PEK locus shows copy number variations (CNVs) amongB. nigra genomes
The PEK locus contained multiple duplicated genes, including a cluster of TIR-NBS-LRR, a class of NLR intracellular receptors. NLRs are often organized in genomic clusters as the result of tandem duplications, unequal crossing over and gene conversion (Kuang et al. 2004). Thus, we suspected that the locus may be highly dynamic and polymorphic among B. nigra genomes. Indeed, we found extensive copy number variations (CNVs) for some of the genes when comparing the available B. nigra genomes NI100, C2, and Sangam (not shown). Specifically, the TNLs were present in two copies in NI100, four copies in C2, and seven copies in Sangam. Similarly, we found CNVs also forBn MAP1D which is present in two copies in NI100, three copies in C2, and four copies in Sangam. Collectively, our data showed that thePEK locus is highly polymorphic among available B. nigragenomes.

Discussion

Hypersensitive response induced by Pieris spp. egg deposition is an appealing model system to study the interaction between plants and insect eggs as well as an effective plant defence trait to chewing herbivores. Here, we report for the first time that P. brassicaeegg-killing HR-like cell death in B. nigra segregates as a Mendelian dominant trait underlined by a single locus. Through BSA-seq and fine-mapping, we identified the PEK locus on a ~50kb interval on the proximal arm of B. nigrachromosome B3. We found a cluster of TIR-NBS-LRR (TNLs) intracellular receptors which are the only genes associated with plant immunity and are considered likely candidate genes. So far, no TNL receptor genes have been shown to be involved in immunity responses against insects. It is intriguing to further understand whether they directly recognize an egg-derived component and/or co-act downstream of egg recognition.
Segregation of the HR phenotype throughout our crossing scheme supported the evidence for a Mendelian trait underlined by a single dominant locus. As we crossed two plants from heterogenous wild B. nigraaccessions, we observed phenotypic segregation of different morphological traits in the F1. Segregation of HR was consistent with a single dominant locus originating from a heterozygous donor resistant (R) plant. In fact, we showed a 1:1 segregation ratio of F1 and BC1 populations, followed by a 3:1 segregation ratio of F2, BC1S1 and BC1S2 derived from selfings of heterozygous resistant plants. Accordingly, selfing of plants without HR (S plants) resulted in progenies that were also unable to develop HR. We successfully identified the PEK locus using a BSA-seq approach which was already proven to be advantageous for quickly identifying single Mendelian loci in genetic populations with little recombination (Liu, Yeh, Tang, Nettleton, & Schnable, 2012), as well as in highly heterozygous species (Dakouri et al., 2018; Prodhomme et al., 2019). So far, HR has been frequently associated with monogenic qualitative resistance to bacteria, fungi, nematodes, and viruses (Kourelis & van der Hoorn, 2018). In plant-insect interactions, however, HR seemed less prominent as defense response and mostly occurring against cell content feeders such as aphids, gall midges or planthoppers (Botha et al., 2005; Himabindu, Suneetha, Sama, & Bentur, 2010; Klingler et al., 2009; Stuart, Chen, Shukle, & Harris, 2012). It is thus remarkable that an HR cell death evolved to target insect eggs and, also, that it is underlined by a single major effect locus as previously shown mainly for HR-based resistance traits against pathogens.
Through recombinant analysis, we fine mapped the PEK locus to a ~50 kb region. Further fine-mapping in BC1S2 populations did not increase the resolution into the locus. PEK contains eleven genes, among which a cluster of intracellular receptors of the TNL type. The other genes within the PEK locus were annotated either as “unknown function”, as an unspecified “membrane proteins” (BniB03g15430.C2) or were orthologs of a methionine aminopeptidase 1D (MAP1D, AT4G37030). MAP1D is an enzyme responsible for the cleavage of the initiator Methionine residue at the N-terminal of proteins (Ross, Giglione, Pierre, Espagne, & Meinnel, 2005). MAPs have been indicated as first step required for the stabilization and/or degradation of chloroplasts proteins (Apel, Schulze, & Bock, 2010), but a putative involvement in plant defense is yet to be proven. The involvement of MAP proteins in plant immunity has not been proven. However, given the involvement of TNLs in perception and signalling of plant immunity against pathogens (Cui, Tsuda, & Parker, 2015; Monteiro & Nishimura, 2018), and that cloned R genes providing resistance based on HR are often NLRs (Kourelis & van der Hoorn, 2018), we consider these TNLs as the main candidate genes for egg-induced HR.
How can we explain a putative role for a TNL intracellular receptor within the plant-egg interaction? So far, TNLs and other NLRs have been associated with qualitative disease resistance traits, often based on HR, and indeed most of the cloned resistance (R) genes are NLRs (Kourelis & van der Hoorn, 2018). Their main function is to activate defense upon perception of “effector proteins”, a diversified array of proteins generally injected inside plant cells by pathogens and piercing-sucking insects to modulate and/or suppress the initial plant pattern-triggered immunity (PTI) response (Basu, Varsani, & Louis, 2018; Toruño, Stergiopoulos, & Coaker, 2016). NLRs either directly or indirectly detect these effectors at different cellular localizations to trigger so-called effector-triggered immunity (ETI) which often leads to HR (Monteiro & Nishimura, 2018). If the TNLs within the PEKlocus are responsible for egg-induced HR, hypothetical “egg effectors” should be able to diffuse from the egg glue through the cell wall (Fig. 4). Interestingly, ongoing efforts suggest that the egg-associated elicitor of HR may not be a protein (Caarls et al. in prep). Alternatively, the TNLs within PEK may not be involved in egg recognition but rather in sensing perturbations of cellular homeostasis (Cui et al., 2015). For example, the TNL SNC1 of A. thalianaactivates upon misregulation of MPK3/6 signalling and unregulated SA accumulation (Wang et al., 2013). Furthermore, certain autoimmune phenotypes in which cell death is regulated by sphingolipids appears to be monitored also by a TNL (Berkey, Bendigeri, & Xiao, 2012; Palma et al., 2010).