Figure 4. Proposed model of plant perception and immune response to Lepidoptera eggs (Mamestra brassicae and Pierisspp.) in Brassica spp. and A. thaliana. General elicitors or egg-associated molecular patterns (EAMPs), that are conserved among Lepidoptera eggs, e.g. PCs, induce a weak plant immune response resembling PTI (Caarls et al. 2023; Stahl et al. 2020). The recognition is mediated by different LecRKs in A. thaliana(Gouhier-Darimont, Stahl, Glauser, & Reymond, 2019; Groux et al., 2021). A putative effector present in Pieris eggs instead induces an ETI and following HR-like cell death in some Brassica spp, putatively mediated by TNLs within the PEK locus.
While waiting for functional validation of the PEK locus, a hypothetical role for TNLs in the perception and/or modulation of the plant response to Pieris spp. eggs could provide an extra step in the PTI-ETI model for eggs (Fig. 4). Insect eggs induce signalling pathways of PTI response similarly to the recognition of PAMPs from bacteria, fungi, and other biotic stresses (Jones & Dangl, 2006; Reymond, 2021). According to this model, egg elicitors that are conserved across insect species, also known as egg-associated molecular patterns (EAMPs), are detected by plant cell surface receptors (PRRs). An example of such conserved elicitors/EAMPs are the phosphatidylcholines (PCs), phospholipids which are present in all insect eggs (Stahl et al., 2020). PCs are, however, not sufficient to elicit HR in B. nigra and the elicitor(s) of HR seem to rather be present in a water-soluble phase from the glue-like secretion surrounding the eggs (Caarls et al., 2023). So far, at least two PRRs, LecRK-I.1 and LecRK-I.8, were found to mediate Pieris egg-induced PTI and HR-like phenotypes in A. thaliana (Gouhier-Darimont et al., 2019; Groux et al., 2021). Thus, LecRKs could potentially also modulate the PTI phenotypes that were induced in A. thaliana by eggs of P. brassicae, Spodoptera littoralis and Drosophila melanogaster (Bruessow et al., 2010) and in Brassica nigra by eggs of Pieris spp. and Mamestra brassicae (Caarls et al., 2023). Pieris spp. eggs induce a visible HR, while we observed no HR under eggs of generalists, e.g. M. brassicae or non-adapted Pierid butterflies (Caarls et al., 2023; Griese et al., 2021). In this scenario, one or more Pieris egg “effector” compounds, could be detected by unknown plant receptors, such as the PEK locus, and trigger a second immune response leading to a macroscopic HR-like cell death which acts as a defence trait.
Previously, we showed that B. nigra expresses a strong HR in response to Pieris spp. eggs resulting in egg-killing and that this response is consistently stronger compared to the responses observed in other Brassicaceae species (Griese et al., 2021). From a plant breeding perspective, the molecular markers flanking thePEK locus may be sufficient to attempt the introgression of egg-killing HR-like cell death into elite Brassica crops lines, as interspecific crosses between Brassica crop cultivars and crop wild relatives (CWR) from secondary/tertiary gene pools are sometimes done for other resistance traits (Hu et al., 2021; Katche, Quezada-Martinez, Katche, Vasquez-Teuber, & Mason, 2019; Lv, Fang, Yang, Zhang, & Wang, 2020).
From a scientific perspective, the characterization of genetic diversity of loci containing PRR or NLR receptors across a broad phylogenetic context helped to resolve the macroevolutionary history of pathogen and insect resistance traits and generate hypothesis on putative functional domains (Snoeck, Guayazan-Palacios, & Steinbrenner, 2022; Wang et al., 2021). In this study, we found within PEK locus many SNPs and/or InDels between our parental accessions. Additionally, we found copy number variants (CNVs) at the locus between B. nigra reference genomes, but the identification of the casual variants will likely require de novo assembly of the locus within our plant material. The high diversification in causal polymorphisms at NLR loci is well described (Dolatabadian & Fernando, 2022) and it resulted from evolution through a massive expansion and lineage diversification that allows adaptation to a multitude of biotic stresses (Shao, Xue, Wang, Wang, & Chen, 2019). Consequently, NLR loci are hardly ever well represented by a single plant genome and, further, are likely to be misassembled (Barragan & Weigel, 2021). It is thus fundamental to characterize the genomic context of PEK locus across the Brassicaceae to study whether the locus’ genetic diversity across the plant family correlates with interspecific variation in HR phenotype. Such study may potentially reveal different types of polymorphisms at the PEK locus, as the interspecific variation in HR may have arisen from differences in life-history traits of plant species and/or exposure to insect eggs. For example, the weak type of HR developed byA. thaliana could be the result of lower selective pressure by the herbivore since as a short-day flowering species it is not a host ofPieris spp. (Harvey, Gols, Wagenaar, & Bezemer, 2007) and only occasionally of Anthocaris cardamines (Wiklund, 1984). Conversely, B. rapa crop morphotypes show a weak HR (Bassetti, 2022) as the result of a different mechanism, for example negative selection during domestication as can occur with inducible defence traits in other crops (Turcotte, Turley, & Johnson, 2014; Whitehead, Turcotte, & Poveda, 2017).
In conclusion, here we report that intraspecific variation for HR induced by P. brassicae eggs is associated with a single locus inB. nigra . Through classical forward genetics we identified thePEK locus, which contains a cluster of TNL receptors. The locus seems highly polymorphic between the known B. nigra genomes and we expect this to be the case also for our accessions. This implies the need for improved genome assembly before future fine-mapping. This future work will enable cloning and functional testing of the firstB. nigra gene involved in defense against insect eggs.

Acknowledgements

We are grateful to Dr. Charlotte Prodhomme and Corentin Clot for insightful discussion on the CoSSA pipeline settings for k- mer based genetic mapping. Thanks to Dr. Chengcheng Cai and Dr. Robin van Velzen for discussing whole genome sequencing data analysis. Further, we are grateful to Dr. Isobel Parkin and Dr. Kumar Paritosh for access to early versions of B. nigra genomes. We are grateful to the employees of Unifarm (WUR) for rearing and caring of the plants used in the experiment. We thank Pieter Rouweler, André Gidding, and the late Frans van Aggelen for rearing of Pieris brassicae. This research was made possible by support of the Dutch Technology Foundation TTW, which is part of the Dutch Research Council (NWO) (NWO/TTW VIDI grant 14854 to N.E.F.).

Availability of data

The datasets supporting the conclusions of this article are temporary available at this Zenodo repository (https://doi.org/10.5281/zenodo.8131352). All sequencing raw data have been deposited in the European Nucleotide Achieve (ENA) under accession number PRJEB64240.