The plastidial metabolite 2-C-methyl-D-erythritol-2,4-cyclodiphosphate modulates defence responses against aphids

Abstract

Feeding by insect herbivores such as caterpillars and aphids induces plant resistance mechanisms that are mediated by the phytohormones jasmonic acid (JA) and salicylic acid (SA). These phytohormonal pathways often crosstalk. Besides phytohormones, methyl-D-erythriol-2,4-cyclodiphosphate (MEcPP), the penultimate metabolite in the methyl-D-erythritol-4-phosphate pathway, has been speculated to regulate transcription of nuclear genes in response to biotic stressors such as aphids. Here, we show that MEcPP uniquely enhances the SA pathway without attenuating the JA pathway. Arabidopsis mutant plants that accumulate high levels of MEcPP (hds3) are highly resistant to the cabbage aphid (Brevicoryne brassicae), whereas resistance to the large cabbage white caterpillar (Pieris brassicae) remains unaltered. Thus, MEcPP is a distinct signalling molecule that acts beyond phytohormonal crosstalk to induce resistance against the cabbage aphid in Arabidopsis. We dissect the molecular mechanisms of MEcPP mediating plant resistance against the aphid B. brassicae. This shows that MEcPP induces the expression of genes encoding enzymes involved in the biosynthesis of several primary and secondary metabolic pathways contributing to enhanced resistance against this aphid species. A unique ability to regulate multifaceted molecular mechanisms makes MEcPP an attractive target for metabolic engineering in Brassica crop plants to increase resistance to cabbage aphids.

Keywords: Arabidopsis; aphid resistance; indole glucosinolates; phloem-sucking herbivores; phytohormone signalling; retrograde signalling; secondary metabolites.

Figure 1

Scheme of methyl‐D‐erythritol‐4‐phosphate (MEP) pathway in Arabidopsis thaliana. The main MEP pathway is indicated in black. The MEP‐related pathway is indicated in grey. Abbreviation: DXS, 1‐deoxy‐D‐xylulose‐5‐phosphate synthase; DXP, 1‐deoxy‐D‐xylulose 5‐phosphate; DXR, 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase; MEP, methyl‐D‐erythritol‐4‐phosphate; MCT, 2‐C‐ methyl‐D‐erythritol‐4‐phosphate cytidylytransferase; CDP‐ME, 4‐(cytidine 5′‐diphospho)‐2‐C‐methyl‐D‐erythritol; CMK, 4‐(cytidine 5′‐diphospho)‐2‐C‐methyl‐D‐erythritol kinase; CDP‐MEP, 4‐(cytidine 5′‐diphospho)‐2‐C‐methyl‐D‐erythritol‐2,4‐cyclodiphosphate; MDS, 2‐C‐methyl‐D‐erythritol‐2,4‐cyclodiphosphate synthase; MEcPP, 2‐C‐methyl‐D‐erythritol‐2,4‐cyclodiphosphate; HDS, 2‐(E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate synthase; HMBPP, 2‐(E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate; HDR, (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate reductase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; ME, methyl‐D‐erythritol, ME‐1‐glc, 2‐C‐methyl‐d‐erythritol‐O‐1‐β‐d‐glucopyranoside, ME‐4‐glc, 2‐C‐methyl‐d‐erythritol‐O‐4‐β‐d‐glucopyranoside

Figure 2

Effects of MEcPP on plant resistance to the cabbage aphid Brevicoryne brassicae in Arabidopsis. Impact of MEcPP on plant resistance to the cabbage aphid was determined by bioassay and the relative transcript expression of selected genes. (a) Average of aphid progeny (mean ± SE; n = 20) after 9 days of feeding on WT, hds3, or 35S:HDS (hds3) plants. The kinetics of relative transcript expression of (b) HDS, (c) the SA biosynthetic marker gene ICS1, (d) the SA‐responsive marker gene PR1, and (e–f) genes induced by B. brassicae aphid infestation (ERF and PAD4) from leaf tissue of each plant type. Values represent average expression ± SE (n = 5) of each gene relative to the relative transcript expression of the ELONGATION FACTOR‐1a (EF1a) gene. Aphid progeny and relative transcript expression were compared among WT, hds3, and 35S:HDS (hds3) plants by one‐way ANOVA followed by Tukey's honestly significant difference (HSD) posthoc test. Different letters indicate significant differences among plant genotypes for each time point separately (P ≤ 0.05) [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

Plant resistance against the specialist caterpillar Pieris brassicae, and relative transcript expression of selected genes in phytohormonal signalling pathways. (a) Fresh body mass (mean ± SE; n = 20) of P. brassicae caterpillars after 4, 7, and 9 days of feeding on WT, hds3, or 35S:HDS (hds3) plants. Time course of expression of (b) HDS, (c) a salicylic acid (SA) biosynthetic gene (ICS1), (d) jasmonic acid (JA) biosynthetic gene (AOS), (e) a JA‐responsive marker gene (VSP2), and (f) a JA‐inducible transcription factor gene (ERF1). The value at each time point represents mean ± SE (n = 5) of HDS, ICS1, AOS, VSP2, and ERF1 transcript levels relative to those of the ELONGATION FACTOR‐1a (EF1a) gene. Body mass and transcript expression were compared among WT, hds3, and 35S:HDS (hds3) plants by one‐way ANOVA followed by Tukey's honestly significant difference (HSD) posthoc test. Different letters indicate significant differences among plant genotypes for each time point separately (P ≤ 0.05)

Figure 4

Feeding by Brevicoryne brassicae aphids induces MEcPP‐ and MEcPP‐related metabolite levels in Arabidopsis. The levels of (a) MEcPP, (b) MEP, and (c) ME were quantified in undamaged (control) and aphid‐damaged WT, hds3, and 35S:HDS (hds3) leaves by HPLC/MS. The levels (mean ± SE; n = 5) of MEcPP‐ and MEcPP‐related metabolites were compared within each plant type by Student's t test; asterisks represent significant difference (P ≤ 0.05). Abbreviations: DM, dry matter; MEcPP, 2‐C‐methyl‐D‐erythritol‐2,4‐cyclodiphosphate; MEP, methyl‐D‐erythritol‐4‐phosphate; ME, methyl‐D‐erythritol [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

Levels of selected glucosinolates in WT, hds3, and 35S:HDS (hds3) leaves of uninfested control plants and plants infested with Brevicoryne brassicae aphids for 7 days. Aliphatic and indolic glucosinolates were quantified by HPLC/MS. Levels (means ± SE; n = 5) of (a) 4MSOB, (b) 4MTB, (c) I3M, and (d) 4MO‐I3M in undamaged leaves or leaves of plants after 7 days of aphid feeding. The levels of 4MSOB, 4MTB, I3M, and 4MO‐I3M were compared among WT, hds3, and 35S:HDS (hds3) plants by one‐way ANOVA followed by Tukey's honestly significant difference (HSD) posthoc test. Different letters indicate significant differences among plant genotypes, lower case letters for control plants and upper case letters for aphid‐infested plants (P ≤ 0.05). Abbreviations: DM, dry matter; 4MSOB, 4‐methylsulfinylbutyl GLS; 4MTB, 4‐methylthiobutyl GLS; I3M, indole‐3‐yl methyl GLS; 4MO‐I3M, 4‐methoxy‐indole‐3‐yl‐methyl GLS [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Relative transcript abundance of selected genes involved in indole glucosinolate metabolism, after Brevicoryne brassicae aphid feeding or after exogenous application of MEcPP. Relative transcript expression (means ± SE; n = 5) of (a) a regulator of indolic GLS (a) MYB51 transcription factor and (b) CYP81F2, coding for a cytochrome P450 monooxygenase that catalyses the conversion of I3M into 4MO‐I3M for WT, hds3, and 35S:HDS (hds3) plants at designated time points of aphid infestation are presented. Transcript expression levels of (c) MYB51 transcription factor and (d) CYP81F2 in wild type leaf tissues was determined at 2 hr after exogenous application of synthetic MEcPP or ME. Transcript expression levels after aphid infestation or after exogenous application of MEcPP are compared by one‐way ANOVA followed by Tukey's honestly significant difference (HSD) posthoc test. Different letters indicate significant differences among plant genotypes (P ≤ 0.05). Abbreviation: MEcPP, 2‐C‐methyl‐D‐erythritol‐2,4‐cyclodiphosphate; ME, methyl‐D‐erythritol; DM, dry matter [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 7

Differential transcript expression of PAD3 and TPS11 in hds3 mutant plants. Relative transcript levels (means ± SE; n = 5) of (a) camalexin biosynthetic gene (PAD3), (b) a key gene in trehalose metabolisms (TPS11). Transcript expression were determined in WT, hds3, and 35S:HDS (hds3) leaves. They were compared among plant types at each time point by one‐way ANOVA followed by Tukey's honestly significant difference (HSD) posthoc test. Different letters indicate significant differences among plant genotypes (P ≤ 0.05). Abbreviations: DM, dry matter; PAD3, PHYTOALEXIN DEFICIENT3; TPS11, TREHALOSE‐6‐PHOSPHATE SYNTHASE11 [Colour figure can be viewed at http://wileyonlinelibrary.com]