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.