Introduction
Insect pests represent a major threat for global food security (Oerke,
2006). Pests control largely relies on the use of pesticides which, on
the other side, are increasingly reported to have harmful ecological
effects (Forister et al., 2019). Future crop yield stability will thus
depend on plant breeding efforts to improve crop resistance traits
against pests (Crall et al., 2018). Hypersensitive response (HR) is one
of the most studied traits associated with plant defence against
pathogens and pests and it is often underlined by single resistance (R)
genes (Wagner et al., 2021). So far only a handful of R genes have been
identified against insect herbivores, mostly consisting of cell surface
receptors (pattern recognition receptors, PRRs) or intracellular
receptors (nucleotide-binding leucin rich-repeat, NLRs) (Kouretlis &
van der Hoorn, 2018). Resistance traits against insects based on R genes
are mostly limited to piercing-sucking insects such as gall midges
(Bentur et al., 2016; Harris et al., 2012), or phloem-feeding insects
such as aphids (Botha, Li, & Lapitan, 2005; Dogimont, Chovelon,
Pauquet, Boualem, & Bendahmane, 2014; Klingler, Nair, Edwards, &
Singh, 2009; Nicolis & Venter, 2018; Rossi et al., 1998; Sun et al.,
2020), whiteflies (Nombela, Williamson, & Muñiz, 2003) and planthoppers
(Tamura et al., 2014; Liu et al., 2015; Zhao et al., 2016). Remarkably,
reports on resistance genes to chewing insects are even more scarce,
with only a few studies showing that PRR surface receptors also mediate
defenses against chewing caterpillars (Gilardoni, Hettenhausen, Baldwin,
& Bonaventure, 2011; L. Hu et al., 2018; Steinbrenner et al., 2020).
Indeed, immunity against feeding insect herbivores appears to be mainly
controlled by polygenic quantitative traits (Kliebenstein 2017).
Given the lack of effective R genes against chewing insects,
resistance mechanisms targeting insect eggs have been proposed as a
complementary defence strategy (Fatouros, Cusumano, Danchin, & Colazza,
2016; Tamiru, Khan, & Bruce, 2015). Clearly, the recognition and
killing of insect eggs is advantageous to plants as it prevents the
destructive feeding by the hatching larvae (Hilker & Fatouros, 2015,
2016). The investigation of egg-killing traits thus represents an
alternative and unexplored source of novel R genes to increase
crop resistance to pests (Fatouros et al., 2016).
Cabbage white butterflies, such as the gregarious Pierisbrassicae and the solitary P. rapae (Lepidoptera:
Pieridae), are pests of crucifer crops (Brassica spp.) and a
serious agricultural challenge (Kumar, 2017; Ryan et al., 2019).Pieris eggs induce a HR-like cell death in their host plants of
the Brassicaceae family resembling a HR induced by pathogens (Caarls et
al., 2023; Griese et al., 2021; Shapiro & De Vay, 1987). Under field
conditions, a severe cell death reduces egg survival up to more than
40% on black mustard B. nigra (Griese et al., 2021). Therefore,
the egg-induced cell death represents a trait with a high potential as a
novel plant defense against eggs, hence reducing the impact of
successive larval stages. While it is known that plants respond toPieris eggs with a salicylic acid (SA)-dependent immune response
(Bonnet et al., 2017; Bruessow, Gouhier-Darimont, Buchala, Metraux, &
Reymond, 2010; Caarls et al., 2023; Little, Gouhier-Darimont, Bruessow,
& Reymond, 2007), the genes involved in detection and activation of
egg-induced HR remain unknown.
A few recent studies began to investigate the genetic basis ofPieris spp. egg-induced HR-like cell death using the model
species A. thaliana (Groux et al., 2021) and the crop B.
rapa (Bassetti et al., 2022), which both benefit from extensive
resources for classical forward genetics. A genome-wide association
study (GWAS) in A. thaliana identified two loci, an L-typelectin receptor-like kinase-I.1 (LecRK-I.1 ) and a putative
Ca2+ channel glutamate receptor 2.7(GLR2.7 ) (Groux, 2019; Groux et al., 2021). Further, a QTL
mapping in B. rapa identified three QTLs Pbc1-3 associated
with cell death size (Bassetti et al., 2022). The QTLs included many
genes involved in plant immunity, but they underlined large genomic
regions yet to be fine-mapped. A partial overlap among the loci
identified in both plant species was suggested, given thatBra LecRK-I.1 is included within Pbc3 (Bassetti et al.,
2022). Overall, the study of plant-insect egg molecular interaction is a
relatively recent field, and more research is clearly needed to
understand to which extent the genetic regulation of Pierisegg-plant interaction is conserved between plant species.
Egg-induced HR-like cell death observed in A. thaliana andB. rapa manifests as a light necrosis that has no or little
effect on egg survival (Groux, 2019; Groux et al., 2021). In contrast,
egg deposition on B. nigra triggers a severe HR, spreading from
the leaf abaxial up to the adaxial side, which correlates with a
substantial killing of different Pieris eggs (Caarls et al.,
2023; Fatouros et al., 2014; Griese et al., 2021; Griese et al., 2020).
Previously, we found that the severity and occurrence of egg-induced
cell death in B. nigra differs between plants of the same
accession (Caarls et al., 2023) and between accessions (Caarls et al.,
2023; Griese, Dicke, Hilker, & Fatouros, 2017; Pashalidou, Fatouros,
Loon, Dicke, & Gols, 2015), suggesting the existence of natural
variation for egg-induced HR. A treatment with a solution of compounds
derived from the eggs, an egg wash, was shown to mimic egg-induced
responses in plants (Caarls et al., 2023) and it provides suitable
treatment to screen large genetic populations. In summary, B.
nigra represents an ideal plant species to study the genetics of
egg-induced HR because the phenotype is strong, stable, easy to score,
varies between accessions, and has a proven egg-killing effect.
In this study, we investigated the inheritance and genetic basis ofP. brassicae egg-induced HR-like cell death in B. nigra .
Given the lack of advanced genetic populations, we crossed plant
material collected from the field. We found variation in the response
between and within field-collected B. nigra accessions, and then
used crosses between strong- and low-responding individual plants to
study the inheritance of the trait. We found that Pierisbutterfly egg-induced HR B. nigra segregates as a Mendelian
trait, and then performed genetic mapping through bulk-segregant
analysis paired with whole genome sequencing (BSA-seq). Recombinant
analysis was used to further fine-map the genetic region and identify a
single dominant locus of ~50 kb which we namedPieris e gg-k illing (PEK). Within PEK, a gene
cluster of TIR-NBS-LRRs, a type of NLR receptors was present, showed
copy number variants (CNVs) between different B. nigragenomes.
Material and
methods
Plant
materials
The inheritance of the HR-like cell death was studied using B.
nigra accessions collected from a local population in the floodplain of
the Rhine River near Wageningen, The Netherlands (N51.96, E05.68). The
accessions SF3-O1, SF19-O1, SF25-O1, SF29-O1, SF47-O1 and SF48-O1
originated from one multiplication by open pollination (“O1”) of
accessions used in previous studies (Griese, Dicke, Hilker, & Fatouros,
2017). The accessions DG1, DG12 and DG29 originated from open pollinated
wild plants collected in 2018. A single DG1 plant showing no HR in
response to eggs was selfed to obtain accession DG1-S1. A single DG1-S1
plant was then crossed with a single SF48-O1 plant to obtain an F1
population (Fig. 2a). Single plants from the F1 that
showed HR-like cell death were backcrossed to other DG1-S1 plants to
obtain segregating backcross families (BC1). Selfing of
individual plants was done to generate
BC1-S1 and
BC1-S2 populations.
Plants were grown in a greenhouse under standardized conditions (21° day
/ 18° night, RH 50 - 70%, LD 16:8 h). Seeds were vernalized at 4° C for
two days to induce even germination and then were sown in small trays
with sowing soil (Lentse potgrond, Lent, The Netherlands). Seedlings
were transplanted one week after germination into 17 cm diameter pots
with potting soil (Lentse potgrond, Lent, The Netherlands). Plants were
grown for five weeks before treatment with P. brassicae egg wash.