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, 178 (3), 1310-1331

Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1

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Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1

Erik J Slootweg et al. Plant Physiol.

Abstract

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

Figures

Figure 1.
Figure 1.
Assessing the role of the hydrophobic interface between the N- and C-terminal two-helix segments of the Rx1 CC domain. A, The two halves of the Rx1 CC interact in a coimmunoprecipitation assay. Combinations of coexpressed CC fragments H1-H2 (amino acids 1–45) and H3-H4 (amino acids 45–116) fused to 4xMyc or 4xHA affinity tags were subjected to anti-c-Myc immunoprecipitation. Extracts of leaves expressing the single strands were included as controls for aspecific binding and protein stability. The blots show the cell extract used as input and the result of the immunoprecipitation as detected with anti-c-Myc and anti-hemagglutinin (HA) antibodies. B, Targeted mutagenesis of hydrophobic residues in the heptad repeats. A set of constructs was made in which three apolar residues per α-helix were exchanged for Glu (E). The groups of three substitutions were named Z1 to Z4 in correspondence with the predicted helix in which they are positioned. Hydrophobic positions of the heptad repeat are indicated in yellow in the CC structure and the amino acid sequence. The substituted residues are indicated. C, Effects of mutations Z1 to Z4 on the intramolecular interaction of the Rx1 CC domain. Mutant versions of H1-H2-4Myc were coexpressed with wild-type (wt) H3-H4-4HA or vice versa. The H1-H2-4Myc constructs were pulled down by anti-Myc antibodies, and the coimmunoprecipitated H3-H4-4HA constructs were visualized by anti-HA immunoblot. Some of the lanes have been rearranged to align input and corresponding immunoprecipitation results; this is indicated by solid lines on the immunoblot. D, Mutant versions of H1-H2-4Myc and H3-H4-4HA were transiently expressed with Rx1 NB-ARC-LRR-GFP (amino acids 142–937) in the presence of the coat protein (CP106) of PVX to assess the cell death response. The combinations of the Rx1 segments are indicated in a schematic drawing. Images were taken 5 d post infiltration (dpi). To assess PVX resistance, variants of H1-H2 and H3-H4 were coinfiltrated in N. benthamiana leaves with the NB-ARC-LRR-GFP and a PVX:GFP amplicon. Virus accumulation was determined by anti-PVX CP ELISA of leaf extracts at 5 dpi with alkaline phosphatase-conjugated antibodies. p-Nitrophenol accumulation was detected via its A405. As a control (ctrl), PVX:GFP was expressed in the absence of Rx1.
Figure 2.
Figure 2.
Effects of mutations Z1 to Z4 on the functionality of full-length Rx1, either as the separate groups of mutations or in the combinations of Z1 with Z4 (Rx1 Z14) and Z2 with Z3 (Rx1 Z23). A, Rx1 constructs were coexpressed with the CP of PVX to assess their ability to induce elicitor-dependent cell death. The constructs were coexpressed with GFP to detect autoactivity. Images of the cell death response at 5 dpi are shown. The level of cell death was quantified by measuring the absorption of light at 655 nm by chlorophyll in a leaf extract (see “Materials and Methods”). A stronger cell death leads to lower chlorophyll levels. The error bars represent se (n = 8). Different letters represent significant differences (one-way ANOVA with a posthoc Tukey’s test, P < 0.05). B, Rx1-mediated resistance was tested by coexpressing the Rx1 constructs with a PVX:GFP amplicon and subsequent detection of the CP of PVX with an alkaline phosphatase-conjugated antibody in an ELISA at 5 dpi. The error bars represent se (n = 8). Statistical analysis is as in A.
Figure 3.
Figure 3.
Ala substitution of aromatic and hydrophobic surface residues in helix 1 and helix 4 of the CC. A, Two groups of mainly aromatic residues in the CC (highlighted in magenta in the structure and amino acid sequence) were substituted for Ala (A). In helix 1, Y3A and M10A were combined and named S1. In helix 4, the substitutions W90A, F93A, and F94A were introduced, and this combination was referred to as S4. Both groups of substitutions were introduced in several constructs, including the H1-H2 and H3-H4 CC strands and full-length Rx1. B, Effects of the S1 and S4 mutations on the interaction between the H1-H2 and H3-H4 strands of the CC. Anti-HA immunoprecipitation was performed to study the interaction between coexpressed wild-type (wt) and mutated (S1 and S4) versions of the H1-H2-4Myc and H3-H4-4HA constructs. Expression of only H1-H2-4Myc or H3-H4-4HA was used as a negative control. C, The effects of the S1 and S4 mutations on Rx1 functioning were tested for the transcomplementation of H1-H2 and H3-H4 with the NB-ARC-LRR, as shown by a schematic drawing. Resistance was tested by coexpressing the Rx1 constructs with the PVX:GFP amplicon in N. benthamiana followed by an anti-PVX CP ELISA with extracts of the infiltrated leaf material. Error bars indicate the sd of six samples. The ability of the constructs to induce cell death (HR) was assessed by coexpression of the complementary Rx1 fragments with the avirulent PVX elicitor CP106. D, The effects of S1 and S4 on the functioning of full-length Rx1 were tested in transient PVX resistance and cell death assays. Mutant constructs (S1, S4, or the combination S14) and wild-type Rx1 were coexpressed with a PVX:GFP amplicon to test for resistance. A leaf infiltrated with PVX:GFP, but not Rx1, was included as a control. Error bars indicate the sd (n = 6). To assess the ability of the mutants to induce a cell death response, the full-length Rx1 constructs were coexpressed with the avirulent PVX CP, and images of the response were taken at 3 dpi. As a control for the autoactive cell death response, the Rx1 constructs were coexpressed with GFP.
Figure 4.
Figure 4.
A and B, Immunoprecipitation assay to test if the loss of interaction between H1-H2 and H3-H4 due to mutations (Z1–Z4, S1, and S4) causes the strands to dissociate in the complete CC and expose internal binding surfaces. HA-tagged versions of the wild-type (wt) and mutant Rx1 CC (amino acids 1–142) were coexpressed with wild-type H1-H2-4Myc (amino acids 1–45; A) or H3-H4-4Myc (amino acids 45–116; B), as indicated by schematic overviews of the constructs. The CC constructs were immunoprecipitated with antibodies against the HA tag. Coimmunoprecipitation of the interacting H1-H2 or H3-H4 strands was detected by anti-Myc immunoblotting. C, Complementation of the loss of function caused by the Z1, Z2, and Z4 mutations via coexpression of wild-type H1-H2 or H3-H4 strands. Full-length Rx1 mutant constructs displaying decreased elicitor-dependent cell death (Z1, Z2, and Z4) or decreased PVX resistance (Z1 and Z4) were coexpressed with H1-H2-GFP, H3-H4-GFP, or GFP to investigate if the presence of the wild-type strands could restore the functionality of Rx1. Combinations in which Rx1-mediated cell death was reconstituted are indicated by blue asterisks. These three combinations also were tested in the absence of the CP to determine if the coexpressed CC fragment induces an autoactive response (row of images at bottom). Resistance was assessed by the detection of PVX in an ELISA (error bars represent the sd; n = 8). The CC strands or GFP were coexpressed with PVX:GFP in the absence of Rx1 as a negative control.
Figure 5.
Figure 5.
Effects of the mutations in the CC on the domain interactions of Rx1. A, Effects of the mutations Z1 to Z4, S1, and S4 on the interaction between the CC and the NB-ARC-LRR of Rx1. A Myc-tagged construct of the Rx1 NB-ARC-LRR (amino acids 144–937) was coexpressed with 4xHA-tagged constructs of the wild-type (wt) and mutant CC. The NB-ARC-LRR was pulled down using anti-Myc antibodies, and the coimmunoprecipitation of the CC constructs was detected via an anti-HA immunoblot. B, Immunoprecipitation assay testing the effect of the CC mutations on the interaction between the CC-NB-ARC and LRR of Rx1. 4Myc-tagged CC-NB-ARC constructs were coexpressed with 4HA-LRR. Immunoprecipitation was performed with anti-Myc antibodies, and the coprecipitation of the HA-tagged LRR was visualized by anti-HA immunoblot. C, Complementation of functionality for full-length Rx1 mutants by coexpression of the wild-type CC. The full-length Rx1 constructs carrying the combined Z14 or Z23 mutations were coexpressed with wild-type versions of the individual CC strands (H1-H2 and H3-H4) or the complete CC (shown in the schematic drawing at top). The PVX CP was coexpressed to test if the presence of the CC or CC strands could restore the ability of the Rx1 mutant to initiate a cell death response. The combinations in which a cell death occurred are marked with white asterisks. These combinations were tested with GFP instead of the CP to test for autoactivity (row of images at bottom). D, Interaction study to test if mutations in the CC disrupt the interaction between the CC and NB-ARC-LRR in the full-length Rx1 protein and, thereby, make the CC-binding surface on the NB-ARC-LRR accessible for coexpressed CC constructs. GFP-4HA-tagged constructs of full-length Rx1 (wild type, Z14, Z1, S1, and S4) were coexpressed with a wild-type CC-4Myc construct. The full-length Rx1 constructs were immunoprecipitated by anti-HA antibodies, and the coimmunoprecipitation of the CC was detected via anti-Myc immunoblot. The truncated NB-ARC-LRR served as a positive control for this interaction. HA-tagged CC and HA-tagged GFP constructs were used as negative controls.
Figure 6.
Figure 6.
Effects of the mutations in the CC on the interaction with RanGAP2 and the subcellular localization of Rx1. A, Coimmunoprecipitation of HA-tagged versions of the Rx1 CC variants Z1 to Z4, S1, S4, and the wild type (wt) with the N-terminal WPP domain of RanGAP2 (Rg2-ΔC-GFP). Equal loading of the input material is shown by the Coomassie Brilliant Blue-stained Rubisco (CBB). B, Effects of the presence of the RanGAP2 WPP domain on the interaction between H1-H2 and H3-H4. Anti-HA immunoprecipitation of H1-H2-4HA coexpressed with H3-H4-4Myc in the presence or absence of mCherry-tagged RanGAP2 WPP domain (Rg2-ΔC-mCh) is shown. Two exposures (30 and 120 s) are shown for the anti-Myc immunoblot with the results of the anti-HA immunoprecipitation to show the two bands of different intensity. C, Full-length Rx1 constructs (wild type, Z1–Z4, S1, and S4) with a C-terminal GFP fusion were imaged using confocal microscopy after 2 d of expression in N. benthamiana leaves. The images show nuclei (n) and surrounding cytoplasm in representative cells. Chlorophyll autofluorescence is shown in red. Bar = 10 μm for all images. The ratio of GFP fluorescence intensity in the cytoplasm and nucleus was determined in seven to 12 cells for each construct. The graph shows the average cytoplasmic intensity/nuclear intensity ratio (IC/IN). The error bars represent the se. Higher values indicate a more cytoplasmic localization profile. D, Coexpression of Rx-GFP variants with RanGAP2 constructs to test the effect on the localization of Rx. Rx-GFP (wild type, S1, and S4) was coexpressed with either full-length RanGAP2-mCherry (Rg2-mCh) or with Rg2-ΔC-mCh-NLS, a construct in which the RanGAP2 WPP domain was tagged to a nuclear localization signal. The localization of wild-type Rx is affected by the coexpression of these constructs: RanGAP2 sequesters it in the cytoplasm, and Rg2-ΔC-mCh-NLS targets it to the nucleus. The GFP intensities from the Rx-GFP constructs were determined for the nucleus and cytoplasm, and the average ratios of the intensities are plotted (n = 9; error bars denote the se). E, PVX resistance assay. Full-length Rx (wild type, S1, and S4) was coexpressed with either mCherry as a control or with Rg2-ΔC-mCh-NLS and an avirulent PVX amplicon. Previously, we showed that targeting full-length Rx to the nucleus led to a partial loss of resistance (Slootweg et al., 2010). The level of virus after 5 d was determined by an anti-PVX CP ELISA. Error bars represent the se (n = 9). Student’s t test was used to determine if coexpression of Rg2-ΔC-mCh-NLS resulted in a significantly higher virus level than coexpression with free mCherry (*, P < 0.5 and **, P < 0.05).
Figure 7.
Figure 7.
Blue Native gel analysis of the complex formed by the Rx1 CC and RanGAP2 in the cell. A, Blue Native gel analysis of Rx1 CC constructs (wild type [wt], S1, and S4) coexpressed with RanGAP2-GFP (right two images) or expressed alone (left two images). HA-tagged GFP (4HA-GFP) was included as a control and was detected with the anti-HA and anti-GFP antibody. The mass given for the RanGAP2, Rx1-CC, and GFP constructs represents the mass of a monomer, and for Rubisco the approximate mass of the complex is given. The behavior of the proteins on this gel is determined by the mass of the complex they are part of and by their shape. The blots were aligned to each other using the Rubisco complex and the 4HA-GFP, which is present on each immunoblot. B, SDS-PAGE analysis of the samples used in A demonstrating that the banding patterns on the Native Blue blot are not due to protein degradation or modifications. RanGAP2-GFP and the CC-4HA constructs run as single bands on SDS-PAGE. A Coomassie Brilliant Blue (CBB)-stained blot is included as a control for equal loading. The dashed vertical lines indicate the positions of the marker lane on the immunoblots.
Figure 8.
Figure 8.
Exploring suppressive effects of the coexpressed CC domain on the functionality of wild-type Rx1. A, Schematic view of the potential mechanisms behind the functional complementation and the dominant negative phenotypes that could occur upon coexpression of the wild-type CC domain with mutant or wild-type Rx, respectively. A red cross indicates a loss of signaling of the full-length protein, and the green V indicates a state in which the protein is able to signal. B, Assay to test if coexpression of the CC of Rx1 suppresses the resistance mediated by Rx1, leading to higher PVX levels in a transient PVX resistance assay. Coexpression of GFP or the CC domain of Bs2 was used as negative controls. PVX accumulation was determined via an anti-PVX ELISA. Error bars present the se (n = 8), and letters denote significantly (P < 0.05) different groups (one-way ANOVA with posthoc Tukey’s test). C, To determine if the S1 or S4 mutation affected the suppressive effect seen for the wild-type Rx1 CC, CC constructs harboring these mutations were coexpressed with wild-type Rx1 in a transient PVX assay. Coexpression of GFP served as a negative control. Error bars present the se (n = 12), and letters denote significantly (P < 0.05) different groups (one-way ANOVA with posthoc Tukey’s test). D, Coexpression of the wild type and S1 and S4 variants of the CC-NB-ARC with wild-type Rx1 in a transient PVX assay to test if the mutations affect the suppressive effect of the CC-NB-ARC. Coexpression of GFP was used as a negative control. Error bars present the se (n = 8), and letters denote significantly (P < 0.05) different groups (one-way ANOVA with posthoc Tukey’s test). E, Coexpression of the N- and C-terminal CC segments with the full-length Rx1 in a transient PVX resistance assay to test if the suppressive effect is caused by a specific region in the CC. Error bars present the se (n = 8), and letters denote significantly (P < 0.01) different groups (one-way ANOVA with posthoc Tukey’s test). F, Coexpression of PVX:GFP, Rx1, and the CC segments. The virus accumulation was visualized via the GFP expressed from the PVX genome. Lower GFP levels are apparent on leaves in which H1-H2 is coexpressed. G, To test if the reduction of PVX levels was a direct effect of the H1-H2 segment on the virus or if it was due to an enhancement of Rx1 activity, the CC segments were coexpressed with PVX:GFP in the absence and presence of Rx1. PVX levels were compared on an anti-GFP immunoblot via the GFP expressed by PVX. All samples were loaded undiluted and 8× diluted to make a better comparison possible between the samples with high and low GFP concentrations.

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