Die Another Way: An EDS1-SAG101 Complex Mediates TNL Immunity in Solanaceous Plants

Disease resistance (R) receptors detect pathogen effector-mediated host manipulation and induce effector-triggered immunity that is often associated with programmed cell death. R proteins are generally conserved across plants, consisting of nucleotide binding site and leucine-rich-repeat domains alongside a variable N-terminal coiled-coil or Toll-like interleukin receptor (TIR) domain, referred to as CNL or TNL receptors, respectively (Adachi et al., 2019). Genetic dissection of TNL immunity in Arabidopsis (Arabidopsis thaliana) has identified a strict dependency on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), which forms heteromeric complexes with sequence-related PHYTOALEXIN DEFICIENT4 (PAD4) or SENESCENCE ASSOCIATED GENE101 (SAG101) that vary in their ability to contribute to immune signaling (Cui et al., 2015). Two complementary studies offer new insights into the evolutionary history and functional diversity of EDS1 family genes in angiosperms (see figure). Gantner et al. (2019) take a functional genomic approach to dissect EDS1-dependent TNL immunity through a combination of CRISPR/Cas-mediated gene editing and Agrobacterium tumefaciens-mediated transient expression-based rescue experiments in the model angiosperm Nicotiana benthamiana. By contrast, Lapin et al. (2019) begin with an evolutionary perspective, interrogating 52 green plant genomes for EDS1 family member homologs that the authors functionally investigate in Arabidopsis (family Brassicaceae) and Nicotiana (family Solanaceae). Evolution and Functional Diversification EDS1-Related Genes. (Top) Phylogenetic analysis of EDS1 family homologs identified by interrogating 52 green plant genomes reveals evolutionary relationships and distinct EDS1 and PAD4 clades specific to angiosperm (flowering) versus conifer (non-flowering) seed plants, whereas a single SAG101 lineage specific to certain angiosperms is observed. Adapted from Lapin et al. (2019), Figure 1. (Bottom) Functional complementation/rescue of XopQ-triggered cell death in the N. benthamiana pad4/sag101a/sag101b triple mutant background occurs only with expression of SAG101b homologs (tomato SlSAG101b shown) irrespective of protein fusion tags. Adapted from Gantner et al. (2019), Figure 3. Phylogenetic analysis (see figure) of EDS1 family genes revealed broad conservation of EDS1 and PAD4 orthologs across seed plants (gymnosperms and terrestrial angiosperms), whereas SAG101 homologs appear to be absent from gymnosperms and angiosperm lineages lacking TNL receptors (monocots, dicots belonging to order Caryophyllales; Lapin et al., 2019). This analysis, alongside that of Gantner et al. (2019), identified candidate EDS1, PAD4, and SAG101a/b (gene duplication) homologs in the solanaceous plants Nicotiana benthamiana and Solanum lycopersicum (tomato). Using complementary sets of protein–protein interaction assays, both groups identified strong conservation of intra-species heterodimer complex formation between EDS1 and PAD4/SAG101 homologs within Arabidopsis or tomato (Gantner et al., 2019; Lapin et al., 2019). In addition, expanded yeast two-hybrid analyses revealed occasional inter-species EDS1-PAD4 interactions in angiosperm models other than Arabidopsis (Lapin et al., 2019). CRISPR/Cas-generated eds1 family mutants, tested alone or in combination, revealed a conserved role for NbEDS1 and, intriguingly, a specific requirement for NbSAG101b in TNL (Roq1-mediated) immunity/cell death activated by the Xanthomonas campestris pv vesicatoria effector XopQ (Gantner et al., 2019; Lapin et al., 2019). This is in contrast to Arabidopsis, where the EDS1-PAD4 complex predominantly contributes to TNL-based effector-triggered immunity (Wagner et al., 2013). A generalizable role for NbEDS1-NbSAG101b in TNL immunity was further supported by its requirement for cell death signaled through additional truncated (autoactivated) or full-length TNL receptors (DM2hTIR, RPS4TIR, and N; Gantner et al., 2019). Cross-species complex transferability assays revealed that SlEDS1-SlPAD4 coexpression rescues Arabidopsis eds1/pad4/sag101 mutants defective in RPP4- or RPP2-mediated TNL immunity to avirulent Hyaloperonospora arabidopsidis, whereas AtEDS1-AtPAD4/AtSAG101 could not rescue Nicotiana eds1 family mutants. Gantner and colleagues speculated that an additional Arabidopsis factor was lacking; Lapin et al. (2019) revealed this factor to be the AtNRG1 helper NLR. This indicated a requirement for matching (coevolved) signaling proteins to function in TNL immunity and host cell death. Evolutionarily and structurally guided analysis of the EDS1-SAG101 complex interface further delineated protein domains and amino acid residues essential for heterodimer formation and TNL immunity in N. benthamiana. Functional analysis of AtPAD4-AtSAG101 gene chimeras (coexpressed with AtEDS1 and AtNRG1; Lapin et al., 2019), or NbSAG101a-NbSAG101b chimeras (Gantner et al., 2019), highlighted the importance of a surface-exposed α-helical region located in the conserved EP domain. Together, these studies reveal fine-tuned constraints on EDS1 family complexes that coevolved to regulate TNL-based immunity in seed plants. Moreover, the contrasting roles of PAD4 and SAG101 in establishing functional heterodimer complexes with EDS1 in different angiosperms further underscores the importance of understanding how evolution has shaped plant immune networks, which may ultimately guide future works aimed at mechanistically unraveling EDS1-mediated immunity.