Analysis of the ZAR1 Immune Complex Reveals Determinants for Immunity and Molecular Interactions

Abstract

Plants depend on innate immunity to prevent disease. Plant pathogenic bacteria, like Pseudomonas syringae and Xanthomonas campestris, use the type III secretion system as a molecular syringe to inject type III secreted effector (T3SE) proteins in plants. The primary function of most T3SEs is to suppress immunity; however, the plant can evolve nucleotide-binding domain-leucine-rich repeat domain-containing proteins to recognize specific T3SEs. The AtZAR1 NLR induces strong defense responses against P. syringae and X. campestris The P. syringae T3SE HopZ1a is an acetyltransferase that acetylates the pseudokinase AtZED1 and triggers recognition by AtZAR1. However, little is known about the molecular mechanisms that lead to AtZAR1-induced immunity in response to HopZ1a. We established a transient expression system in Nicotiana benthamiana to study detailed interactions among HopZ1a, AtZED1, and AtZAR1. We show that the AtZAR1 immune pathway is conserved in N. benthamiana and identify AtZAR1 domains, and residues in AtZAR1 and AtZED1, that are important for immunity and protein-protein interactions in planta and in yeast (Saccharomyces cerevisiae). We show that the coiled-coil domain of AtZAR1 oligomerizes, and this domain acts as a signal to induce immunity. This detailed analysis of the AtZAR1-AtZED1 protein complex provides a better understanding of the immune signaling hub controlled by AtZAR1.

Figure 1.

Coexpression of HopZ1a and AtZED1 in N. benthamiana leads to a strong HR dependent on NbZAR. A. tumefaciens carrying constructs expressing Empty Vector (EV), HopZ1a, HopZ1a^C/A, AtZED1, and/or AtZAR1 were syringe infiltrated into N. benthamiana leaves. Leaves with an HR are indicated to the left of the red boxes, and ion leakage was measured 3 h (T0) and 24 h (T21) after dexamethasone induction. The error bars indicate the se from six repetitions. The letters beside the bars indicate significance groups, as determined by a one-way ANOVA comparison followed by a Tukey’s post hoc test (P value ≤ 0.05). The experiment was performed at least three times with similar results. A, HopZ1a-HA or HopZ1a^C/A-HA was coexpressed with EV-5xMyc or AtZED1-5xMyc in N. benthamiana leaves. B, HopZ1a-HA or HopZ1a^C/A-HA was coexpressed with AtZED1-3xFlag, and EV-5xMyc or AtZAR1-5xMyc in N. benthamiana leaves silenced for GUS or NbZAR genes. C, Immunoblots of CoIP assays for the interaction between AtZAR1 or NbZAR1 proteins with AtZED1. AtZAR1, NbZAR1, or NbZAR1^CC-NB with a 5xMyc tag was coexpressed with AtZED1 containing a 3xFlag tag in N. benthamiana leaves. Western-blot analysis (WB) was performed on the crude extract (input) or the immunopurified fractions (IP) from anti Myc beads. An asterisk indicates the band corresponding to the protein of interest, if multiple bands were observed. The molecular masses of the proteins are: AtZED1-3xFlag 42 kD, AtZAR1-5xMyc 107 kD, NbZAR1 5xMyc 106 kD, and NbZAR1^CC-NB-5xMyc 55 kD. The experiments were repeated two times with similar results.

Figure 2.

The AtZAR1^LRR region is necessary for interaction and HR in N. benthamiana. A, Schematic representation of AtZAR1 protein domains and truncations. The colored boxes correspond to the following domains: yellow is the CC domain, blue is the nucleotide-binding-Apaf1-R-CED4 (NB-ARC) domain, gray is the linker region, and green is the Leu-rich repeat (LRR) domain. All constructs were expressed under the control of a dexamethasone-inducible promoter and with a C-terminal 5xMyc tag. The molecular masses are shown for the fusion with a 5xMyc tag. B, Immunoblots of CoIP assays for the interaction between AtZAR1 deletions and AtZED1. AtZAR1 or the AtZAR1^Δ1 to AtZAR1^Δ4 deletions with a 5xMyc tag were coexpressed with AtZED1 containing a 3xFlag tag in N. benthamiana leaves. Western-blot analysis (WB) was performed on the crude extract (input) or the immunopurified fractions (IP) from anti Myc beads. An asterisk indicates the band corresponding to the protein of interest, if multiple bands were observed. EV is Empty Vector. The experiments were repeated at least three times with similar results. C, AtZAR1^Δ1 is unable to complement NbZAR1-VIGS. A. tumefaciens carrying constructs expressing HopZ1a-HA or HopZ1a^C/A-HA was coexpressed with AtZED1-3xFlag, and EV-5xMyc, AtZAR1-5xMyc, or AtZAR1^Δ1 in N. benthamiana leaves silenced for GUS or NbZAR genes. The HR is shown 24 h after dexamethasone induction and is indicated with an asterisk. The scale bar is 1 cm. D, AtZAR1^Δ1 is unable to complement NbZAR1-VIGS. The electrolyte leakage level for a set of coinfiltrations in NbZAR-VIGS plants was measured 3 h (T0) and 24 h (T21) after dexamethasone induction. The error bars indicate the se from six repetitions. The letters beside the bars indicate significance groups, as determined by a one-way ANOVA comparison followed by a Tukey’s post hoc test (P value ≤ 0.05). The experiment was performed at least three times with similar results.

Figure 3.

Multiple domains of AtZAR1 interact with AtZED1. A, Immunoblots of CoIP assays for interactions between AtZAR1 domains and AtZED1. AtZAR1, AtZAR1^CC, AtZAR1^NB, or AtZAR1^LRR with a 5xMyc were coexpressed with AtZED1 containing a 3xFlag tag in N. benthamiana leaves. Western-blot analysis (WB) was performed on the crude extract (input) or the immunopurified fractions (IP) from anti Myc beads. An asterisk indicates the band corresponding to the protein of interest, if multiple bands were observed. EV, Empty vector. The experiments were repeated at least three times with similar results. B, AtZED1 was constructed as a fusion with the DNA-binding domain and tested for interaction against AtZAR1^CC2, AtZAR1^CC, AtZAR1^CC-NB3, AtZAR1^LRR, or EV as a fusion to the activation domain in the LexA yeast two-hybrid system. HopF2_PtoDC3000 and its chaperone ShcF2_PtoDC3000 were used as positive controls, because they are known to strongly interact (Shan et al., 2004). The experiment was performed twice with similar results. C, Bimolecular fluorescence complementation assay of AtZED1-AtZAR1^CC interactions. A. tumefaciens carrying AtZAR1^CC, AtZED1, or EV as a fusion to the cYFP or the nYFP was mixed in equivalent optical densities and pressure-infiltrated into the leaves of N. benthamiana. Top, the YFP channel alone. Bottom, a merge of the YFP channel and bright field (BF). Leaf sections were imaged using a Zeiss LSM710 confocal scanning microscope 24 to 48 h after induction. The red arrowheads show the nuclei. Bar = 50 μm. The experiment was performed twice with similar results.

Figure 4.

AtZAR1^CC-YFP induces constitutive HR when inducibly expressed in N. benthamiana. A. tumefaciens carrying constructs expressing Empty Vector (EV) or AtZAR1 domains with a C-terminal YFP fusion was syringe infiltrated into N. benthamiana leaves. The experiments were performed at least three times with similar results. A, For each construct, the percentage of leaves with a strong HR (black bars), medium HR (hatched bars), weak HR (dotted bars), or no HR (white bars) is shown. B and C, Electrolyte leakage was measured 15 (T0) and 36 (T21) hours after dexamethasone induction. The error bars indicate the se from six repetitions. The letters beside the bars indicate significance groups, as determined by a one-way ANOVA comparison followed by a Tukey’s post hoc test (P value ≤ 0.05). C, Electrolyte leakage for AtZAR1^CC-YFP or EV-YFP was grouped into four phenotypic categories based on the macroscopic HR (none, weak, medium, and strong) observed 36 h after dexamethasone induction.

Figure 5.

AtZAR1^CC oligomerizes in yeast and in planta. A, AtZAR1^CC or AtZAR1^CC2 was constructed as a fusion with the DNA-binding domain and the activation domain, and tested for interaction against themselves or empty vector (EV) in the LexA yeast two-hybrid assay. HopF2_PtoDC3000 and its chaperone ShcF2_PtoDC3000 were used as positive controls, because they are known to strongly interact (Shan et al., 2004). The experiment was performed twice with similar results. B, Bimolecular fluorescence complementation assay of AtZAR1^CC interactions. A. tumefaciens carrying AtZAR1^CC-YFP, AtZAR1^CC, and EV as a fusion to the cYFP or nYFP was mixed in equivalent optical densities and pressure-infiltrated into the leaves of N. benthamiana. Left, the YFP channel alone. Right, a merge of the YFP channel and bright field (BF). Leaf sections were imaged using a Zeiss LSM710 confocal scanning microscope 24 to 48 h after induction. The red arrowheads show the nuclei. Bar = 50 μm. The experiment was performed twice with similar results.

Figure 6.

Atzar1 mutants are susceptible to P. syringae carrying HopZ1a, and AtZAR1^G645E is unable to interact with AtZED1. A, AtZAR1 sequence schematic showing the three main domains. The colored boxes correspond to the following domains: yellow is the CC domain, blue is the nucleotide-binding-Apaf1-R-CED4 (NB-ARC) domain, gray is the linker region, and green is the leucine-rich repeat (LRR) domain. The amino acid changes induced by the mutation are indicated above the schematic. The P816L substitution was previously described in Wang and colleagues (2015b). B, Half-leaves of Arabidopsis Col-0, Atzar1-1, Atzar1^V202M, Atzar1^S291N, Atzar1^L465F, Atzar1^G645E, or Atzar1^S831F were infiltrated with 10 mm MgCl₂ or PtoDC3000 carrying empty vector (EV) or HopZ1a. The bacteria were pressure-infiltrated into the leaves at 5 × 10⁷ cfu/mL. Photos were taken 20 h after infiltration. The number of leaves showing an HR is indicated below the leaf. HRs are marked with an asterisk. Bar = 1 cm. C, PtoDC3000 carrying HopZ1a was syringe-infiltrated at ∼1 × 10⁵ cfu/mL into the leaves of Arabidopsis Col-0, Atzar1-1, or Atzar1 point mutants, and bacterial counts were determined 1 h postinfiltration (day 0) and 3 d postinfection (day 3). One-factor ANOVA using a general linear model (GLM) followed by multiple comparisons of means using Tukey’s post hoc test was performed to determine significant differences between plant genotypes, and significant differences are indicated by the letters above the bars. Error bars indicate sd from the mean. The experiment was repeated three times with similar results. D, Immunoblot showing the coimmunopurification of AtZAR1 with AtZED1 from N. benthamiana tissues. AtZAR1 with a 5xMyc epitope tag was coexpressed with AtZED1 or AtZED1 with a 3xFlag epitope tag in N. benthamiana. Western-blot analysis (WB) was performed on the crude extract (input) or the immunopurified fractions (IP) from anti Myc beads. An asterisk indicates the band corresponding to the protein of interest, if multiple bands were observed. This experiment was repeated three times with similar results.

Figure 7.

AtZED1^D231N is impaired in the HopZ1a-induced HR. A. tumefaciens carrying constructs expressing empty vector (EV) or HopZ1a (A) or HopZ1a^C/A (B) coexpressed with AtZED1 or AtZED1 point mutants was syringe infiltrated into N. benthamiana leaves. Leaves with an HR are indicated to the left of the red boxes, and ion leakage was measured 3 h (T0) and 24 h (T21) after dexamethasone induction. The error bars indicate the se from six repetitions. The letters beside the bars indicate significance groups, as determined by a one-way ANOVA comparison followed by a Tukey’s post hoc test (P value ≤ 0.05). The experiment was performed at least three times with similar results.

Figure 8.

AtZED1 point mutants are impaired in their interactions with AtZAR1 and HopZ1a. A, Immunoblot showing the coimmunopurification of AtZAR1 with AtZED1 transiently expressed in N. benthamiana tissues. AtZAR1 or Empty vector (EV) tagged with 5xMyc epitope was coexpressed with AtZED1 or AtZED1 tagged with 3xFlag tag epitope in N. benthamiana. Western-blot analysis (WB) was performed on the crude extract (input) or the immunopurified (IP) fractions from anti Myc beads. An asterisk indicates the band corresponding to the protein of interest, if multiple bands were observed. The experiment was repeated three times with similar results. B, Immunoblot showing the coimmunopurification of AtZAR1^LRR with AtZED1 transiently expressed in N. benthamiana tissues. AtZAR1^LRR or EV tagged with 5xMyc epitope was coexpressed with AtZED1 or AtZED1 tagged with 3xFlag tag epitope in N. benthamiana. WB was performed on the crude extract (input) or the IP fractions from anti Myc beads. An asterisk indicates the band corresponding to the protein of interest, if multiple bands were observed. The experiment was repeated two times with similar results. C and D, Bimolecular fluorescence complementation assay of AtZED1 or AtZED1 mutants with AtZAR1^CC (C) or HopZ1a^C/A (D). A. tumefaciens carrying AtZAR1^CC (C), or HopZ1a (D) as a fusion to the nYFP was mixed in equivalent optical densities with A. tumefaciens carrying AtZED1, AtZED1 mutants, or EV as a fusion to the cYFP and pressure-infiltrated into the leaves of N. benthamiana. Top, YFP channel alone. Bottom, a merge of the YFP channel and bright field (BF). Leaf sections were imaged using a Zeiss LSM710 confocal scanning microscope 24 to 48 h after induction. The red arrowheads show the nuclei. Bar = 50 μm. The experiment was performed twice with similar results.