Alkamides activate jasmonic acid biosynthesis and signaling pathways and confer resistance to Botrytis cinerea in Arabidopsis thaliana

Abstract

Alkamides are fatty acid amides of wide distribution in plants, structurally related to N-acyl-L-homoserine lactones (AHLs) from Gram-negative bacteria and to N- acylethanolamines (NAEs) from plants and mammals. Global analysis of gene expression changes in Arabidopsis thaliana in response to N-isobutyl decanamide, the most highly active alkamide identified to date, revealed an overrepresentation of defense-responsive transcriptional networks. In particular, genes encoding enzymes for jasmonic acid (JA) biosynthesis increased their expression, which occurred in parallel with JA, nitric oxide (NO) and H₂O₂ accumulation. The activity of the alkamide to confer resistance against the necrotizing fungus Botrytis cinerea was tested by inoculating Arabidopsis detached leaves with conidiospores and evaluating disease symptoms and fungal proliferation. N-isobutyl decanamide application significantly reduced necrosis caused by the pathogen and inhibited fungal proliferation. Arabidopsis mutants jar1 and coi1 altered in JA signaling and a MAP kinase mutant (mpk6), unlike salicylic acid- (SA) related mutant eds16/sid2-1, were unable to defend from fungal attack even when N-isobutyl decanamide was supplied, indicating that alkamides could modulate some necrotrophic-associated defense responses through JA-dependent and MPK6-regulated signaling pathways. Our results suggest a role of alkamides in plant immunity induction.

Figure 1. Overview of N-isobutyl decanamide responsive genes in Arabidopsis seedlings.

Number of genes (vertical axis) Up-regulated (red) and Down-regulated (blue) by N-isobutyl-decanamide treatment at 1, 3, 7, and 14 d.a.t. (A). Edwards-Venn diagrams showing common or distinct responsive genes identified at every time evaluated (B). The number of genes up- or down-regulated in a single condition is shown in bold letters. The number of genes regulated at all sampled-times are shown in bold italic font. Agglomerative hierarchical clustering of differentially expressed genes at every sampled times (C). Clustering was performed using the Smooth correlation and average linkage clustering in GeneSpring GX 7.3.1 software (Agilent Technologies®). Blue color indicates Down-regulated, red Up-regulated and white unchanged values, as shown on the color scale at the right side of the figure.

Figure 2. Functional classification of Arabidopsis genes differentially expressed in response to N-isobutyl decanamide treatment.

Categorization of 1,281 genes regulated by treatment was obtained according to the Munich Information Center for Protein Sequences classification (MIPS) classification using FunCat database (http://mips.helmholtz-muenchen.de/proj/funcatDB/) and Arabidopsis annotation. Statistically significant categories were identified by using a hypergeometric method and Bonferroni correction with a cutoff of p-value >0.05 (A). MIPS defense-related subcategories significantly represented (B). Percentages relate to total differentially regulated genes per sampled times. The average transcriptional change of all induced genes in overrepresented functional categories was also calculated along time of treatment (C).

Figure 3. Effects of N-isobutyl decanamide on defense-related metabolite production and PR1 expression.

Arabidopsis seedlings were grown for 6 days on N-isobutyl decanamide-free medium and transferred to control plates with or without N-isobutyl decanamide for 7 additional days. Salicylic acid (SA) accumulation was determined by measuring free and conjugated SA by GC-MS (A). Benzoic acid was used as internal standard, data are means of three independent experiments ± SD. Transgenic Arabidopsis line carrying PR1∶GUS was stained for GUS expression (B). Detection of hydrogen peroxide (H₂O₂) was made by staining leaves from control and treated seedlings with DAB (C). Images were captured with a Nomarski microscope. Nitric oxide (NO) from leaves of control and treated seedlings was detected by analyzing fluorescent signal of DAF-2DA with a confocal microscope (D). All photographs are representative individuals from at least 9 seedlings analyzed.

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.

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 ± 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 µM N-isobutyl decanamide, and then stained for GUS expression (B). Quantitative real-time PCR (qRT-PCR) analysis of nine JA-responsive genes using C_T 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 ± SE from three independent biological replicates, and there were four technical replicates for qRT-PCR assay.

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 µM N-isobutyl decanamide containing plates (black squares), transferred to decanamide-free plates and then inoculated with a 10 µl droplet of B. cinerea spores (5×10⁵ conidiospores/ml). The percentage of leaves with necrotic symptoms at 3, 4 and 5 days after inoculation was determined (A). Bars mean ± 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 ± 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).

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 µl 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 µM N-isobutyl decanamide containing plates, dipped into a B. cinerea inoculum of 5×10⁵ 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.