Figure 5.

NbADR1 physically associates with the preformed NbPAD4-NbEDS1 complex upon Pst DC3000 ΔHopQ1 infection. A) Co-IP assay showed the interaction of NbEDS1 with NbPAD4. The NbPAD4-6HA or XopQ-6HA (as a negative control) was transiently coexpressed with NbEDS1-3flag or the GFP-flag control in Nicotiana benthamiana leaves. The OD₆₀₀ for each Agrobacterium was adjusted to 0.3. The total proteins were immunoprecipitated with the anti-Flag agarose beads, and the IP product proteins were detected by immunoblotting using the anti-flag or anti-HA antibody. Ponceau-S staining of Rubisco was used as a loading control. B) BiFC assay showed the interactions of NbEDS1 with NbPAD4 in epidermal cells of N. benthamiana leaves. The N-terminal fragment of yellow fluorescent protein (nYFP)-fused NbEDS1, the C-terminal fragment of YFP (cYFP)-fused NbPAD4, and the controls β-glucuronidase (GUS)-nYFP and GUS-cYFP were transiently coexpressed in N. benthamiana leaves, respectively. The OD₆₀₀ for each Agrobacterium was adjusted to 0.3. YFP fluorescence was detected under the confocal microscope with identical gain settings (laser, 514 nm, 6%; collection bandwidth, 526 to 588 nm; pinhole, 1.20 AU; master gain, 660.0) at 30 h after coexpression. Scale bars represent 20 μm. C) The homology model of the NbEDS1-NbPAD4 protein complex based on the structure of the Arabidopsis AtEDS1-AtPAD4 complex. The key residues predicted for NbEDS1-NbPAD4 interface were marked with orange in NbEDS1. D) Co-IP assay showed that the interaction of NbEDS1 with NbPAD4 is largely disrupted by the “LTVIV” mutation (L259E, T262F, V263E, I266E, and V267E) within NbEDS1. The OD₆₀₀ for each Agrobacterium was adjusted to 0.3. The total proteins were immunoprecipitated with the anti-Flag agarose beads, and the IP product proteins were detected by immunoblotting using the anti-flag or anti-HA antibody. Ponceau-S staining of Rubisco was used as a loading control. E) Stomatal apertures in leaves of N. benthamiana eds1 with transient expression of GFP-Flag, NbEDS1-3Flag, or NbEDS1-LTVIV-3Flag, after 1 h of flood treatment with mock (10 mM MgCl₂) or Pseudomonas syringae pv. tomato (Pst) DC3000 ΔHopQ1 (OD₆₀₀ = 0.4). The OD₆₀₀ for each Agrobacterium was adjusted to 0.5. Data are means ± Sd; n = 50 stomata. Letters indicate significant differences by 1-way ANOVA analysis (Tukey's post hoc test, P < 0.05). F) Co-IP assay showed that NbADR1 recruits NbPAD4 and NbEDS1 upon Pst DC3000 ΔHopQ1 spray infection. The YFP-NbADR1 and GFP were transiently coexpressed with XopQ-6HA, NbEDS1-3Flag, or NbPAD4-6HA variants in N. benthamiana leaves, respectively, and the leaves were subsequently inoculated with Pst DC3000 ΔHopQ1 (OD₆₀₀ = 0.4). The OD₆₀₀ for each Agrobacterium was adjusted to 0.3 (except that for YFP-NbADR1 was 0.6). The total proteins were extracted from the leaves at 0, 3, or 6 h after treatment and were immunoprecipitated with the anti-GFP agarose beads. The IP product proteins were detected by immunoblotting using the anti-Flag or anti-HA antibody. Ponceau-S staining of Rubisco served as a loading control. Asterisks indicate the bands of GFP or YFP-ADR1. All these experiments were repeated 3 times with similar results. IB, immunoblotting; IP, immunoprecipitation.