Oviposition by pierid butterflies triggers defense responses in Arabidopsis

Abstract

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::beta-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.

Figure 1.

A, Comparison of transcript profiles between oviposition by P. brassicae (24–72 h, three replicates each) and herbivory by P. rapae (challenge for 5 h by 3rd-instar larvae, seven replicates). B, Comparison of transcript profiles between oviposition, infection by P. syringae AvrRPM1 for 24 h (three replicates, data from AtGenExpress), or during lesion formation in the mutant acd2-2 (three replicates). Diagrams represent the numbers of induced (normal) or repressed (italic) genes in each category. All selected genes have an expression ratio ≥ 2 or ≤ 0.5 and a P value < 0.05. The total number of differentially expressed genes in each experiment is indicated outside the diagram. Genes induced after oviposition and any of the other treatments are listed in Supplemental Tables S1, S4, and S5.

Figure 2.

Real-time quantitative PCR analysis of selected egg-inducible genes. Relative expression levels of PR1 gene (PR1, At2g14610), a trypsin inhibitor gene (At1g73260), and a chitinase gene (At2g43570) were analyzed 72 h after oviposition by P. brassicae. Leaf samples were collected at the site of oviposition (egg), next to the egg batch (near), in egg-free distal leaves (distal), and in leaves from plants that were not in contact with butterflies (CTL). Expression levels were normalized with respect to the housekeeping gene EIF4A1 (At3g13920) and displayed relative to the expression control samples (expression value set at 1). Data bars represent the mean levels of transcripts (±se, n = 3). b.d., Below detection.

Figure 3.

Role of signaling mutants in response to P. brassicae eggs. Expression changes were monitored with CATMA microarrays 72 h after oviposition in the jasmonate-insensitive Arabidopsis mutant coi1-1 (A) and in the SA-deficient mutant sid2-1 (B). Expression ratios of mutant plants are plotted against expression ratios of wild-type plants. Pink dots represent genes that are differentially regulated (P < 0.05) between mutant and wild-type plants. Values are the mean of three independent replicates. Genes induced by oviposition in wild-type or mutant plants are listed in Supplemental Table S2.

Figure 4.

Time course of expression changes after oviposition by P. brassicae (solid line) and P. rapae (dotted line). Microarray data from six representative genes are shown: short-chain alcohol dehydrogenase (SAG13, At2g29350), trypsin inhibitor (At1g73260), chitinase (At2g43570), Cys proteinase (At3g49340), PR Bet v1 allergen protein (At1g70830), and hevein-like protein (HEL/PR4, At3g04720). Values are the mean (±se) of three independent experiments.

Figure 5.

sem analyses of eggs of P. brassicae (A and B) and P. rapae (C) showing the meniscus (arrows) formed by the glue that connects the egg to the plant surface. d to I, Cellular changes in response to P. brassicae eggs 3 d after oviposition. E, Leaves were stained with trypan blue to visualize cell death. G, Callose deposition (arrow) was detected by staining leaves with aniline blue and viewed under a fluorescent microscope. H, Close-up view of callose deposition at the site of egg batch deposition; some eggs were left on the leaf to better localize the site of oviposition. I, Accumulation of H₂O₂ (arrow) at the site of oviposition was revealed by DAB staining. Images taken before removing the eggs are presented to show the correspondence between egg batch location and cellular responses (D for E and F for G). Magnification bars = 1 mm in A, and D to I; 100 μm in B and C.

Figure 6.

Activation of the defense gene PR1 by egg deposition. Arabidopsis transgenic lines containing the PR1∷GUS reporter gene were visualized for GUS staining assay. Oviposition by P. brassicae for 48 h (A and B), and 72 h (C and D), and by P. rapae for 72 h (E and F, single egg indicated by an arrow). Application of P. brassicae crude egg extract activated PR1∷GUS expression (G and H, left side), but not when the extract was boiled (G and H, right side). Treatment with 1 μL (I, top) and 2 μL (I, bottom) of crude egg extract stored at −20°C was also effective. Application of egg masses dissected from P. brassicae activated PR1∷GUS expression (J). GUS staining in response to application of solid (K, top arrow) or liquid (K, bottom arrow) P. brassicae crude accessory gland extract was only observed for the liquid phase (L, bottom arrow). Images of leaves before staining are presented to show colocalization of egg deposition (A, C, and E) or crude extract application (G and K) and GUS staining. Magnification bars = 5 mm.