Alkamide-Regulated Transcriptional Networks Figure 5. Levels of JA and their corresponding transcripts are enhanced by N-isobutyl decanamide. N-isobutyl decanamide-dependent accumulation of JA was determined by GC-MS from three biological replicate samples (A), data are means of three independent experiments 6 SD, asterisks denote a significant difference from control seedlings (P#0.05). Transgenic Arabidopsis seedlings expressing GUS under the regulation of the JA-induced LOX2 promoter (LOX2:GUS) were grown for 7 days on solidified medium supplied with the solvent (control) or with 30 and 60 mM N- isobutyl decanamide, and then stained for GUS expression (B). Quantitative real-time PCR (qRT-PCR) analysis of nine JA-responsive genes using CT value of ACT2/7 as internal expression reference (C). Relative expression values were normalized with endogenous levels from each transcript in Col-0 control seedlings. Bars represent 6 SE from three independent biological replicates, and there were four technical replicates for qRT-PCR assay. doi:10.1371/journal.pone.0027251.g005 incubated for 24 hours with 30 mM N-isobutyl decanamide or with the solvent as control and then, inoculated with a droplet of 56105 spores/ml B. cinerea spores on the surface. WT and eds16 leaves showed decreased disease symptoms when treated with N-isobutyl decanamide, unlikely jar1, mpk6 and coi1-1 leaves, which presented symptoms similar to solvent-treated controls, and, therefore, were not responsive to the alkamide-activated resistance (Figure 7A & B). Similarly, fungal growth, assessed by quantitative PCR amplifica- tion of Actin A DNA of B. cinerea [53], was significantly enhanced in the jar1, mpk6 and coi1-1 mutant leaves, but restricted in the WT and eds16 with the N-isobutyl decanamide treatment (Figure 7C). In order to qualitatively analyze the damage caused by the pathogen, infection was estimated by recording a range of severity of disease symptoms (from no symptoms, to severe tissue maceration). For this purpose, we placed leaves from WT and mutant plants on 0.7% agar plates supplied with solvent or N- isobutyl decanamide for 24 hours. Then, leaves were immersed into B. cinerea spores solution and transferred to N-isobutyl decanamide-free agar plates. As shown in Figure 7D, three d.a.i., lesion severity was higher in the jar1, mpk6 and coi1-1 mutants than in WT leaves, even when they received N-isobutyl decanamide pre-treatment. As expected in the WT, disease symptoms decreased when the alkamide was supplied. Unlike JA-related mutants, eds16/sid2-1 leaves displayed reduced injuries with N-isobutyl decanamide pretreatment, as compared to those observed for the corresponding untreated controls (Figure 7D). All together, these results show that N-isobutyl decanamide- induced resistance to B. cinerea in Arabidopsis WT leaves is due to the induction of defense programs that require an intact JA signaling pathway. Discussion Lipids, besides being important structural molecules in living systems, function as modulators of a multitude of signal transduction pathways evoked by environmental and develop- mental stimuli. Alkamides belong to a novel class of lipid signals that regulate morphological processes in plants [23]. Recent findings provided evidence of a widespread distribution of structurally related lipid amide signals in evolutionary distant organisms, including the animal, fungal and plant-produced NAEs and the bacterial quorum-sensing AHL regulators [54,33]. PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27251