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, 20 (3), 739-51

The Coiled-Coil and Nucleotide Binding Domains of the Potato Rx Disease Resistance Protein Function in Pathogen Recognition and Signaling

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The Coiled-Coil and Nucleotide Binding Domains of the Potato Rx Disease Resistance Protein Function in Pathogen Recognition and Signaling

Gregory J Rairdan et al. Plant Cell.

Abstract

Plant genomes encode large numbers of nucleotide binding and leucine-rich repeat (NB-LRR) proteins, some of which mediate the recognition of pathogen-encoded proteins. Following recognition, the initiation of a resistance response is thought to be mediated by the domains present at the N termini of NB-LRR proteins, either a Toll and Interleukin-1 Receptor or a coiled-coil (CC) domain. In order to understand the role of the CC domain in NB-LRR function, we have undertaken a systematic structure-function analysis of the CC domain of the potato (Solanum tuberosum) CC-NB-LRR protein Rx, which confers resistance to Potato virus X. We show that the highly conserved EDVID motif of the CC domain mediates an intramolecular interaction that is dependent on several domains within the rest of the Rx protein, including the NB and LRR domains. Other conserved and nonconserved regions of the CC domain mediate the interaction with the Ran GTPase-activating protein, RanGAP2, a protein required for Rx function. Furthermore, we show that the Rx NB domain is sufficient for inducing cell death typical of hypersensitive plant resistance responses. We describe a model of CC-NB-LRR function wherein the LRR and CC domains coregulate the signaling activity of the NB domain in a recognition-specific manner.

Figures

Figure 1.
Figure 1.
Deletion Analysis of the Rx CC Domain. (A) Schematic diagram of Rx CC deletion constructs. The entire Rx CC domain (amino acids 1 to 144) is shown, with each deletion construct analyzed in this study represented by a gray bar that spans the amino acid sequence remaining. All CC derivatives were expressed as a fusion with a C-terminal EGFP:HA tag and were tested for their ability to complement Rx NB-ARC-LRR. The CC:EGFP:HA derivatives were coexpressed by agroexpression (see Methods) in N. benthamiana leaves with Rx NB-ARC-LRR plus PVX CP and assessed for cell death 2 d later. Black bars marked with asterisks indicate the constructs that were able to complement NB-ARC-LRR in this assay. The boxed sequence illustrates the N3-C4 construct lacking both N- and C-terminal residues (used in [D]). (B) HR phenotype of CC deletion mutants. The full CC domain (CC), selected Rx CC derivatives (C1, C2, and C3), or empty vector (EV) were coexpressed with wild-type Rx NB-ARC-LRR and PVX CP in N. benthamiana leaves and assessed for their ability to elicit an HR. Leaves were photographed at 3 d after infiltration. (C) PVX resistance phenotypes of CC deletion variants. The Rx CC constructs described above were coexpressed with wild-type Rx NB-ARC-LRR in wild-type N. benthamiana leaves with a dilute suspension of Agrobacterium carrying a binary vector encoding an infectious PVX:GFP clone. PVX:GFP accumulation was evaluated by visualization of GFP fluorescence at 7 d after infiltration. (D) Interactions of the CC derivatives (illustrated in [A]) with NB-ARC-LRR. N. benthamiana leaves were expressed via agroexpression with both Rx NB-ARC-LRR:Myc and one of the Rx CC:EGFP:HA derivatives. Two days later, protein extracts were subjected to immunoprecipitation (IP) with anti (α)-HA antibody–conjugated beads, and the immunoprecipitated or total protein extracts were immunoblotted (IB) with the indicated antibodies. Results obtained in the HR and PVX resistance assays presented above are compiled at bottom.
Figure 2.
Figure 2.
NAAIRS Scanning Mutagenesis of the Rx CC Domain. (A) Functional analysis of NAAIRS substitutions. The minimal sequence of the Rx CC domain sufficient to induce an HR (construct C2 in Figure 1A) was systematically mutated by replacing consecutive blocks of six residues with the amino acid sequence NAAIRS for analysis within the context of the full-length Rx clone. The lines below the Rx sequence underscore the amino acids that were replaced by NAAIRS in Rx variants, designated A to N. Plus signs indicate that a CP-dependent HR was seen within 48 h of agroexpression in N. benthamiana leaves; W indicates that an HR was observed, but at a significantly later time compared with the wild type, typically presenting a visibly weaker response at 72 to 96 h after infiltration. (B) Functional analysis of NAAIRS internal deletions. The minimal sequence of the Rx CC domain sufficient to induce an HR was systematically mutated by replacing consecutive blocks of 12 residues with the amino acid sequence NAAIRS within the context of the full (amino acids 1 to 144) CC:HA construct. Each internal deletion is underscored and indicated by a number below. Plus signs indicate that a CP-dependent HR was seen within 48 h of coexpression of the indicated variants with NB-ARC-LRR in N. benthamiana leaves; W indicates that an HR was observed, but at a significantly later time compared with the wild type, typically presenting a visibly weaker response at 72 to 96 h after infiltration. (C) NB-ARC-LRR binding properties of NAAIRS internal deletions. NB-ARC-LRR binding by NAAIRS variants was assessed by coexpressing the indicated CC:HA variant with NB-ARC-LRR:Myc in N. benthamiana leaves followed by immunoprecipitating (IP) the CC:HA variant with anti (α)-HA antibody–conjugated beads. The immunoprecipitates or total proteins were then immunoblotted (IB) with the indicated antibodies.
Figure 3.
Figure 3.
The EDVID Motif. (A) Identification of a conserved EDVID motif. N-terminal sequences from characterized CC-NB-LRR resistance proteins were aligned to the region of Rx defined by internal deletion 7 in Figure 2B. Amino acids are grouped by color according to conserved side chain chemical properties. Residues conserved among CC domains are indicated below the alignment. A consensus sequence encompassing the EDVID motif (underlined) is shown below the sequence. Residues are colored according to shared physicochemical properties. (B) Effects of the HR phenotype of site-directed mutations within the EDVID motif. Individual amino acids within the Rx EDVID motif (TEDMVD in Rx) were mutated individually or in combinations as underlined. The resulting mutants were coexpressed in the context of either full-length Rx or the Rx CC:HA plus NB-ARC-LRR in N. benthamiana leaves and assessed for their ability to elicit a CP-dependent HR. HR reactions were seen within 24 h (+++), 48 h (++), 72 h (+), or not at all (−). nd, not done. (C) PVX resistance phenotype of EDVID variants. The same constructs used in (B) were coexpressed in N. benthamiana leaves with a dilute suspension of Agrobacterium possessing a binary vector containing an infectious PVX:GFP clone. Incomplete resistance to PVX:GFP was indicated by the visualization of GFP fluorescence at 7 d after infiltration. As a control, the PVX:GFP was coexpressed with EGFP, which is not excited by the 365-nm light used to visualize PVX:GFP. (D) NB-ARC-LRR binding by EDVID mutants. The indicated CC:HA derivatives were coexpressed with NB-ARC-LRR:Myc in N. benthamiana leaves followed by immunoprecipitation (IP) of the CC:HA variant with anti (α)-HA antibody–conjugated beads. Immunoprecipitates and input proteins were subsequently immunoblotted (IB) with the indicated antibodies.
Figure 4.
Figure 4.
Delineation of a RanGAP2 Binding Region. (A) Interaction of St RanGAP2 with CC deletion fragments. St RanGAP2:FLAG was transiently expressed in N. benthamiana leaves along with EGFP:HA, CC:EGFP:HA, or one of the N- and C-terminal deletion derivatives of Rx CC:EGFP:HA as depicted in Figure 1A. Protein extracts were subjected to anti (α)-FLAG immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. (B) Interaction of St RanGAP2 with CC internal deletions/mutants. St RanGAP2:FLAG was transiently expressed in N. benthamiana leaves along with EGFP:HA, CC:HA, internal deletion derivatives of Rx CC:HA as depicted in Figure 2B, or a mutant derivative (TEDMVD to TAAMVA) of CC:HA. Protein extracts were subjected to anti-FLAG and anti-HA immunoprecipitation followed by immunoblotting with the indicated antibodies.
Figure 5.
Figure 5.
Mutations within the NB, ARC, and LRR Domains Impair Binding of the Rx CC Domain. (A) The CC does not bind NB or NB-ARC fragments. Rx CC:Myc was coexpressed with NB-ARC-LRR:HA, NB:EGFP:HA, NB-ARC:HA, or EGFP:HA followed by immunoprecipitation (IP) with anti-Myc (αMyc) antibody–conjugated beads. The immunoprecipitates and total extracts were immunoblotted (IB) with the indicated antibodies. (B) and (C) CC binding of Rx NB-ARC-LRR:HA variants. The indicated mutants (see text) or Gpa2/Rx chimera (GGRRR) NB-ARC-LRR:HA constructs (described in Methods) were coexpressed in N. benthamiana leaves with CC:Myc. Immunoprecipitation with anti-Myc antibody–conjugated beads was performed 2 d later. The immunoprecipitates and total extracts were subsequently immunoblotted with the indicated antibodies. The ability to produce an HR within 48 h when the various construct combinations were coexpressed in N. benthamiana leaves with or without CP is indicated by a plus sign. W indicates that an HR was observed, but at a significantly later time compared with the wild type, typically presenting a visibly weaker response at 72 to 96 h after infiltration. (D) Inactive Rx LRR mutants bind Rx CC-NB-ARC. Inactive LRR:Myc constructs identified by error-prone PCR mutagenesis (see Supplemental Table 1 online) were coexpressed with Rx CC-NB-ARC:HA in N. benthamiana leaves and immunoprecipitated with anti-Myc antibody–conjugated beads 2 d later. The immunoprecipitates or total extracts were then immunoblotted with the indicated antibodies. The ability to produce a CP-dependent HR within 48 h when the same construct combinations were coexpressed in N. benthamiana leaves is indicated by a plus sign. (E) Effect of LRR mutations on CC binding. NB-ARC-LRR:HA constructs incorporating the same LRR mutations as in (D) were coexpressed in N. benthamiana leaves with CC:Myc and immunoprecipitated 2 d later with anti-HA antibody–conjugated beads. The immunoprecipitates and total protein were then immunoblotted with the indicated antibodies.
Figure 6.
Figure 6.
The Rx NB Domain Is Capable of Initiating an HR. (A) Relative expression levels of Rx-derived fragments. The indicated Rx fragments (as described in the text) were expressed in N. benthamiana leaves, and proteins were extracted for anti-HA immunoblotting at 2 d after infiltration (top panels). Equal loading was determined by Coomassie blue staining of total protein on the same blot (bottom panels). Expression of full-length Rx:HA was likewise compared with that of Rx NB:EGFP:HA. Proteins in the left and right sets of panels were separated on different percentage SDS-PAGE gels for optimum resolution. (B) Expression of Rx fragments in N. tabacum leaves. The indicated Rx-derived fragments were transiently expressed in N. tabacum via agroexpression. The resulting response was photographed at 2 d after infiltration. (C) Rx fragment signaling in Eds1- and Sgt1-silenced plants. N. benthamiana plants expressing the Rx transgene were silenced with TRV vectors carrying either no insert (TV:00) or sequences derived from N. benthamiana Sgt1 or Eds1 genes. Three weeks after infection with the silencing constructs, Rx derivatives were agroexpressed in silenced leaves. The positions of expression of Rx fragments are represented schematically in the panel at left. Three days after agroexpression, leaves were photographed (top row), cleared with ethanol, and photographed again (bottom row). (D) Expression of Rx NB:EGFP:HA in Sgt1-silenced plants. N. benthamiana plants were silenced with TRV vectors carrying fragments of either the GUS gene or Nb Sgt1, as indicated. Three weeks later, pBIN61 (empty vector) and pBIN61-NB:EGFP:HA were agroexpressed in silenced leaves. Two days later, protein extracts were subjected to anti (α)-HA immunoblotting. Equal loading was determined by Coomassie blue staining of total protein on the same blot.

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