Insect eggs trigger systemic acquired resistance against a fungal and an oomycete pathogen

Abstract

Plants are able to detect insect eggs deposited on leaves. In Arabidopsis, eggs of the butterfly species Pieris brassicae (common name large white) induce plant defenses and activate the salicylic acid (SA) pathway. We previously discovered that oviposition triggers a systemic acquired resistance (SAR) against the bacterial hemibiotroph pathogen Pseudomonas syringae. Here, we show that insect eggs or treatment with egg extract (EE) induce SAR against the fungal necrotroph Botrytis cinerea BMM and the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. This response is abolished in ics1, ald1 and fmo1, indicating that the SA pathway and the N-hydroxypipecolic acid (NHP) pathway are involved. Establishment of EE-induced SAR in distal leaves potentially involves tryptophan-derived metabolites, including camalexin. Indeed, SAR is abolished in the biosynthesis mutants cyp79B2 cyp79B3, cyp71a12 cyp71a13 and pad3-1, and camalexin is toxic to B. cinerea in vitro. This study reveals an interesting mechanism by which lepidopteran eggs interfere with plant-pathogen interactions.

Keywords: Botrytis cinerea; Pieris brassicae; herbivore interactions; indolic metabolism; insect eggs; plant; systemic acquired resistance (SAR).

Fig. 1

Oviposition and treatment with egg extract (EE) reduce Botrytis cinerea BMM infection. (a) Experimental design. (b–d) Effect of 5 d‐pretreatment with Pieris brassicae oviposition (Ovi) (b), P. brassicae EE (c), or Spodoptera littoralis EE (d) on B. cinerea growth. Lesion perimeter in distal leaves was measured 3 d after inoculation. Inoculated plants without pretreatment were used as controls (CTL). (e) Plants were pretreated with either P. brassicae EE or a 5 µg µl⁻¹ solution of phosphatidylcholine (PC)‐mix from chicken egg. Respective controls consisted of CTL plants or plants treated with a mock solution (Mock). (f) Egg extract pretreatment reduced B. cinerea growth in distal leaves when compared to CTL plants grown separately (intraplant systemic acquired resistance (SAR)). No difference in B. cinerea growth was observed when EE‐treated and untreated neighbor plants (NB) were in the same pot (interplant SAR). Means ± SE of three independent experiments are shown (n = 8–30 per experiment). Significant differences between control and treated plants are indicated (linear mixed model; ***, P < 0.001). Lowercase letters indicate significant difference at P < 0.05 (linear mixed model and post‐hoc general linear hypothesis test with Tukey contrasts). Dots indicate individual values.

Fig. 2

Egg extract‐induced systemic acquired resistance depends on the salicylic acid pathway. (a–c) Plant genotypes were pretreated with Pieris brassicae egg extract (EE) for 5 d and further infected with Botrytis cinerea BMM for 3 d. Lesion perimeter was measured in control (CTL) and distal leaves from EE‐treated plants (EE). The double mutant ics1 ics2 was homozygous for ics1 (^−/−) and heterozygous for ics2 (^−/+). (d) Local leaves (1°) were untreated (CTL), treated with EE for 5 d (EE) or infiltrated with Pseudomonas syringae pv tomato DC3000 (Pst) for 2 d. Distal leaves (2°) were then inoculated with B. cinerea spore suspension (B.c.) for 3 d before lesion perimeter measurement. Means ± SE of three independent experiments are shown (n = 8–28 per experiment). Lowercase letters indicate significant difference at P < 0.05 (linear mixed model and post‐hoc general linear hypothesis test with Tukey contrasts). Dots indicate individual values.

Fig. 3

Egg extract (EE)‐induced systemic acquired resistance depends on the N‐hydroxy‐pipecolic acid pathway. (a) Plant genotypes were pretreated with Pieris brassicae EE for 5 d and further infected with Botrytis cinerea BMM for 3 d. Lesion perimeter was measured in control (CTL) and distal leaves from EE‐treated plants (EE). Means ± SE of three independent experiments are shown (n = 8 per experiment). Lowercase letters indicate significant difference at P < 0.05 (linear mixed model and post‐hoc general linear hypothesis test with Tukey contrasts). (b) Plant genotypes were pretreated with P. brassicae EE for 5 d and further infected with B. cinerea. H₂O or 1 mM pipecolic acid (Pip) was applied to the soil 1 d before infection, and lesion perimeter measurements were recorded 3 d after infection. Means ± SE of three independent experiments are shown (n = 6–8 per experiment). For each genotype, different lowercase letters indicate significant differences at P < 0.05 (ANOVA followed by Tukey’s Honest Significant Difference test). Dots indicate individual values.

Fig. 4

Establishment of egg extract (EE)‐induced systemic acquired resistance requires tryptophan‐derived compounds. (a) Simplified scheme of biosynthesis of tryptophan derivatives and position of biosynthesis (red) and regulatory (blue) genes tested in this study. Brackets indicate an unstable intermediate. Several arrows indicate multiple steps. 4‐OH‐ICN, 4‐hydroxy‐ICN; IAN, indole‐3‐acetonitrile; IAOx, indole‐3‐acetaldoxime; ICA, indole‐3‐carboxylic acid; ICHO, indole‐3‐carbaldehyde; ICN, indole carbonyl nitrile; L‐Trp, tryptophan. (b–f) Plant genotypes were pretreated with Pieris brassicae EE for 5 d and further infected with Botrytis cinerea BMM for 3 d. Lesion perimeter was measured in control (CTL) and distal leaves from EE‐treated plants (EE). Means ± SE of three independent experiments are shown (n = 8–21 per experiment). Lowercase letters indicate significant difference at P < 0.05 (linear mixed model and post‐hoc general linear hypothesis test with Tukey contrasts). Dots indicate individual values. The ‘tmyb’ label on the x‐axis in part (c) represents myb34 myb51 myb122.

Fig. 5

Camalexin accumulation after egg extract and Botrytis cinerea BMM treatment. Plant genotypes were pretreated with Pieris brassicae egg extract (EE) for 5 d, and distal leaves were further treated with a mock solution or infected with B. cinerea. Untreated plants were used as controls (CTL). (a,b) Camalexin concentrations were measured in distal leaves. Means ± SE of three independent experiments are shown (n = 10–12 per experiment). For each time point, different letters indicate significant differences at P < 0.05 (ANOVA followed by Tukey’s Honest Significant Difference test). (c) In vitro growth inhibition assay. Radial growth of a B. cinerea colony growing on potato dextrose agar plates supplemented with different concentrations of camalexin or indole‐3‐carboxylic acid (ICA) was measured after 24 h of incubation. Solid line, mean; shaded band, SE (n = 12). This experiment was repeated twice with similar results.

Fig. 6

Pieris brassicae larval development is inhibited in Botrytis cinerea BMM‐infected plants. Plants were sprayed with a suspension of B. cinerea spores (B.c.) or mock solution (control; CTL). Freshly hatched P. brassicae were then placed on plants for a total of 12 d. Newly infected plants were placed every 3 d, in order to have sufficient material for the larvae to feed on. Larval weight was recorded after 6 and 12 d. Means ± SE are shown (n = 22–43). Significant differences between control and infected plants are indicated (Welch’s two sample t‐test; ***, P < 0.001; ns, not significant). This experiment was repeated twice with similar results. Dots indicate individual values.

Fig. 7

Egg extract‐induced systemic acquired resistance reduces Hyaloperonospora arabidopsidis Noco2 infection. Effect of 1 d‐pretreatment with Pieris brassicae egg extract (EE) on H. arabidopsidis infection in distal leaves was measured 8 d after inoculation. Inoculated plants without pretreatment were used as controls. Percentage of systemically infected plants (a) or number of spores on systemic leaves (b) were quantitated. Mean values ± SE of three independent experiments are shown (n = 4–5 per experiment). Significant differences between control and treated plants are indicated ((a) Pearson's chi‐squared test; (b) Welch’s two sample t‐test; ***, P < 0.001; ns, not significant).