Alkamide-Regulated Transcriptional Networks Figure 6. N-isobutyl decanamide confers protection against Botrytis cinerea attack. Leaves from 20 day-old plants grown in soil were pre-incubated 24 h on solvent (control, white squares), or 30 mM N-isobutyl decanamide containing plates (black squares), transferred to decanamide-free plates and then inoculated with a 10 ml droplet of B. cinerea spores (56105 conidiospores/ml). The percentage of leaves with necrotic symptoms at 3, 4 and 5 days after inoculation was determined (A). Bars mean 6 SE of 30 inoculated leaves from two independent experiments. Tissue damage caused by B. cinerea was measured at 5 days after inoculation (B). Data points represent average lesion size 6 SE from 30 independent leaves, asterisks denote a significant difference from control leaves (P#0.05) as determined by t test. Representative inoculated leaves at 5 days after inoculation were imaged (C, top panels) and trypan blue-stained, inoculation sites are shown (C, bottom panels). doi:10.1371/journal.pone.0027251.g006 The central idea of this work was to explore the transcriptional responses in Arabidopsis to alkamides and ascertain its relevance in activating long-term defense responses. Toward this goal, we performed a global transcription profile of Arabidopsis response to N-isobutyl decanamide (Figure S1). Our results establish that exogenous application of N-isobutyl decanamide triggers profound physiological changes in Arabidopsis, with activation of developmental and defense- and stress-related genes (Table S1; Figures 2 & 3). Interestingly, treatment with N- isobutyl decanamide resulted in an increase in the endogenous levels of JA (Figure 5), a lipid phytohormone known to be central in activation of plant defense responses to a range of biotic challengers, including herbivores, insects, necrotrophic fungi and oomycetes [55,56,57]. These results are consistent with previous reports showing that NAEs induce LOXs activity and JA accumulation [58,59]. NAEs comprise a group of bioactive signaling lipids naturally present from fungi to plants to mammals that share structural and functional relationship with alkamides. In mammals, anandamide (NAE 20:4) acts as an endogenous ligand for cannabinoid receptors and plays different physiological roles including the modulation of neurotransmission in the central nervous system [60], synchronization of embryo development [61] and vasodilation [62]. Interestingly, arachidonic acid (AA, 20:4) a precursor of anandamide in mammals has been shown to posses important signaling roles in plant stress and defense networks trough production of JA and the activation of JA-dependent transcripts [8]. These results indicate that lipid signals biochem- ically related to alkamides and NAEs could regulate the same or similar signaling pathways. Previously, Teaster and coworkers [63] conducted microarray analyses to identify transcriptional targets of plant NAE 12:0 in 4 d-old Arabidopsis seedlings. We found a set of 171 differentially expressed genes by N-isobutyl decanamide, whose expression was also reported as regulated by NAEs, including ABA-responsive genes (At3g02480, At5g53820) and germin-like genes (At5g38910, At5g39550, At5g39180, At5g39110, At5g39190) (Table S1). Our results indicate that although important differences in plant age, concentrations of compounds and time of exposure already exists when comparing our expression analysis results with those reported for NAE 12:0, common genes were found to be up- regulated by the two compounds, indicating commonalities in the transcriptional responses elicited by NAE 12:0 and N-isobutyl decanamide. One of the first indications that plant fatty acid amides indeed participate in plant-pathogen interactions was the observation that NAEs accumulated in the growth media of tobacco suspension cells and leaves after application of the fungal elicitor xylanase. Indeed, exogenous NAE application triggered the expression of the PHENYLALANINE AMMONIA LYASE gene (PAL), which has been implicated in plant defense against PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27251