An EDS1-SAG101 Complex Is Essential for TNL-Mediated Immunity in Nicotiana benthamiana

Abstract

Heterodimeric complexes containing the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) are regarded as central regulators of plant innate immunity. In this context, a complex of EDS1 with PHYTOALEXIN DEFICIENT4 (PAD4) is required for basal resistance and signaling downstream of immune receptors containing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs) in Arabidopsis (Arabidopsis thaliana). Here we analyze EDS1 functions in the model Solanaceous plant Nicotiana benthamiana (Nb). Stable Nb mutants deficient in EDS1 complexes are not impaired in basal resistance, a finding which contradicts a general role for EDS1 in immunity. In Nb, PAD4 demonstrated no detectable immune functions, but TNL-mediated resistance responses required EDS1 complexes incorporating a SENESCENCE ASSOCIATED GENE101 (SAG101) isoform. Intriguingly, SAG101 is restricted to those genomes also encoding TNL receptors, and we propose it may be required for TNL-mediated immune signaling in most plants, except the Brassicaceae. Transient complementation in Nb was used for accelerated mutational analyses while avoiding complex biotic interactions. We identify a large surface essential for EDS1-SAG101 immune functions that extends from the N-terminal lipase domains to the C-terminal EDS1-PAD4 domains and might mediate interaction partner recruitment. Furthermore, this work demonstrates the value of genetic resources in Nb, which will facilitate elucidation of EDS1 functions.

Figure 1.

Occurrence and Expression of EDS1 Family Genes in Solanaceae. (A) Phylogenetic clustering of putative EDS1 family proteins from Solanaceae and control species. The tree was midpoint rooted. At, Arabidopsis thaliana; Ca, Capsicum annuum (SAG101 orthologs from cultivars Zunla and CM334 shown); Cc, Coffea canephora; Ma, Musa accuminata; Mg, Mimulus guttatus; Pi, Petunia inflata; Nb, Nicotiana benthamiana; Sl, Solanum lycopersicum; Sm, Solanum melongena. (B) Expression of EDS1 family genes in N. benthamiana. Plants were challenged with virulent (Xcv ΔxopQ) or avirulent (Xcv 85-10) Xanthomonas campestris pv vesicatoria bacteria or mock treated (MgCl₂). RNA was extracted at indicated time points, and expression of EDS1 family genes measured by quantitative RT-PCR. Displayed data originates from normalization to PP2A expression, and similar results were obtained when using Elongation Factor 1-α (EF1α) for normalization. Data points represent means of four biological replicates with se shown.

Figure 2.

Complex Formation and Localization of Tomato EDS1 Family Proteins. (A) Protein localization in living cells detected by confocal laser scanning microscopy. Indicated proteins (from tomato) were transiently coexpressed as GFP fusions together with SlEDS1 in N. benthamiana leaf tissues by agroinfiltration, and protein localization was analyzed 3 dpi. Localization of single proteins and integrity of fluorophore fusions is shown in Supplemental Figure 3. Scale bar = 20 µm. (B) Formation of complexes by tomato EDS1 proteins. Indicated proteins were transiently (co)expressed in N. benthamiana by agroinfiltration. At 3 dpi, extracts were used for StrepII purification, and total extracts and eluates analyzed by immunoblotting. Ponceau staining is shown as loading control.

Figure 3.

Immune Responses of N. benthamiana Mutant Lines Deficient in EDS1 Family Genes or Roq1 (A) Recognition of XopQ in different mutant lines. Indicated N. benthamiana lines were challenged with XopQ-translocating P. fluorescens bacteria (top; infiltrated at OD₆₀₀ = 0.2) or Xcv strain 85-10 bacteria (bottom; infiltrated at OD₆₀₀ = 0.4). Phenotypes were documented at 4 dpi. Similar results were obtained in three independent experiments, and multiple plants of the indicated genotypes were infiltrated in each experiment. (B) Bacterial growth of Xcv bacteria on mutant lines. Indicated lines were infected with Xcv strain 85-10 or a corresponding mutant strain lacking XopQ (ΔxopQ). Means and sd of four biological replicates are shown. Letters indicate statistically significant differences as determined by one-way ANOVA and Fisher LSD post hoc test (P < 0.01). (C) Bacterial growth in eds1 and roq1 mutant lines. As in (B), but means and SD of eight biological replicates are shown for days 3 and 6. The roq1 mutant line was a T₁ line segregating for two disruptive alleles at the Roq1 locus (Supplemental Fig. 4F). (D) Reconstitution of XopQ detection in the pss triple mutant line. By agroinfiltration, XopQ was expressed alone or in combination with PAD4, SAG101a, or SAG101b (from tomato and fused to a StrepII and 4 × c-myc tag or GFP). Phenotypes were documented 5 dpi. (E) Immunodetection of fusion proteins expressed in (D). Samples were taken 3 dpi from a second infiltration on the same leaf shown in (D). Ponceau staining of the membrane is shown as loading control.

Figure 4.

Genetic Dependencies of TNL-Type Immune Receptors in N. benthamiana and Arabidopsis. (A) EDS1-dependent cell-death induction requires SAG101b in N. benthamiana. Inducers of presumably EDS1-dependent cell death (DM2h^TIR – DM2h_(1-279); RPS4^TIR – RPS4_(1-234)_E111K [Swiderski et al., 2009]; XopQ – XopQ-myc; p50 + N – p50-Cerulean + N-Citrine [Burch-Smith et al., 2007]) were expressed in different N. benthamiana lines, as indicated (left), or coexpressed with PAD4, SAG101a or SAG101b (from tomato and fused to a 4xmyc-TwinStrep tag) in the pss mutant line (right). Phenotypes were documented 5 dpi. (B) Functionality and genetic dependency of Roq1 in Arabidopsis. A T-DNA construct coding for Roq1 under control of an RPS6 promoter fragment and an ocs terminator was transformed into the indicated Arabidopsis lines. Four-week-old control and T₁ plants were infected with Pst DC3000 bacteria (syringe infiltration, OD₆₀₀ = 0.001). Symptom development was documented 3 dpi. At least eight independent T₁ plants were tested for each genotype per replicate, and the experiment was conducted three times with similar results.

Figure 5.

Cross-Species Transfer of EDS1 Family Genes. (A) Arabidopsis EDS1-PAD4-SAG101 proteins cannot functionally replace EDS1-SAG101b in N. benthamiana. Indicated proteins were expressed (by agroinfiltration) either in eds1 or pss mutant lines, and phenotypes were documented 7 dpi. Arabidopsis proteins were expressed with or without an epitope tag, and images originate from untagged proteins (Supplemental Figure 6 shows details on T-DNA constructs and protein detection). (B) Tomato EDS1-PAD4 can function in TNL signaling in Arabidopsis. Col eds1-2 pad4-1 double mutant was transformed with constructs for expression of EDS1 and PAD4, either from Arabidopsis or tomato and with or without an epitope tag (Supplemental Figure 6C) and under control of Arabidopsis promoter fragments. Segregating T₂ populations were selected with BASTA, and 3-week-old plants infected with H. arabidopsidis isolate Cala2. True leaves were used for Trypan Blue staining 7 dpi. At least four independent T₂ populations were tested for each construct with similar results. Lines expressing untagged proteins were used for infection assays. Lines expressing epitope-tagged proteins were used for immunodetection (Supplemental Figure 6D). fh, free hyphae; hr, hypersensitive response.

Figure 6.

EDS1-SAG101b Heterocomplexes Are the Functional Modules in N. benthamiana TNL Signaling. (A) Homology model of the tomato EDS1-SAG101b complex used for transient reconstitution of TNL-signaling in mutant N. benthamiana tissues. The N-terminal (Nb) and C-terminal (Cb) domains of SAG101b are depicted in dark and light green, respectively. The inset shows the symmetrically arranged helices of EDS1 and SAG101b forming the N-terminal interaction interface. Amino acids targeted by mutagenesis are shown as sticks and are highlighted in pink (EDS1) and orange (SAG101b), respectively. (B) Conservation of surface-exposed amino acids in SlEDS1. SlSAG101b is shown in ribbon presentation (green). EDS1 residues functionally interrogated by mutagenesis are marked. (C) Interaction of EDS1 variants with SAG101b. Indicated proteins were (co)expressed in N. benthamiana by agroinfiltration and tissues used for StrepII purification at 3 dpi. (D) Functionality of EDS1 variants affected in heterocomplex formation. Indicated variants (as in [C], with C-terminal 6xHA) were coexpressed with XopQ-myc in eds1 mutant plants, and plant reactions were documented 7 dpi. (E) Interaction of SAG101b variants with EDS1. As in [C], but SAG101b-StrepII variants were coexpressed with EDS1. (F) Functionality of SAG101b variants affected in heterocomplex formation. SAG101b-StrepII variants were coexpressed with XopQ-myc in pss mutant plants, and plant reactions were documented 7 dpi.

Figure 7.

Identification of Nonfunctional EDS1-SAG101b Complex Variants. (A) Interaction of EDS1 variants with SAG101b. Indicated proteins were (co)expressed in N. benthamiana by agroinfiltration. Tissues were used 3 dpi for StrepII purification. (B) Immune activities of EDS1 variants. Indicated EDS1 variants (with C-terminal 6xHA tag) were transiently coexpressed with XopQ-myc in eds1 mutant plants by agroinfiltration. Plant reactions were documented 7 dpi. (C) Structural basis for SlSAG101a-SlSAG101b chimeric proteins. EDS1 and SAG101 both contain an N-terminal hydrolase-like and a C-terminal EP domain. In the heterodimer, an N-terminal interface is formed by the hydrolase-like domains, and a C-terminal interface is formed by the EP domains. For chimeras, the N terminus of SAG101b (aa 1-322) or SAG101a (aa 1-339) was fused with the C terminus of SAG101a (aa 340-581) or SAG101b (aa 323-567), respectively. (D) Heterocomplex formation by SAG101 chimeric proteins. SAG101 chimeras and native SAG101 isoforms (with a C-terminal StrepII tag) were coexpressed with EDS1-6xHA by agroinfiltration. Tissues were used 3 dpi for StrepII-purification. (E) Functionality of SAG101 chimeric proteins. Indicated proteins were expressed together with XopQ-myc in pss mutant plants by agroinfiltration. Plant reactions were documented 7 dpi.