An EDS1-SAG101 complex functions in TNL-mediated immunity in Solanaceae

Abstract

EDS1 (Enhanced disease susceptibility 1) forms mutually exclusive heterodimers with its interaction partners PAD4 (Phytoalexin-deficient 4) and SAG101 (Sensecence-associated gene 101). Collectively, these complexes are required for resistance responses mediated by nucleotide-binding leucine-rich repeat-type immune receptors (NLRs) possessing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs). Here, immune functions of EDS1 complexes were comparatively analyzed in a mixed species approach relying on Nicotiana benthamiana (Nb), Solanum lycopersicum (Sl) and Arabidopsis thaliana (At). Genomes of most Solanaceae plants including Nb and Sl encode for two SAG101 isoforms, which engage into distinct complexes with EDS1. By a combination of genome editing and transient complementation, we show that one of these EDS1-SAG101 complexes, and not an EDS1-PAD4 complex as previously described in At, is necessary and sufficient for all tested TNL-mediated immune responses in Nb. Intriguingly, not this EDS1-SAG101 module, but mainly Solanaceae EDS1-PAD4 execute immune functions when transferred to At, and TNL functions are not restored in Nb mutant lines by expression of At EDS1 complexes. We conclude that EDS1 complexes do not represent a complete functional module, but co-evolve with additional factors, most likely protein interaction partners, for their function in TNL signaling networks of individual species. In agreement, we identify a large surface on SlEDS1 complexes required for immune activities, which may function in partner recruitment. We highlight important differences in TNL signaling networks between At and Nb, and genetic resources in the Nb system will be instrumental for future elucidation of EDS1 molecular functions. regions with insertions. Sequence conservation was calculated using the rate4site algorithm (Pupko et al., 2002) and mapped at the surface of structural models using pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). Structural models and analysis thereof are provided in Appendix S1. N., Metz, M., Holub, E., Staskawicz, B.J., Daniels, M.J., and Parker, J.E. (1998). Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R genemediated signaling pathways in Arabidopsis. . Dissecting virulence function from recognition: cell death suppression in Nicotiana benthamiana by XopQ/HopQ1-family effectors relies on EDS1dependent immunity. Plant J 91, 430-442. References Aarts,

A coevolved EDS1-SAG101-NRG1 module mediates cell death signaling by TIR-domain immune receptors

“…Several tested Arabidopsis TNLs recognizing oomycete pathogen strains, a TNL autoimmune allele of Suppressor of Npr1-1, Constitutive1 (SNC1) and a TNL pair CHS3/CSA1 displayed varying dependence on AtNRG1 signaling in immunity (Castel et al, 2018; Wu et al, 2018b). By contrast, all so far tested TNL pathogen resistance and cell death responses in N. benthamiana signaled via NbEDS1a, NbNRG1 and NbSAG101b , but did not require NbPAD4 (Adlung et al, 2016; Qi et al, 2018; Gantner et al, 2019). Similarly, we find a dependency of TNL ( Roq1 ) immunity and cell death responses to Xcv bacteria on NbEDS1a and NbSAG101b but not NbPAD4 (Figure 3).…”

“…We used Agrobacteria-mediated transient expression of proteins in N. benthamiana in combination with simultaneous Xcv infiltration or XopQ-myc agroinfiltration of leaf sectors to monitor TNL Roq1 resistance and cell death, respectively (Figure 3, Supplemental Figure 3). For the assays (setup outlined on Figure 3A), WT N. benthamiana and reported Nbeds1a, Nbpad4 single and Nbeds1a pad4 double mutants (Ordon et al, 2017), as well as N. benthamiana pad4 sag101a sag101b triple mutant (abbreviated to Nb-pss , (Gantner et al, 2019)) were used. We first tested whether agroinfiltration interferes with Roq1 resistance to Xcv growth (Supplemental Figure 3A).…”

“…This also shows that FLAG- Sl EDS1 is functional in N. benthamiana Roq1 -dependent Xcv growth restriction. To test activities of Nb SAG101a or Nb SAG101b individually in Roq1 immunity, an Nbeds1a pad4 sag101a sag101b quadruple mutant ( Nb-epss ) was selected from a cross between Nbeds1a with Nb-pss (Ordon et al, 2017; Gantner et al, 2019). Transient co-expression of Nb SAG101b-GFP but not Nb SAG101a-GFP with functional FLAG- Sl EDS1 (Figure 3C) restored resistance to Xcv growth in Nb- epss , although not completely to the level of WT N. benthamiana (Figure 3D).…”

“…We next tested effects of individually mutating two At EDS1 EP domain amino acids F419E and H476F which are on the At EDS1 EP domain α-helical coil surface closest to the At SAG101 patch found to be necessary for Roq1 cell death (Figure 6C). Mutations at the Sl EDS1 position F435, which corresponds to At EDS1 F419, impaired Sl EDS1 function in Roq1 cell death (Gantner et al, 2019). Alongside the two At EDS1 mutants, we tested two At EDS1 variants that are non-functional in Arabidopsis TNL immunity: At EDS1 LLIF with weak EDS1-partner N-terminal binding (Figure 1C, 6C, (Wagner et al, 2013; Cui et al, 2018)), and At EDS1 R493A with impaired EDS1-PAD4 heterodimer signaling (Bhandari et al, 2019).…”

“…In dicotyledonous species, activated intracellular TNL receptors converge on the non-NLR lipase-like protein EDS1, which transduces signals to downstream defense and cell death pathways to stop pathogen growth (Wiermer et al, 2005; Adlung et al, 2016; Qi et al, 2018; Gantner et al, 2019). In Arabidopsis, At EDS1 functions in a heterodimer with one of its partners, At PAD4, to transcriptionally mobilize anti-microbial defense pathways and bolster SA-dependent programs that are important for basal and systemic immunity (Rietz et al, 2011; Wagner et al, 2013; Cui et al, 2017; Bhandari et al, 2019).…”