Alkamide-Regulated Transcriptional Networks Figure 4. JA-related pathways are transcriptionally induced by N-isobutyl decanamide. Simplified representation of JA biosynthetic pathway, genes and metabolites are illustrated (A). Fold-change values of JA-biosynthetic and –responsive pathway genes differentially expressed with N-isobutyl decanamide treatment (A, B). Data from microarray expression profiles are shown in the color scale from blue to red. doi:10.1371/journal.pone.0027251.g004 15% and 60% of infected leaves showed necrotic lesions at fourth and fifth d.a.i., respectively. It is important to note that lesions in control leaves five d.a.i. were of about 6 mm in diameter, whereas in alkamide-treated leaves, the lesions had a diameter between 0.8 and 1.5 mm (Figure 6B). Visual inspection showed that after 5-d of inoculation, solvent-treated leaves inoculated with the pathogens presented generalized necrotic lesions spanning half or more the surface of the leaf, while N-isobutyl decanamide-treated leaves manifested significantly reduced symptoms (Figure 6C). We monitored hyphal growth of the pathogens by direct microscopic observation of stained mycelium in infected leaves. We found that disease symptoms in solvent-treated leaves at day 3 after inoculation were accompanied by prolific mycelium growth. In contrast, N-isobutyl decanamide treatment inhibited fungal growth over leaf surfaces, as compared to the control (Figure 6C). On the basis of these findings, it can be concluded that N-isobutyl decanamide treatment renders enhanced resistance to B. cinerea in Arabidopsis leaves. To determinate if the reduced leaf damage and fungal growth inhibition observed in alkamide-treated leaves could be the result of a direct toxic effect of N-isobutyl decanamide on the fungal pathogen tested, we evaluated the antifungal activity of N-isobutyl decanamide on B. cinerea mycelial growth by inoculating mycelia disks on Petri plates containing PDA media supplemented with the solvent or with increasing concentrations of alkamide. Although 120 mM N-isobutyl decanamide inhibited mycelium growth by approximately 15%, any lower concentration had no significant effect (Figure S4). Indicating that plant defense responses elicited by N-isobutyl decanamide, and no an antifungal activity were responsible of pathogen proliferation over inoculated leaves. JA signaling is required for the N-isobutyl decanamide-induced resistance to B. cinerea To test whether JA signaling is involved in the N-isobutyl decanamide-induced increased resistance to necrotrophic fungal infection of Arabidopsis leaves, we evaluated the responses of Arabidopsis JA-related mutants jasmonic acid resistant1 (jar1), coronatine insensitive1 (coi1-1), a mutant defective at the MITOGEN-ACTIVAT- ED PROTEIN KINASE6 (MPK6) locus, which has been found to be critical in defense responses to B. cinerea [22], and the SA-related mutant enhanced disease symptoms16 (eds16/sid2-1). Fully developed leaves from Col-0 wild-type (WT) and mutant plants were pre- PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27251