated and grown on agar plates containing 0.26 MS medium. Plates were placed vertically at an angle of 65u to allow root growth along the agar surface and to allow unimpeded growth of the hypocotyl into the air. For plant growth, we used a plant growth cabinet (Percival Scientific AR95L, Perry, IA), with a photoperiod of 16 h of light, 8 h of darkness, light intensity of 300 mmol/m-2/s-1 and temperature of 22uC. After grown for 6 days, plants were transferred to control or N-isobutyl decanamide containing solid MS medium for different times. Homozygous coi1-1 seedlings were selected by screening a heterozygous population in agar solidified MS medium supplied with 5 mM JA (Sigma Chemical Co., St. Louis), seedlings resistant to root inhibition were transferred to soil baskets and leaves from 20 d-old were detached for in vitro pathogenicity assays. Alkamide-Regulated Transcriptional Networks Synthesis of N-isobutyl decanamide N-isobutyl-decanamide was obtained by catalytic reduction of affinin, the most abundant alkamide present in Heliopsis longipes (Gray) Blake (Asteraceae) roots as described before [23]. Experimental design and microarray platform For microarray analyses a dye balanced modified loop design was implemented. Four biological replicates representing each sampling point were obtained by pooling in a 1:1 proportion shoot and root purified RNA from 120 randomly chosen seedlings. This experiment involved a total of sixteen sets of microarray hybridizations, including direct and dye swap comparisons between treatments as well as across time points for the same treatment. This design allowed us to determine differences in gene expression between N-isobutyl decanamide-treated and control seedlings, and whether the differences were time dependent. The Arabidopsis Oligonucleotide Array version 3.0 from The Arizona University was used to carry out this study. Array annotation and composition are available at http://ag.arizona.edu/microarray. RNA isolation, fluorescent labeling of probes, slide hybridization and washing were performed as described previously in [79]. Slides were scanned with an Axon GenePix 4100 scanner at a resolution of 10 mm adjusting the laser and gain parameters to obtain similar levels of fluorescence intensity in both channels. Spot intensities were quantified using Axon GenePix Pro 5.1 image analysis software. All microarray data is MIAME compliant and the raw data has been deposited in the Gene Expression Omnibus database (GEO), accession number GSE12107, as detailed on the MGED Society website http://www.mged.org/Workgroups/MIAME/miame.html. Normalization and data analysis Raw data were imported into the R 2.2.1 software (http:// www.R-project.org). Background correction was done using the method ‘‘substract’’ whereas normalization of the signal intensities within slides was carried out using the ‘‘printtiploess’’ method [80] using the LIMMA package (www.bioconductor.org). Normalized data were log2 transformed and then fitted into mixed model ANOVAs [81] using the Mixed procedure (SAS 9.0 software, SAS Institute Inc., Cary, NC, USA) with two sequenced linear models considering as fixed effects the dye, time, N-isobutyl-decanamide treatment and time 6 N-isobutyl-decanamide treatment. Array and array 6 dye were considered as random effects. The type 3 F- tests and p-values of the time 6 N-isobutyl-decanamide treatment and N-isobutyl-decanamide treatment were also carried out. Model terms were explored and significance levels for those terms were adjusted for by the False Discovery Rate (FDR) method [82]. Estimates of the expression differences were calculated using the mixed model. Based on these statistical analyses, the spots with tests with an FDR less or equal to 5% and with changes in signal intensity between N-isobutyl decanamide treatment and control seedlings of 2.0-fold or higher were considered as differentially expressed. Expression analysis by qRT-PCR Total RNAs were isolated from Arabidopsis plants using TRIzol reagent (Invitrogen). Primer design (Tm, 60–65uC) was per- formed using Primer Express Software, Version 3 (Applied Biosystems); full sequences from each primer are shown in Table S3. cDNA templates for PCR amplification were prepared from all samples by using reverse specific primers and SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Each reaction contained cDNA template from PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27251