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First published online December 1, 2006; 10.1104/pp.106.090837 Plant Physiology 143:784-800 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Oviposition by Pierid Butterflies Triggers Defense Responses in Arabidopsis1,[W],[OA]Department of Plant Molecular Biology, University of Lausanne, 1015 Lausanne, Switzerland
Insect eggs represent a threat for the plant as hatching larvae rapidly start with their feeding activity. Using a whole-genome microarray, we studied the expression profile of Arabidopsis (Arabidopsis thaliana) leaves after oviposition by two pierid butterflies. For Pieris brassicae, the deposition of egg batches changed the expression of hundreds of genes over a period of 3 d after oviposition. The transcript signature was similar to that observed during a hypersensitive response or in lesion-mimic mutants, including the induction of defense and stress-related genes and the repression of genes involved in growth and photosynthesis. Deposition of single eggs by Pieris rapae caused a similar although much weaker transcriptional response. Analysis of the jasmonic acid and salicylic acid mutants coi1-1 and sid2-1 indicated that the response to egg deposition is mostly independent of these signaling pathways. Histochemical analyses showed that egg deposition is causing a localized cell death, accompanied by the accumulation of callose, and the production of reactive oxygen species. In addition, activation of the pathogenesis-related1::  -glucuronidase reporter gene correlated precisely with the site of egg deposition and was also triggered by crude egg extract. This study provides molecular evidence for the detection of egg deposition by Arabidopsis plants and suggests that oviposition causes a localized response with strong similarity to a hypersensitive response.
Plants under attack from herbivorous insects develop an array of defenses that are aimed at slowing the growth or development of the aggressor and specifically tailored to protect themselves from further attack. Numerous studies have revealed that, in addition to constitutive defenses represented by trichomes, thick secondary cell walls, or poisonous compounds, plants are equipped with inducible defenses that can be grouped into indirect (production of volatile odor blends to attract natural enemies of the attacker encountered) and direct responses (production of antidigestive proteins, toxic secondary compounds, and enzymes that affect insect growth and development; Karban and Baldwin, 1997  ; Walling, 2000; Dicke et al., 2003). In Arabidopsis (Arabidopsis thaliana), challenge with chewing herbivores has been shown to provoke substantial transcriptional changes largely controlled by the plant hormone jasmonic acid (JA; Reymond et al., 2004; De Vos et al., 2005).
One important aspect of plant/insect interaction that has received less attention is the potential recognition of insect egg deposition. Eggs represent a future threat for the plant and the anticipation of damage by a preactivation of defenses could provide an advantage to the host. Both direct and indirect responses to oviposition have been observed in plants (Hilker and Meiners, 2006
Indirect defenses induced by egg deposition comprise the release of volatiles or the modification of the surface chemistry by egg-laden leaves resulting in the attraction of egg parasitoids. Twigs of Scots pine (Pinus sylvestris) emit volatiles after oviposition by the pine sawfly Diprion pini, attracting the egg parasitoid Chrysonotomyia ruforum. This response was shown to be both local and systemic and could be mimicked by JA treatment (Hilker et al., 2002 There is thus evidence that plants are capable of detecting the presence of insect eggs and that they respond by activating direct and indirect defenses. However, there is no information on the molecular changes that take place in the host following oviposition. In this study, we investigated the response of Arabidopsis to oviposition by pierid butterflies and analyzed whole-genome transcript profiles in egg-laden leaves for a period of 3 d. The combined results of transcriptome analysis and visualization of defense-associated markers reveal that oviposition triggers a local response similar to a programmed cell death (PCD) accompanied by the activation of many defense-related genes.
Expression Changes in Response to Pieris brassicae Eggs
After mating, female Pieris brassicae butterflies lay eggs on the underside of leaves of Brassicaceae family plants. Approximately 10 to 40 eggs are gently deposited close to each other without visible physical damage. From 4 to 6 d after oviposition, eggs start to hatch, young larvae eat the egg chorion, and then start to eat the leaf around the oviposition site. We wanted to know whether molecular changes take place in Arabidopsis during the first days after oviposition, before egg hatching and thus before larvae start to feed on the plant. Using near full-genome microarrays, we investigated transcriptional changes 24, 48, and 72 h after oviposition by P. brassicae. On average, butterflies laid one to two egg batches per plant and we collected five leaf discs of approximately 5 mm in diameter at the appropriate times. RNA was extracted, amplified, labeled, and hybridized to Complete Arabidopsis Transcriptome MicroArray (CATMA) microarrays containing 22,473 gene-specific tags (Allemeersch et al., 2005 Eggs laid by P. brassicae triggered large changes in gene expression. A total of 303 genes were induced 24 h after oviposition, 416 after 48 h, and 671 after 72 h (Supplemental Table S1). In addition, oviposition caused a down-regulation of 53, 123, and 426 genes after 24, 48, and 72 h, respectively (Supplemental Table S1). This represents a maximal induction of approximately 4% and a repression of approximately 2% of the transcriptome. Analysis of the time course showed that the 56% of the genes that are induced at 72 h are already up-regulated at 24 or 48 h and that the expression of most genes gradually increased or stayed steady over the 3 d of measurements, indicating that the response is more likely due to the continuous presence of the eggs and not to a touch response to egg deposition. The majority of down-regulated genes (68%) was only repressed 72 h after oviposition (Supplemental Figs. S1–S3). It is possible that we have missed some early changes in gene expression but we observe that the response to oviposition corresponds to consistent and long-lasting transcriptional changes. In addition, we might have underestimated the number of differentially regulated genes after oviposition by using distal leaves as control tissue. As mentioned above, the rationale was to use leaves that had been in contact with butterflies as a control to eliminate the possibility of identifying genes responding to touch or butterfly derived chemicals. The limitation of this experimental design is that genes induced after oviposition at similar levels in oviposited leaves and in distal leaves will not be detected. However, one hybridization using leaf samples from plants that did not contain eggs indicated that the number of genes induced systemically is low (Supplemental Fig. S4).
We examined the potential function of differentially expressed genes and classified them according to gene ontology (GO) terms using the tools for GO annotations at The Arabidopsis Information Resource (http://www.arabidopsis.org; Berardini et al., 2004
The overlap between oviposition- and herbivory-repressed genes was even smaller (Fig. 1A ; Supplemental Table S1). Of the 10 genes repressed by both treatments, seven are annotated as expressed proteins with unknown function. The other three include an expansin (ATEXPA8, At2g40610), a germin (At5g20630), and a putative zinc-finger protein (At2g25900).
Looking into the list of induced genes in more details we identified several markers of hypersensitive response (HR), which is a particular case of PCD. HR is an induced response triggered by the specific recognition of bacterial pathogens, viruses, fungi, and nematodes and is characterized by a localized cell death at the site of infection that prevents the progression of the disease (Kombrink and Somssich, 1995 ). We thus compared the expression profile after oviposition by P. brassicae with the profile observed after an HR caused by the bacterial pathogen Pseudomonas syringae AvrRPM1 (data from AtGenExpress; http://www.weigelworld.org/resources/microarray), and with the expression changes triggered by lesion formation in acd2-2. This mutant has spontaneous spreading cell death lesions (Mach et al., 2001). We observed an important overlap between the three transcriptome signatures; 50% of the egg-induced genes, respectively, 69% of the repressed genes, were also differentially regulated during either a bacterial HR or in leaves developing spontaneous lesions, indicating that eggs trigger a transcriptional response sharing a lot in common with those observed during the programmed death of plant cells (Fig. 1B; Supplemental Table S1). Interestingly, we observed that most of the 75 genes induced both by oviposition and insect herbivory (Fig. 1A) were also induced in acd2-2 mutant (62/75) and in P. syringae infected plants (64/75), indicating that these genes are likely markers of a general stress response. Similarly, the majority of the 10 genes repressed both by oviposition and insect herbivory were also down-regulated in acd2-2 mutants (8/10) and by P. syringae treatment (8/10; Supplemental Table S1).
Egg deposition by P. brassicae caused the up-regulation of many defense-related genes (Table II
). Classical marker genes that accumulate during HR were induced, including pathogenesis-related (PR) genes PR2, PR3, PR4, and PR5, and regulators of innate immunity, like EDS1, PAD4, and SAG101. Some avirulence- or HR-responsive genes of unknown function were also up-regulated. Genes related to PCD included the antiapoptosis BAX-INHIBITOR-1 (Matsumura et al., 2003
In addition, we observed that 41 receptor-like kinases (RLKs) were induced by eggs from 24 to 72 h after oviposition. RLKs are transmembrane proteins containing an intracellular kinase domain and a variable extracellular domain thought to interact with different extracellular ligands. There are more than 600 RLKs in Arabidopsis (Shiu and Bleecker, 2001 ) and the role of only a few of them has been characterized, notably in development, growth, symbiosis, or defense (Zipfel and Felix, 2005). RLKs are known to be involved in the perception of pathogen-derived elicitors that activate defense responses. Recent studies with bacterial elicitors have shown that they induce more than 100 RLKs (Zipfel et al., 2004, 2006). By comparing our data with those of Zipfel et al. (2006) we found that 22 of the oviposition-induced RLKs were also induced after treatment with the bacterial elicitors flg22 or elf26 (Table III
).
Genes down-regulated by P. brassicae eggs consisted of genes involved in cell wall metabolism, cuticle biosynthesis, and fatty acid synthesis. This was accompanied by the repression of many genes that participate in photosynthetic activities (Table II).
The up-regulation of two representative genes by P. brassicae oviposition was verified by quantitative real-time PCR after 72 h. A chitinase gene (At2g43570) was highly induced at and near the oviposition site, but only very weakly in the distal leaf (Fig. 2 ). The expression of a trypsin inhibitor gene (At1g73260) showed a highly localized response to P. brassicae eggs, as the gene was more than 1,000-fold more expressed at the egg site than in the near, distal, or control samples. We also analyzed the expression of PR1 since it is a known defense marker gene involved in innate immunity. A probe for this gene was lacking in the current CATMA microarray. We found that PR1 is highly induced (60- to 70-fold compared to control plants) 72 h after oviposition, both at the site of oviposition and in a region surrounding the egg batch (Fig. 2). There was also a small induction in the distal leaf.
Role of JA and SA Pathways in Egg Responses
We next determined the contribution of known signaling pathways involved in defense in Arabidopsis. Given the importance of JA and SA in plant defense responses (Reymond and Farmer, 1998
We did not identify enrichment of particular functional categories among the genes that were differentially regulated by oviposition between coi1-1 and wild-type plants or between sid2-1 and wild-type plants. Moreover, most of the genes differentially regulated between mutant and wild-type plants were not the same in coi1-1 and in sid2-1 plants (Supplemental Table S2). An interesting exception consisted of two genes encoding NPR1-interacting proteins (NIMIN-1, At1g02450; NIMIN-2, At3g25882) that were significantly more induced by oviposition in coi1-1 and less induced in sid2-1. These proteins have been shown to modulate PR gene expression by negatively regulating distinct functions of NPR1 (Weigel et al., 2005 ). NPR1 plays a crucial role in the pathway cross talk between JA and SA (Spoel et al., 2003). We identified three germins in the list of genes induced 2-fold more in coi1-1 plants. Germins have oxalate oxidase activity leading to hydrogen peroxide (H2O2) production. Genes involved in cell wall remodeling exhibited also a similar expression bias, including a pectin methyl esterase (At3g47380), two endoxyloglucan transferases (At2g14620 and At4g18990), and a cellulase (At1g65610). Proteases and protease inhibitors were also enriched in this category, including two aspartyl proteases (At3g12700 and At5g37540), a Cys proteinase (At5g43060), a protease inhibitor (At3g22620), and a serpin (At2g26390; Supplemental Table S2).
Two known PR genes (PR2, At3g57260; HEL, At3g04720) and an avirulence-responsive gene (AIG2, At3g28930) were induced 2-fold more by oviposition in sid2-1 than in wild-type plants. On the opposite, five WRKY transcription factors were not or less induced by oviposition in sid2-1 plants (Supplemental Table S2). Accordingly, WRKY transcriptions factors have been implicated in the control of SA-dependent defense gene induction (Ulker and Somssich, 2004
To gain more insight in the specificity of egg-induced expression changes, we extended our analysis to a species with a different oviposition behavior. In contrast to P. brassicae, P. rapae butterflies lay only one egg per site. We analyzed transcript profiles of Arabidopsis leaves 24 to 72 h after oviposition by P. rapae. Each Arabidopsis leaf contained approximately one to two single eggs and we collected leaf discs of approximately 5 mm at the site of oviposition. Samples from three independent experiments for each time point were hybridized to the microarrays. We observed that single egg deposition by P. rapae triggered a much weaker response than that by P. brassicae; only 23, 45, and 59 genes were up-regulated after 24, 48, and 72 h, respectively. Of those, 25 genes were induced in at least two time points, indicating that although the effect was small these changes were not due to random experimental variability. Furthermore, 15 of these 25 genes were also induced after oviposition by P. brassicae (Supplemental Table S3). In general, P. brassicae eggs caused a larger induction than P. rapae eggs but we also identified some genes specifically induced by P. rapae eggs, including a PR Bet v1 allergen protein (At1g70830) and others showing a quantitatively similar response to each treatment, including the hevein-like gene HEL/PR4 (Fig. 4 ). Only four genes were down-regulated 72 h after oviposition. However, three of these genes, including one germin-like protein (At5g20630) and two Gly-rich proteins (At1g04800 and At4g29020) were also down-regulated after oviposition by P. brassicae (Supplemental Table S3).
Oviposition Induces Cellular Changes Associated with Cell Death A careful examination of egg-laden Arabidopsis plants did not reveal any physical damage. Furthermore, we could not see obvious phenotypical changes, nor any sign of infection at the site of oviposition during the days between oviposition and hatching. Scanning electron microscope images of leaves containing P. brassicae and P. rapae eggs showed that eggs are fixed to the plant surface with a glue forming a visible meniscus surrounding the base of the egg, without apparent modification of the leaf epidermis (Fig. 5, A–C ). Since the analyses of expression changes after oviposition identified many genes associated with PCD, we carried out histochemical experiments to analyze known cellular changes associated with these responses. First, trypan blue was used to stain dead cells in leaves 72 h after oviposition by P. brassicae. We observed a strong staining corresponding closely to the site where the egg batch was deposited (Fig. 5, D and E). As trypan blue is a dye that enters permeable dead or damaged cells, a control experiment was done where leaves were stained just after oviposition. In this case, we only saw a faint circular staining corresponding to the contact point between each egg and the leaf surface (data not shown), indicating that careful detachment of the eggs before the staining procedure was not damaging the plant surface and hence was not causing the strong cell death response observed after 3 d.
Second, we examined callose deposition in egg-laden leaves. The accumulation of autofluorescent phenolic compounds and deposition of callose are markers associated with lesion formation in response to pathogen invasion (Koga et al., 1988 ) and lesions found in lesion-mimic mutants (Dietrich et al., 1994). Leaves were stained with aniline blue 72 h after P. brassicae oviposition and viewed under a fluorescent microscope. We observed a strong accumulation of callose closely associated with the site of egg batch deposition (Fig. 5, F–H).
Third, PCD is often preceded by an early production of reactive oxygen species (Levine et al., 1994
Fourth, having observed that the defense gene PR1 was highly induced by P. brassicae eggs (Fig. 2) we selected this gene as a marker to further study the response to egg deposition. We obtained a transgenic Arabidopsis line containing the PR1 promoter coupled to the
To carry out an initial study on the nature of the elicitor(s) responsible for activating PR1::GUS expression, we collected P. brassicae eggs deposited on Arabidopsis leaves, gently crushed them in an Eppendorf tube and painted a few microliters of crude extract on a reporter plant. After 72 h, plants were stained for GUS activity and clearly showed activation of the PR1 promoter at the site of application (Fig. 6, G and H, left side). When the extract was boiled for 3 min before application, no activity could be detected (Fig. 6, G and H, right side). We then found that the supernatant of centrifuged crude egg extract (10,000g, 1 min) kept a strong activity after storage at –20°C (Fig. 6I). This provides a useful information for the future isolation of active fractions. To confirm that eggs, or compounds associated with eggs, are responsible for the induction of PR1, we dissected female butterflies and removed eggs from the ovaries. A portion of the eggs (equivalent to a normal batch or about 30 eggs) was applied to a leaf. After 72 h, a strong GUS staining was observed at the site of application (Fig. 6J). Female accessory glands produce a cement that allows the deposited eggs to be glued to the plant surface. We obtained P. brassicae accessory glands (Dr. J.J.A. van Loon, Wageningen University) and tested whether gland extracts can activate PR1::GUS. The supernatant from accessory glands that were crushed and centrifuged caused GUS staining 72 h after application while the solid pellet did not (Fig. 6, K and L). The staining was, however, less intense than with crushed eggs or with dissected ovaries. All experiments were repeated several times with similar results.
Egg-Induced Responses
In this study, we show that Arabidopsis plants are able to detect the presence of eggs of the pierid butterflies P. brassicae and P. rapae, and trigger a response that has strong similarities with an HR. In the case of pathogenesis, HR is elicited after the recognition of specific pathogen-derived molecules by plant-specific resistance genes and triggers a localized response that is characterized by a restricted necrosis at the site of infection, the production of reactive oxygen species, the accumulation of secondary metabolites, and the induction of PR genes. Cells directly below the oviposition site stained strongly with trypan blue, indicating that these cells were negatively affected by the presence of the eggs and were undergoing cell death, though not to the extreme result of developing a necrotic zone around the eggs. Indeed, there was no physical difference between the leaf surface where eggs were recently removed and other parts of the leaf. However, the plant was responding strongly as was further evidenced by the very specific accumulation of callose at the oviposition site, along with the induction of a callose synthase gene. Callose has been implicated in plant defense, for example in the wound response where it acts as a plug to seal the wound site. It is also believed that callose is rapidly deposited as a physical barrier to impede microbial and fungal attack (Stone and Clarke, 1992
Another supporting factor of a defensive response by the plant was the production of H2O2 at the oviposition site. H2O2 is often produced as a result of external biotic and abiotic stimuli and has been shown to play a role in the control of HR (Levine et al., 1994
Until now, only two studies have reported that insect eggs can trigger an HR-like response in a plant. Eggs of P. brassicae and Pieris napi caused the apparition of a necrotic zone on B. nigra plants, increasing egg mortality (Shapiro and Devay, 1987
In addition, we observed a down-regulation of several expansins, a cellulose synthase, pectin-modifying enzymes, and genes involved in cuticle biosynthesis by P. brassicae eggs, indicating that there might be cell wall remodeling at the site of oviposition. Many photosynthesis-related genes were also repressed, corroborating the previous observation of a net decrease of photosynthetic activity in Scots pine after egg deposition by D. pini (Schroder et al., 2005
We showed that a compound present in the supernatant of crude egg extracts activated PR1::GUS expression, indicating that the response to oviposition was not triggered by a physical damage or by simple contact of the butterfly during egg deposition. Extracts from accessory glands that produce the egg cement had, however, a weaker eliciting activity. Eggshell is constituted of several layers of proteinaceous structures and of a lipidic wax layer while the chemical nature of the egg cement is unknown (Trougakos and Margaritis, 2002
Insect eggs also contain defensive compounds that protect them from predation (Blum and Hilker, 2002
Eggs may contain symbionts that are transmitted by the female and are also exposed to pathogens (Kellner, 2002
The perception of chemical signals at the cell surface is mediated by receptors containing an extracellular ligand-binding domain and a cytoplasmic kinase domain. Arabidopsis contains more than 600 RLKs of which only a few have a defined role (Shiu and Bleecker, 2001
Since the egg shell is composed of proteins and wax, our finding that proteases and lipases are induced by egg deposition could indicate that these enzymes act as a direct defense against the egg. In addition, several chitinase genes were also induced by P. brassicae eggs. Chitin is a major component of insects where it plays a scaffolding role supporting the cuticle as well as peritrophic matrices in the gut epithelium, but it does not seem to be present in the eggshell (Merzendorfer and Zimoch, 2003 In conclusion, we provide here a molecular evidence for the detection of egg deposition by Arabidopsis plants and show that oviposition causes a strong and localized defense response that resembles an HR. It remains to be shown whether this response constitutes a direct defense against the eggs or whether plants have evolved a mechanism to anticipate the threat posed by future feeding larvae.
Plant and Insect Growth Conditions
Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 and mutants were grown as described previously (Reymond et al., 2000
Plants were 6 weeks old at the time of treatment. For insect oviposition, 24 to 30 plants (8–10 plants per time point) were transferred to the greenhouse for 2 to 4 h during which butterflies were allow to lay eggs. On average P. brassicae butterflies were laying one or two batches of 10 to 40 eggs per plant, and P. rapae were laying approximately 20 single isolated eggs per plant. Plants were then placed in a phytotron (10/14 h photoperiod, 100 µmol m–2 s–1, 22°C, 65% relative humidity) for a period of 24, 48, or 72 h before harvesting. Leaf discs of approximately 5 mm in diameter containing all egg batches or single eggs were collected at the appropriate times, the eggs were immediately removed carefully avoiding damage to the surface of the leaf disc, and the leaf samples were frozen in liquid nitrogen for later RNA extraction. As controls, an equal number of leaf discs from a distal egg-free leaf were sampled at each time point. Each experiment was repeated at least three times independently at intervals of several weeks.
CATMA microarrays (Hilson et al., 2004 For hybridization, 40 pmol of the Cy3-labeled sample and 40 pmol of the Cy5-labeled sample were combined with 3x SSC, 0.4% SDS, 18 µg yeast (Saccharomyces cerevisiae) tRNA, and 50% formamide in a final volume of 90 µL. The solution was boiled for 1 min, centrifuged for 1 min, and applied to the microarray under a lifterslip (Erie Scientific). The microarray was sealed in a hybridization cassette (Telechem) under an atmosphere of 3x SSC and submerged in a waterbath at 42°C overnight. The microarray was then washed once for 10 min in 1x SSC/0.1% SDS at 50°C, followed by two washes for 10 min in 0.1x SSC/0.1% SDS at 50°C, one wash for 1 min in 0.1x SSC at 37°C, and dipped several times in 0.1x SSC at room temperature. The microarray was immediately dried by centrifugation for 2 min at 2,600 rpm and scanned with a ScanArray 4000 (Packard BioScience SA). The average fluorescence intensity for each fluor and for each gene was determined using the ImaGene program (BioDiscovery). Median background fluorescence signal around each gene spot was subtracted from each spot and to normalize signal intensities between the Cy3 and Cy5 channels a normalization factor was computed using the Loess method. Signal values < 500 (2–3 times the average background SD) were raised to 500 to avoid extreme expression ratios.
Normalized signal intensities were used to calculate expression ratios. We previously observed that a threshold of 2-fold and a P value < 0.05 strongly increase the chance of identifying differentially expressed genes (Reymond et al., 2004
For the comparison of expression changes between oviposition and infection with Pseudomonas syringae AvrRPM1, ATH1 Affymetrix data from three independent experiments were obtained from the AtGenExpress project at http://www.weigelworld.org/resources/microarray. Expression ratios were calculated from gcRMA-normalized values of control (MgCl2 infiltration) and infected (P. syringae infiltration 24 h) samples. For P. rapae herbivory, CATMA microarray data from seven independent experiments were used. Expression ratios were calculated from normalized values (loess) of unchallenged leaves and leaves challenged for 5 h with third- to fourth-instar P. rapae larvae. For experiments with acd2-2 mutant, CATMA microarray data from three independent experiments were used. Expression ratios were calculated from normalized values (loess) of leaves with no visible lesions and leaves where approximately 10% to 20% of the surface presented necroses. For all experiments, a P value was computed on log ratios with a Student's t test and only genes with an expression ratio All microarray data have been deposited in ArrayExpress under accession numbers E-CAGE-189 (P. rapae oviposition), E-CAGE-190 (P. brassicae oviposition), E-MEXP-911 (P. rapae herbivory), and E-MEXP-912 (acd2-2 mutant).
Leaf discs for quantitative real-time PCR reactions were harvested 72 h after oviposition. All samples were harvested using a scalpel to enable cutting the precise area of the egg batch within 1 mm of the eggs (egg sample). The near sample was the 1 to 3 mm section of leaf surrounding previously cut egg disc. The distal leaf disc sample was cut to a similar size as the egg batch from a leaf without eggs but on the same plant as the eggs. A control sample was also harvested consisting of leaf discs of similar size to the egg leaf discs but on a plant that was not exposed to the butterflies. The eggs were immediately and carefully removed, avoiding damage to the surface of the leaf disc, and the leaf sample was frozen in liquid nitrogen for later RNA extraction.
RNA samples were first reverse transcribed using Superscript II (Invitrogen) based on the manufacturer's instructions with 5 µg total RNA (purified with an RNeasy Qiagen kit followed by a DNaseI treatment) in a final volume of 100 µL. The resulting cDNA samples were stored at –80°C. Real-time PCR was performed in triplicate using FullVelocity SYBR green kit (Stratagene). In a 25 µL reaction volume, 1 µL of the cDNA sample was combined with 3.75 µL of 6-carboxy-X-rhodamine (1/5,000 dilution), 12.5 µL of 2X SYBR, and 2.5 µL of primer mix where both forward and reverse primers are at a 1 µM concentration. The reaction was placed in a MX300 real-time PCR machine (Stratagene) with a temperature profile of 95°C for 5 min, then 40 cycles of 95°C for 10 s and 55°C for 30 s. The following gene-specific primers were used: EIF4A1, At3g13920 forward (5'-CCAGAAGGCACACAGTTTGATGCA-3'), At3g13920 reverse (5'-AGACTGAGCCTGTTGAATCACATC-3'); PR1, At2g14610 forward (5'GTGGGTTAGCGAGAAGGCTA-3'), At2g14610 reverse (5'-ACTTTGGCACATCCGAGTCT-3'); chitinase, At2g43570 forward (5'-GGAGAGTACTGCGACACAGAGAAA-3'), At2g43570 reverse (5'-GGCAGGAACCTGGTCTTGAGCAA-3'); trypsin inhibitor, At1g73260 forward (5'-CCTCGTGGTTGCTGGTCCAAA-3'), At1g73260 reverse (5'-CCTCTCACATAGTCTTGGACGAAA-3'). Relative mRNA abundance was calculated using the comparative
For visualization of cell death, eggs were very carefully removed from leaves 72 h after oviposition avoiding leaf damage and leaves were submerged in trypan blue solution (5 mL lactic acid, 10 mL 50% glycerol, 1 mg trypan blue, and 5 mL phenol) at 30°C for 2 to 3 h. Leaves were then destained in 70% chloral hydrate followed by washes in 70% to 95% ethanol. Callose deposition was detected 72 h after oviposition. Eggs were removed, leaving a few on the border of the egg batch for localization purposes. Ethanol-destained leaves were placed in 0.15 M phosphate buffer pH 8.5 containing 0.01% aniline blue for 1.5 to 3 h. Leaves were examined under a Leica MZ16FA epifluorescence microscope (excitation 340–380 nm, emission 420 nm filter) and images were acquired using a Leica DC300F camera. H2O2 accumulation was measured 72 h after oviposition. After the eggs were removed, the leaves were submerged in a 1 mg/mL solution of DAB and placed in a phytotron (100 µmol m–2 s–1, 20°C and 65% relative humidity) for 8 h. The leaves were then destained by boiling in 95% ethanol for 10 min, and stored at room temperature in 95% ethanol until photographs could be taken.
GUS staining for PR1 expression analyses was performed on leaves 24, 48, and 72 h after oviposition. Eggs were removed and leaves were treated with 90% acetone at room temperature for 30 to 60 min, rinsed in a 50 mM phosphate buffered mixture containing 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.05% Triton X-100. Leaves were then submerged in the same solution with the addition of 0.5 µg/µL 5-bromo-4-chloro-3-indolyl- For PR1::GUS activity after application of P. brassicae egg extract, egg batches were collected on Arabidopsis plants 2 to 3 h after oviposition and crushed with a conical pestle in an Eppendorf tube. A few microliters of crude extract were directly applied on an Arabidopsis leaf with a pipette tip and the plant was left for 72 h before GUS staining. An aliquot of the extract was also boiled for 3 min and painted on a leaf. In another experiment, the crude egg extract was centrifuged (10,000g, 1 min) and the supernatant was stored at –20°C before the application of 1 or 2 µL to a leaf, equivalent to one-third or two-thirds of an egg batch, respectively. For the treatment with female's ovaries, the P. brassicae reproductive system was removed with a scalpel, homogenized, and applied to a leaf. Isolated accessory glands from P. brassicae female butterflies were obtained from J.J.A. van Loon (Wageningen University). Six glands (stored at –80°C for 3 d) were crushed in an Eppendorf tube, and centrifuged (10,000g, 1 min). Leaves were spotted with 2 µL of supernatant (equivalent to one accessory gland) and the plant was left for 72 h before staining. Aliquots of the solid pellet equivalent to one accessory gland were also spotted on the leaves.
JA measurements were done according to previously published protocols (Weber et al., 1997
The following materials are available in the online version of this article.
We gratefully acknowledge Edward E. Farmer for providing financial support for C.G.-D. and for critical reading of the manuscript. We wish to thank Boris Kunstner for maintenance of the plants, Aurore Chételat for help with JA measurements, Dr. Gustavo Bonaventure for help with real-time PCR, Dr. Robin Liechti for establishing the Nomad database, and Natacha Bodenhausen for helpful comments. We also thank Dr. Jane Glazebrook for the coi1-1 nonglabrous mutant, Dr. Christiane Nawrath for the sid2-1 mutant, and Dr. Allan Shapiro for the PR1::GUS plants. Dr. Joop J.A. van Loon kindly provided P. brassicae accessory glands. Received October 4, 2006; accepted November 21, 2006; published December 1, 2006.
1 This work was supported by a genomics grant from the University of Lausanne, by a Secrétariat d'etat à 1'Education et à la Recherche grant (SER 02.0346), and by the Compendium of Arabidopsis Gene Expression project that is funded by the European Commission within its Fifth Framework Programme (grant no, QLK3–CT–2002–02035).  The author responsible for distribution of materials integral to the findings presented in this article in accord with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Philippe Reymond (philippe.reymond{at}unil.ch).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.090837 * Corresponding author; e-mail philippe.reymond{at}unil.ch; fax 0041–21–692–4195.
Allemeersch J, Durinck S, Vanderhaeghen R, Alard P, Maes R, Seeuws K, Bogaert T, Coddens K, Deschouwer K, Van Hummelen P, et al (2005) Benchmarking the CATMA microarray: a novel tool for Arabidopsis transcriptome analysis. Plant Physiol 137: 588–601 Aplin RT, d'Arcy Ward L, Rothschild M (1975) Examination of large white and small white butterflies (Pieris spp.) for presence of mustard oils and mustard oil glycosides. J Entomol Ser A 50: 73–78 Balbyshev NF, Lorenzen JH (1997) Hypersensitivity and egg drop: a no | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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2 or 
0.5 and P value < 0.05 (both criteria had to be met in at least one time point) were considered as differentially regulated by egg deposition. 
