Summary Evolutionary arms‐races between plants and insect herbivores have long been proposed to generate key innovations such as plant toxins and detoxification mechanisms that can drive diversification of the interacting species. A novel front‐line of plant defence is the killing of herbivorous insect eggs. We test whether an egg‐killing plant trait has an evolutionary basis in such a plant–insect arms‐race. Within the crucifer family (Brassicaceae), some species express a hypersensitive response (HR)‐like necrosis underneath butterfly eggs (Pieridae) that leads to eggs desiccating or falling off the plant. We studied the phylogenetic distribution of this trait, its egg‐killing effect on and elicitation by butterflies, by screening 31 Brassicales species, and nine Pieridae species. We show a clade‐specific induction of strong, egg‐killing HR‐like necrosis mainly in species of the Brassiceae tribe including Brassica crops and close relatives. The necrosis is strongly elicited by pierid butterflies that are specialists of crucifers. Furthermore, HR‐like necrosis is linked to PR1 defence gene expression, accumulation of reactive oxygen species and cell death, eventually leading to egg‐killing. Our findings suggest that the plants’ egg‐killing trait is a new front on the evolutionary arms‐race between Brassicaceae and pierid butterflies beyond the well‐studied plant toxins that have evolved against their caterpillars.

Brassica species are characterized by their tremendous intraspecific diversity, exemplified by leafy vegetables, oilseeds, and crops with enlarged inflorescences or above ground storage organs. In contrast to potato tubers that are edible storage organs storing energy as starch and are the vegetative propagation modules, the storage organs of turnips, grown from true seed, are swollen hypocotyls with varying degrees of root and stem that mainly store glucose and fructose. To highlight their anatomical origin, we use the term “hypocotyl-tuber” for these turnip vegetative storage organs. We combined cytological, physiological, genetic and transcriptomic approaches, aiming to identify the initial stages, molecular pathways and regulatory genes for hypocotyl-tuber induction in turnips ( B. rapa subsp. rapa ). We first studied the development of the hypocotyl zone of turnip and Pak choi and found that 16 days after sowing (DAS) morphological changes occurred in the xylem which indicated the early tuberization stage. Tissue culture experiments showed a clear effect of auxin on hypocotyl-tuber growth. Differentially expressed genes between 1 and 6 weeks after sowing in turnip hypocotyls, located in genomic regions involved in tuber initiation and/or tuber growth defined by QTL and selective sweeps for tuber formation, were identified as candidate genes that were studied in more detail for their role in hypocotyl-tuber formation. This included a Bra-FLOR1 paralogue with increased expression 16 DAS, when the hypocotyl starts swelling, suggesting dual roles for duplicated flowering time genes in flowering and hypocotyl-tuber induction. Bra-CYP735A2 was identified for its possible role in tuber growth via trans -zeatin. Weigthed Co-expression Network Analysis (WGCNA) identified 59 modules of co-expressed genes. Bra-FLOR1 and Bra-CYP735A2 were grouped in a module that included several genes involved in carbohydrate transport and metabolism, cell-wall growth, auxin regulation and secondary metabolism that serve as starting points to illuminate the transcriptional regulation of hypocotyl-tuber formation and development.

Brassica plants activate a strong hypersensitive response (HR)-like necrosis underneath eggs of cabbage white butterflies, but their molecular response to eggs is poorly understood. Here, we developed a method to generate egg wash to identify potential insect egg-associated molecular patterns (EAMPs) inducing HR-like necrosis. We found that egg wash, containing compounds from Pieris eggs, induced a similar response as eggs. We show that wash of hatched eggs, of egg glue, and of accessory reproductive glands (ARG) that produce this glue, also induced HR-like necrosis, whereas removal of the glue from eggs resulted in a reduced response. Eggs of Pieris butterflies induced callose deposition, production of reactive oxygen species and cell death in B. nigra and B. rapa leaf tissue, also in plants that did not express HR-like necrosis. Finally, only washes from Pieris eggs induced defence genes and ethylene production, whereas egg wash of a generalist moth did not. Our results indicate that EAMPs are in the egg glue and that the response in B. nigra is specific to Pieris species. Our study expands knowledge on the Brassica-Pieris-egg interaction, and paves the way for identification of EAMPs in Pieris egg glue and corresponding receptor in Brassica spp.

Background Cabbage white butterflies (Pieris spp.) can be severe pests of Brassica crops such as Chinese cabbage, Pak choi (Brassica rapa) or cabbages (B. oleracea). Eggs of Pieris spp. can induce a hypersensitive response-like (HR-like) cell death which reduces egg survival in the wild black mustard (B. nigra). Unravelling the genetic basis of this egg-killing trait in Brassica crops could improve crop resistance to herbivory, reducing major crop losses and pesticides use. Here we investigated the genetic architecture of a HR-like cell death induced by P. brassicae eggs in B. rapa. Results A germplasm screening of 56 B. rapa accessions, representing the genetic and geographical diversity of a B. rapa core collection, showed phenotypic variation for cell death. An image-based phenotyping protocol was developed to accurately measure size of HR-like cell death and was then used to identify two accessions that consistently showed weak (R-o-18) or strong cell death response (L58). Screening of 160 RILs derived from these two accessions resulted in three novel QTLs for Pieris brassicae-induced cell death on chromosomes A02 (Pbc1), A03 (Pbc2), and A06 (Pbc3). The three QTLs Pbc1–3 contain cell surface receptors, intracellular receptors and other genes involved in plant immunity processes, such as ROS accumulation and cell death formation. Synteny analysis with A. thaliana suggested that Pbc1 and Pbc2 are novel QTLs associated with this trait, while Pbc3 also contains an ortholog of LecRK-I.1, a gene of A. thaliana previously associated with cell death induced by a P. brassicae egg extract. Conclusions This study provides the first genomic regions associated with the Pieris egg-induced HR-like cell death in a Brassica crop species. It is a step closer towards unravelling the genetic basis of an egg-killing crop resistance trait, paving the way for breeders to further fine-map and validate candidate genes.

Plants perceive and respond to herbivore insect eggs. Upon egg deposition on leaves, a strong hypersensitive response (HR)-like cell death can be activated leading to egg desiccation and/or dropping. In Brassica spp., including many crops, the HR-like mechanism against eggs of cabbage white butterflies (Pieris spp.) is poorly understood. Using two Brassica species, the crop B. rapa and its wild relative B. nigra, we studied the cellular and molecular plant response to Pieris brassicae eggs and characterized potential insect egg-associated molecular patterns (EAMPs) inducing HR-like cell death. We found that eggs of P. brassicae induced typical hallmarks of early immune responses, such as callose deposition, production of reactive oxygen species and cell death in B. nigra and B. rapa leaf tissue, also in plants that did not express HR-like cell death. However, elevated levels of ethylene production and upregulation of salicylic acid-responsive genes were only detected in a B. nigra accession expressing HR-like cell death. Eggs and egg wash from P. brassicae contains compounds that induced such responses, but the eggs of the generalist moth Mamestra brassicae did not. Furthermore, wash made from hatched Pieris eggs, egg glue, and accessory reproductive glands (ARG) that produce this glue, induced HR-like cell death, whereas washes from unfertilized eggs dissected from the ovaries or removal of the glue from eggs resulted in no or a reduced response. This suggests that there is one or multiple egg associated molecular pattern (EAMP) located in the egg glue a that teresponse in B. nigra is specific to Pieris species. Lastly, our results indicate that the EAMP is neither lipidic nor proteinaceous. Our study expands the knowledge on the mechanism of Brassica-Pieris-egg interaction and is a step closer toward identification of EAMPs in Pieris egg glue and corresponding receptor(s) in Brassica.