Black mustard (B. nigra L.) accessions WUR-01 and WUR-02 used, originated from plants that were collected near the river Rhine in Wageningen, Netherlands (N51.96, E05.68). Brassica rapa L. genotypes used in this study (L58, R-o-18, RC-144) were obtained from the Laboratory of Plant Breeding (WUR). Plants were grown in a greenhouse (18 ± 5°C, 50–70% RH, L16, D8) and were used when three to five  weeks old.

Pieris brassicae L. (Lepidoptera: Pieridae) was reared on Brussels sprouts plants (Brassica oleracea var. gemmifera cv. Cyrus) in a climate room (21 ± 1°C, 50%–70% RH, L16: D8) at the Laboratory of Entomology, Wageningen University. Virgin adult females were obtained by isolating female butterflies immediately after eclosion. Otherwise, twenty females and males could mate in a large cage (60 × 60 × 90 cm) and females were used for oviposition in experiments or oviposition on filter paper (grade 3 hw, Sartorius, Germany) for egg wash production (see section below). The cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae) was reared on Brussels sprouts plants in a climate room (21 ± 1°C, 50%–70% RH, L16: D8) at the Laboratory of Entomology, Wageningen University.

The protocol for preparation of egg washes has been newly developed to prepare egg wash free from leaf-surface related chemical compounds which enables us to conduct chemical analysis and identify possible EAMPs. Full procedure is described in Supplementary Data (Supplementary Figure S1). Pieris brassicae eggs were collected on filter paper pinned underneath a B. oleracea leaf in a cage containing 20 mated females. Egg clutches laid on the paper were cut out and submersed in 1 mL 2-(N-morpholino)ethanesulfonic acid (Mes) buffer per 400 eggs, overnight (16 h) without disturbance. Previously, we have tested different buffers and found that using Mes buffer resulted in the most consistent plant response (data not shown). The solution (egg wash) was pipetted into a new tube the next morning.

To obtain egg-enveloping secretions for wash of only egg glue, 1-day old P. brassicae eggs were collected as above, counted, and then carefully removed from the paper using a brush. The spots of secretions underneath eggs were then cut out and washed overnight (16 h) in 1 mL 20 mM Mes buffer (pH 5.7) per 400 eggs. A wash of the pieces of the same filter paper without secretions was used as control.

To remove egg-enveloping secretions from eggs, eggs on paper were submersed in 1 mL per 400 eggs of either a solution of 1% bleach and 2% Tween-20 or in 250 mM NaPO₄ pH 9.0 for 30 min (two different treatments that were shown to remove egg glue; Jacobs et al., 2013). After treatment, eggs were rinsed once with 1 mL MILLI-Q^® water (Sigma-Aldrich, MO, United States), hereafter “MQ water,” and then 1 mL Mes buffer and then washed overnight (16 h) in 20 mM Mes buffer pH 5.7. Eggs that were used to compare where egg-enveloping secretions were not removed, were submersed in Mes for 30 min, rinsed in MQ and Mes, and then washed.

To study the induction by eggs of different ages, eggs were collected as above on paper, and then kept at room temperature until they were washed at the end of each day, until they hatched (6–7 days after oviposition). After hatching, young caterpillars were carefully removed using a brush. Empty eggshells and associated secretions (on paper) were then washed overnight (16 h).

To collect unfertilized eggs for egg wash, filter paper was pinned underneath a B. nigra leaf of a plant placed in a cage with 10 virgin adult females and then prepared in a similar way as fertilized eggs.

Paper sheets with M. brassicae eggs were obtained from a rearing population from the Laboratory of Entomology (WUR). To obtain egg wash, eggs were counted, spots with eggs were cut out and washed in 1 mL 20 mM Mes buffer (pH 5.7) per 400 eggs. The wash was pipetted off the next morning and frozen at −20°C until use.

For all experiments testing the effects of treating plants with egg washes, 10 μL egg wash (i.e., an equivalent of 25 eggs) was pipetted on the abaxial side of the fourth or fifth emerged leaf of 3–4 weeks old B. nigra plants, unless otherwise specified. Symptoms induced by egg wash were scored 4 days after treatment. To quantify severity, a scoring system from 0 to 4 was adapted from Griese et al. (2017). Severity score 0: no visual response; score 1: brown spots underneath eggs or egg wash spot, only visible at abaxial side leaf; score 2: cell death also visible at adaxial side of leaf, spot smaller than 2 mm diameter, score 3: cell death the size of egg wash spot, and score 4: spreading lesion beyond spot of treatment (Supplementary Figure S1). Score 0 and 1 are classified as “non-HR,” score 2–4 are classified as “HR”.

For experiments with oviposition on plants, one mated female P. brassicae butterfly was placed in a cage with a B. nigra or B. rapa plant and removed when ~10–25 eggs were laid. HR-like cell death was scored 4 days after oviposition, using the same scoring system as with egg wash. For experiments with M. brassicae, five mated female moths were placed together with a B. nigra plant in a cage to allow egg deposition overnight (16 h) and removed in the morning. No visible cell death was observed under M. brassicae eggs as was also reported in our previous studies (Fatouros et al., 2012; Griese et al., 2021).

For histochemical staining of plant tissue underneath eggs, P. brassicae egg-laden plants were sampled 24, 48, and 72 h after oviposition by taking a 10 mm diameter leaf disk of the area surrounding the eggs. Similarly this was done with plants treated with egg wash. Eggs were carefully removed with a brush before proceeding with histochemical staining. Pictures of the leaf disks were taken with a Dino-Lite digital microscope (AnMo Electronics Corporation, Taiwan) before staining (with eggs) and after staining (without eggs). As cell death is generally preceded by production of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion (Torres, 2010), we visualized ROS with different histochemical stainings. 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, MO, United States) was used to stain hydrogen peroxide (H₂O₂). Leaf disks (10 mm Ø) were submersed in 1 mg/mL DAB solution and samples were incubated for 30–60 min in the dark. Nitroblue tetrazolium chloride (NBT; Sigma-Aldrich, MO, United States) was used to stain superoxide radical O₂^•−. For this leaf disks (10 mm Ø) were submersed in 0.2% NBT with 50 mM sodium phosphate buffer (pH 7.5) and samples were incubated 30–60 min in the dark. For visualization of cell death, leaves were submersed in 0.08% trypan blue solution (Sigma-Aldrich, MO, United States), overnight (16 h). For all three stainings, destaining of leaves was performed with 96% ethanol. Pictures of leaf disks before staining (with eggs) and after staining (without eggs) were taken with a Dino-Lite digital microscope (AnMo Electronics Corporation, Taiwan). For staining of callose, leaf disks were first destained and then submersed in 0.01% aniline blue (Sigma-Aldrich, MO, United States) with 150 mM K₂HPO₄ and imaged after at least 2 h of incubation using a DAPI filter on a fluorescence microscope equipped with a DS-5MC camera and NIS elements AR 2.30 software.

In female butterflies, eggs are produced in the ovaries, pass through the common oviduct and are fertilized by sperm released from the bursa copulatrix (BC) into the vagina. Before being expelled through the ovipore, the eggs are covered by secretions released from the accessory reproductive gland (ARG), a paired gland that contains egg-enveloping secretions and glue-like material to attach eggs to leaves (Supplementary Figure S2). For dissection of tissues from the female reproductive tract, namely ARGs and BCs or eggs from the ovarian tubules (hereafter termed “ovarian eggs”), mated P. brassicae females were obtained by pairing a virgin female and virgin male 1 day after eclosion, which were kept together until mating was observed. Virgin females were obtained by keeping them separately shortly after eclosion. Three to four days after eclosion, mated and virgin P. brassicae females were killed by beheading using a scalpel and then dissected. ARGs, BC and ovarian eggs were dissected from females under a stereomicroscope (optical magnification 20×) in 20 mM Mes buffer. Dissected structures were washed overnight (16 h) in 20 mM Mes pH 5.7 using 50 μL per each ARG, 100 μL buffer per each BC or 5 μL buffer per egg. Solution was pipetted off the next morning and frozen until use (modified after Fatouros et al., 2015).

To characterize the chemical nature of EAMP in the egg wash we exposed it to different treatments. Untreated egg wash was used as a control. To study the stability of the elicitor, the egg wash was heated and frozen. For heating, egg wash was made as described above, collected in microcentifuge tubes and boiled in a water bath for 30 min at 95°C. For freezing treatment, egg wash was frozen at −20°C and then thawed for 1 h at room temperature, then frozen again. Freezing–thawing cycles were repeated up to four times, after which, all washes were used to treat plants. To study if the elicitor was an intact protein, a proteinase treatment was performed, a stock of 20 mg/mL Proteinase K (Qiagen, Cat No./ID: 19133) was diluted 100× in 100 μL egg wash (10% volume, pH 7.2) and incubated with egg wash for 30 min at 37°C.

Phosphatidylcholines (PCs) were recently found to be the main components of P. brassicae egg extracts that show a bioactivity in A. thaliana Col-0 resulting in induction of SA and PR1 marker gene as well as ROS accumulation and cell death formation (Stahl et al., 2020). Thus, we tested whether the same PCs used in Stahl et al. (2020) may be associated with development of HR-like cell death by B. nigra. Phospholipids were ordered from Avanti Polar Lipids (Alabaster, Alabama, United States). Specifically, PC(16:1/16:1), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, catalog no. 850358 and PC(18:1/18:1), 1,2-dioleoyl-sn-glycero-3-phosphocholine, catalog no. 850357 were used for experiments. Phospholipid stock solutions were made in 100% MeOH. For phospholipid application, the MeOH was evaporated under a nitrogen-flux and the phospholipids were dissolved in MQ water with 1% DMSO, 0.5% glycerol, 0.1% Tween 20 by sonication. Phospholipids were applied on B. nigra leaves at different concentrations (1, 5 and 10 μg/μL) that were previously shown to have biological activity in A. thaliana (Stahl et al., 2020). An MQ water solution of 1% DMSO, 0.5% glycerol and 0.1% Tween 20 was used as control.

Ethylene production is regularly measured to characterize the induction of early plant immune response by biotic stresses (Fan et al., 2017). Ethylene was measured as previously published (Oome et al., 2014). To measure the plant production of ethylene, in B. nigra, ten plants for each accession (with and without HR-like phenotype) were used. For B. rapa, three plants of each genotype were used. For each plant, leaf disks (3 mm Ø) were sampled from mature leaves of untreated plants and incubated overnight (16 h) in demineralised water. Subsequently, three leaf disks for each biological replicate (a single plant) were randomly chosen and incubated for 5 h in air tight glass vials with in either 400 μL of 20 mM Mes pH 5.7 or 400 μL egg wash (400 eggs/mL in same Mes buffer). After incubation, 1 mL of the headspace of each sample was taken to measure ethylene concentration on a Focus gas chromatograph (Thermo Electron S.p.A., Milan, Italy) equipped with an FID detector and a RT-QPLOT column, 15 m × 0.53 mm ID (Restek, Bellefonte, PA, United States). The system was calibrated with a certified gas of 1.01 μL L⁻¹ (1 ppm) ethylene in synthetic air (Linde Gas Benelux B.V., Schiedam, Netherlands). After sampling leaf disks for the ethylene assay, plants were also treated with P. brassicae egg wash to determine their HR-like cell death phenotype.

To compare gene expression induced by eggs and egg wash, B. nigra plants were treated with either 10 μL of egg wash at lower concentration (400 eggs/mL in Mes buffer) or oviposited with an egg clutch of 10 eggs. Six leaf disks (Ø 6 mm) were sampled with a leaf puncher directly next to the eggs or the egg wash-treated spot at 0, 3, 6, 24, and 48 h, as done in previous research (Fatouros et al., 2014; Griese et al., 2021). For each timepoint, four plants were sampled individually and considered biological replicates. Brassica rapa plants were treated with three single eggs on a single leaf of each plant. Leaf disks (Ø 6 mm) were then harvested next to the eggs at 0, 3, 6, 24, and 96 h after treatment. For each timepoint, three plants were sampled individually and considered biological replicates.

To compare gene expression between P. brassicae and M. brassicae egg wash, B. nigra plants were treated with 10 μL of either egg wash (400 eggs/mL in Mes buffer) or a negative control (Mes buffer). Six leaf disks (Ø 6 mm) were sampled directly next to the egg wash-treated spot 24 h after treatment. Four plants were used for each treatment as biological replicates.

To compare the gene expression of two B. nigra accessions with contrasting ability to develop HR-like cell death, egg wash at higher concentration (~1,000 eggs/mL in demineralised water) was used. Experimental design consisted of two treatments (egg wash, control), two B. nigra accessions (WUR-01 without HR, and WUR-02, developing HR), and three time points after treatment (6, 24, and 48 h after treatment). For each treatment combination, egg wash or control were applied with two droplets of 5 μL on the abaxial side of a single leaf. Leaf disks (Ø 6 mm) were harvested at each time point on the treatment spots and disks from the same treatment on a leaf were pooled. For each treatment combination, a total of 15 plants were used and groups of 3 plants with similar treatments were pooled to compose a total of 5 biological replicates. For each gene expression experiment, samples were snap frozen in liquid nitrogen and stored at −80°C until use.

We investigated to which extent the difference in HR-like phenotype between B. nigra accessions WUR-01 and WUR-02 was related to activation of different phytohormones associated with plant immunity. Thus, we quantified expression of genes induced by SA, such as ICS1, PR1, and PR2 (Little et al., 2007), and genes regulated by JA, i.e., MYC2, VSP1, and VSP2 (Reymond et al., 2004). For experiments on gene expression induced by eggs or egg wash in both B. nigra and B. rapa, RNA extraction was performed according to Oñate-Sánchez and Vicente-Carbajosa (2008). For experiments on gene expression induced by egg wash in two B. nigra accessions, RNA extraction was performed with Direct-zol RNA Miniprep Kit (Zymo Research, CA, United States) following the manufacturer’s protocol. For preparation of cDNA of all experiments, 1 μg of RNA was reverse-transcribed using SensiFAST cDNA synthesis kit (Bioline, United Kingdom) according to the manufacturer’s instructions. Real time qRT-PCR reactions were performed using SensiFAST SYBR No-ROX Kit (Bioline, United Kingdom) in 10 μL reaction volumes, containing 3 μL cDNA and 500 nM of primers on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, CA, United States). The following qRT-PCR program was used: 95°C for 2 min followed by 40 cycles of 95°C for 5 s; primer-specific annealing temperature for 5 s and 72°C for 10 s, with data collection at 72°C, followed by a melt curve analysis. Relative gene expression was calculated with the ΔΔCq method, using GAPDH as reference gene. Primers sequences are available in Supplementary materials (Supplementary Table S1).

All data analysis was carried out in R (R Core Team, 2020). Scoring of HR-like cell death in severity categories were analyzed with a non-parametric method (Kruskal–Wallis test) on HR scores (0, 1, 2, 3, and 4) and different treatments were included as categorical fixed factors. For all other statistical analyses involving comparison of mean values (gene expression, ethylene production), the choice of parametric or non-parametric methods was made after checking the assumptions of normality (Shapiro–Wilk normality test) and homogeneity of variances (Fligner–Killeen test) on the raw data. As parametric methods, Student’s T-test, Welch T-test and ANOVAs followed by Tukey’s honestly significant difference test were used. As non-parametric method, Kruskal–Wallis test followed by pairwise Wilcoxon rank sum test was used. Gene expression data from qRT-PCR were calculated with the ΔΔCt method (Livak and Schmittgen, 2001) and were analyzed on log2-transformed data (parametric test) or on raw data (non-parametric test) specifying time points or treatments as factors.

We first investigated cellular responses against Pieris brassicae oviposition in its natural host, the black mustard Brassica nigra. Reactive oxygen species (ROS) such as superoxide anion (O₂^•−) and hydrogen peroxide (H₂O₂) accumulated underneath eggs at 24 h (Figure 1A). At the same time point, aniline blue staining in B. nigra revealed also callose deposition underneath eggs (Figure 1A). Further, the occurrence of cell death in plant tissue under the eggs was investigated by staining with trypan blue (TB), showing that cell death occurred 72 h after oviposition (Figure 1B). Occasionally, a few TB-stained cells were visible at 48 h (not shown). In B. nigra, we regularly observed a strong, macroscopically visible HR-like cell death that spreads beyond the egg site and stops 96 h after oviposition (Figure 1C). Cellular responses against P. brassicae oviposition were also investigated in B. rapa. ROS accumulation and cell death formation were also detected in B. rapa at 24 h and 72 h after oviposition, respectively (Supplementary Figures S2A,B). Although these early cellular responses appeared similar to what was observed in B. nigra, the macroscopically visible HR-like cell death developed by B. rapa appeared as black necrotic spots that never spread beyond the egg site (Supplementary Figure S2C).

To isolate potential EAMPs and to quickly screen plants for HR-like cell death, we developed a method to dissolve egg-enveloping secretions from P. brassicae eggs (Methods section; Supplementary Figure S1). We then compared visually whether the treatment of leaves with egg wash induced a similar HR-like cell death as the butterfly eggs (Figure 2A). As a control, leaves were treated with only the buffer (no eggs were washed). Symptoms induced by eggs or egg wash were scored after 4 days, and HR-like frequency (proportion of plants showing HR-like) and HR-like severity (mean score of induced symptoms) were compared between the two treatments in B. nigra. HR-like severity after oviposition by eggs or treatment with egg wash did not differ (Kruskal–Wallis: χ² = 1.33, df = 1, p = 0.24; Figure 2B; Supplementary Table S2).

We compared expression of SA-marker gene PR1 in B. nigra oviposited on by P. brassicae butterflies or treated with egg wash. PR1 was significantly upregulated both after oviposition and after treatment with egg wash. In B. nigra, PR1 expression increased at 6 h and was significantly upregulated at 24 h and 48 h after egg deposition (Kruskal–Wallis: χ² = 12.23, df = 3, p = 0.015; Figure 2C). No significant differences in PR1 expression were found between B. nigra plants treated with eggs or egg wash (Figure 2C; Supplementary Table S3). Similarly, in B. rapa, PR1 expression was significantly upregulated by egg deposition at 24 h and showed further increase at 96 h (Kruskal–Wallis: χ² = 11.18, df = 4, p = 0.024; Supplementary Figure S2D).

Next, we tested whether egg wash induced ethylene. Brassica nigra leaves responded with higher ethylene production after incubation with egg wash compared to incubation with control Mes buffer (Figure 2D; Supplementary Table S4). In addition, there was a significant difference in ethylene produced between plants with contrasting HR-like phenotypes. Plants responding with stronger HR-like cell death (score 2 or higher), produced a significantly higher amount of ethylene after incubation with egg wash than plants with no HR-like cell death (Student’s T-test = −4.087, df = 18, p < 0.001; Figure 2D; Supplementary Table S4). Similarly, B. rapa plants developing HR-like cell death, also showed a higher ethylene production upon incubation of leaves with P. brassicae egg wash (Student’s T-test = −3.876, df = 4, p = 0.018; Supplementary Figure S3E). These results suggest that there is an early detection response in plants after contact with egg wash that will ultimately lead to cell death.

All SA marker genes were upregulated in B. nigra plants upon egg wash treatment compared to control. However, the magnitude of expression of SA-related genes was significantly different between the two B. nigra accessions at different time points (Figure 3). In contrast, relative expression of JA-related genes was generally very low and not significantly different between two accessions. SA-marker BnICS1 was significantly expressed at higher levels in plants that showed a visible HR already at 6 h after treatment (Welch’s T-test = −4.901, df = 5.3, p = 0.003). Both, BnPR1 and BnPR2 showed increased expression across the time points, although with a different magnitude between the two accessions. Both genes were already expressed at higher level at 6 h in plants that showed a visible HR (Welch’s T-test = −2.932, df = 5.3, p = 0.03 and Welch’s T-test = −4.850, df = 5.8, p = 0.003 respectively), and the difference with plants not expressing HR increased up to 5-fold (24 h) and 20-fold (48 h) for BnPR1 and 4-fold (24 h) and 5-fold (48 h) for BnPR2. The expression of JA-related genes showed more stable profiles across time points, with a small peak at 24 h in all treatments, but with no significant differences between the two accessions (MYC2, Welch’s T-test = −1.549, df = 5.8 p > 0.05; VSP1, Welch’s T-test = −0.141, df = 3 p > 0.05; VSP2, Welch’s T-test = −0.301, df = 3.5 p > 0.05).

Staining of leaves showed that plant cells did not die underneath M. brassicae eggs as leaves did not stain with trypan blue (Figures 4A,B). Further, M. brassicae eggs induced O₂^•− production in some plants, but weaker than Pieris eggs (Figures 4C,D). While P. brassicae egg wash induced the expression of PR1 after 24 h (ANOVA followed by Tukey, p < 0.01), PR1 expression induced by M. brassicae egg wash was not different from the control treatment (Figure 4C; Supplementary Table S5). In addition, incubation with M. brassicae egg wash induced lower ethylene production compared to P. brassicae egg wash (Kruskal–Wallis: χ² = 21.36, df = 2, p < 0.001; Figure 4D), comparable to the ethylene levels previously observed in B. nigra plants lacking a visible cell death (Figure 2D). Overall, eggs of M. brassicae did not induce cell death and only a weak ROS and ethylene response in B. nigra.

There was no significant difference in eliciting activity of eggs of increasing age, from one-day-old, to five-day-old eggs, although HR-like severity decreased slightly. Egg wash was also prepared from eggshells and secretions that remained on the paper after caterpillars hatched, and this still induced HR-like symptoms (Kruskal–Wallis: χ² = 3.20, df = 5, p > 0.1; Figure 5A; Supplementary Table S6).

A wash of dissected accessory reproductive glands (ARGs) induced HR-like cell death, similar to the positive control P. brassicae egg wash (Figure 5B). On the contrary, neither a wash of mature but unfertilized eggs dissected from the ovary (“ovarian eggs”) nor a wash of the sperm-containing bursa copulatrix (BC) induced symptoms (Figure 5B). HR-like severity was significantly higher in plants treated with egg wash or a wash of ARG, compared to wash of ovarian eggs or bursa copulatrix (Kruskal–Wallis: χ² = 19.833, df = 4, p < 0.001; Supplementary Table S6).

A wash from unfertilized, deposited eggs (containing secretions from the ARG) from virgin butterflies induced a similar response as wash from fertilized, deposited eggs of mated females (Figure 5C). There was no significant effect of the mating status of the female (mated or virgin) on the frequency of HR-like cell death elicited or on HR severity (Kruskal–Wallis: χ² = 2.61, df = 1, p > 0.1). In addition, the wash of ARGs from virgin females induced strong symptoms similar to those of mated females (Figure 5B). These results show that egg fertilization is not necessary for the induction of the HR-like cell death in B. nigra, and that an EAMP resides in the ARG, and is female-derived.

Finally, both egg wash and wash of glue alone induced a severe HR-like cell death, and HR-like severity was significantly lower when B. nigra was treated with a wash of eggs from which the glue was removed (Kruskal–Wallis: H = 26.60, df = 3, p < 0.001; Figure 5D; Supplementary Table S6).

None of the egg wash treatments conducted, i.e., freezing and thawing, proteinase K, or boiling, had an effect on its bioactivity. That the elicitor does not lose the capability to induce HR in B. nigra leaves after these treatments, indicates that it is a small, stable, water-soluble molecule that is likely not a protein (Figure 6; Supplementary Table S7).

Lastly, we tested whether two phosphatidylcholines (PC) could also induce a HR-like responses in B. nigra. We did not observe any visible cell death upon treatment of B. nigra with neither PC16:1/PC16:1 nor PC18:1/PC18:1 (Figure 7; Supplementary Table S8).