Alkamide-Regulated Transcriptional Networks Figure 7. Effect of JA-related mutations on disease resistance response induced by N-isobutyl decanamide. (A–C) Disease symptoms on detached 20 day-old leaves at 4 days after drop inoculated with a 5 ml droplet as described in the legend for Figure 6 from wild-type (Col-0) plants, JA-related mutants jar1, coi1-1 and mpk6, and the SA-deficient mutant eds16/sid2-1. Images show necrotic lesions (A), mean lesion size (B) and, in (C), fungal growth. The data show the qPCR amplification of B. cinerea ActinA relative to the Arabidopsis ACT2/7 gene. (D) Leaves from 20 day-old wild- type and mutant plants were pre-incubated 24 h on solvent (control), 30 or 60 mM N-isobutyl decanamide containing plates, dipped into a B. cinerea inoculum of 56105 spores/ml, transferred to decanamide-free plates and then incubated. Disease symptoms were scored 3 days post-inoculation, graphical representation of disease rating (upper panel) caused in leaves was determined as percentage of leaves showing no symptoms (white bars), chlorosis (grey bars), necrosis (dark grey bars), or severe tissue maceration (black bars). Data values represent one of two independent experiments that gave similar results, 15 leaves were employed per treatment in each assay. doi:10.1371/journal.pone.0027251.g007 pathogens [64,27]. Moreover, the ectopic overexpression of FAAH, a NAE-metabolizing enzyme, renders Arabidopsis seedlings more susceptible to both host and non-host bacterial pathogens [28,65]. However, it remains to be determined whether NAE application can actually confer improved resistance of plants to pathogens. Upon N-isobutyl decanamide treatment, several JA-related genes such as PDFs (At2g43510, At5g44420, At1g75830, At2g26010), VSP2 (At5g24770), JAZ10 and JAZ8 (At5g13210, At1g30135), were induced, with a maximum at day 7 after alkamide treatment (Figure 4), which correlates with the up- regulation of several genes encoding enzymes involved in JA biosynthesis and with a two-fold increased JA level (Figure 4). Similar long-term gene induction patterns and JA increase have been described in Medicago truncatula plants inoculated with the pathogenic soilborne fungus Phymatotrichopsis omnivora, which has a very broad host range and infects almost 2,000 dicotyledonous species. Transcriptomic analysis of this interaction provided evidence that JA production is sustained and prolonged, inducing expression of genes encoding for LOXs, AOC2, OPR3, OPR5, OPR12, and wound-inducible serine proteinase inhibitors (PII) at 3 and 5 days after inoculation [66]. We show that N-isobutyl decanamide treatment conferred protection against B. cinerea attack to Arabidopsis leaves (Figure 6). In contrast to wild-type and the SA-related mutant eds16/sid2-1, all three jar1, coi1 and mpk6 Arabidopsis mutants, whose gene products are involved in JA sensitivity and signaling, failed to resist B. cinerea attack when N-isobutyl decanamide was supplied (Figure 7), suggesting that N-isobutyl decanamide-conferred resistance to necrotrophic fungi requires an intact JA signaling pathway. Responses of plants to necrotrophic pathogens involve multiple intermediates in signal transduction and anti-microbial responses, which includes nitric oxide (NO) and reactive oxygen species (ROS) [67]. The production and accumulation of reactive oxygen species ROS, primarily superoxide (O2-) and hydrogen peroxide (H2O2), during the course of a plant-pathogen interaction has long been recognized. Evidence suggests that the oxidative burst and the cognate redox signaling engaged subsequently, may play a central role in the integration of a diverse array of plant defense responses [68,69]. One well studied effect of oxidative burst is the induction of hypersensitive response (HR) mechanism, where the tissue at the infection site dies and in turn confines the pathogen growth preventing its spreading [70]. In our microarray analysis, we identified several potential components of the ROS signaling pathway, including scavenging enzymes catalases and ascorbate peroxidases, as well as at least 20 cytochrome P450 genes, including the antifungal gene CYP71B15/PAD3, which plays a key role in camalexin production and resistance against necrotrophic pathogens [71] (Figure 4B & Table S1). Activation of these genes correlated with accumulation of hydrogen peroxide (H2O2) and NO in N-isobutyl decanamide-treated leaves (Figure 3). We propose that alkamides might influence plant-pathogen interac- PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27251