Distinct domains in the ARC region of the potato resistance protein Rx mediate LRR binding and inhibition of activation

Abstract

Plant nucleotide binding and leucine-rich repeat (NB-LRR) proteins contain a region of homology known as the ARC domain located between the NB and LRR domains. Structural modeling suggests that the ARC region can be subdivided into ARC1 and ARC2 domains. We have used the potato (Solanum tuberosum) Rx protein, which confers resistance to Potato virus X (PVX), to investigate the function of the ARC region. We demonstrate that the ARC1 domain is required for binding of the Rx N terminus to the LRR domain. Domain-swap experiments with Rx and a homologous disease resistance gene, Gpa2, showed that PVX recognition localized to the C-terminal half of the LRR domain. However, inappropriate pairings of LRR and ARC2 domains resulted in autoactive molecules. Thus, the ARC2 domain is required to condition an autoinhibited state in the absence of elicitor as well as for the subsequent elicitor-induced activation. Our data suggest that the ARC region, through its interaction with the LRR, translates elicitor-induced modulations of the C terminus into a signal initiation event. Furthermore, we demonstrate that physical disruption of the LRR-ARC interaction is not required for signal initiation. We propose instead that this activity can lead to multiple rounds of elicitor recognition, providing a means of signal amplification.

Figure 1.

Alignment of Rx and GPA2. NB-ARC residues mutated in this work are shown in red, and conserved NB-ARC motifs are underlined. Blue lines delineate Rx/GPA2 regions A to E analyzed by sequence exchange in Figure 4. The asterisk marks the site of the largest (amino acids 1 to 382) C-terminal deletion still capable of binding the LRR domain (see Figure 2). Boxed residues highlight sequence differences between Rx and GPA2, with lighter boxes indicating more conservative substitutions. LRRs as originally annotated (Bendahmane et al., 1999) are overlined. Numbering refers to Rx residues.

Figure 2.

Analysis of the Rx NB-ARC Domain. (A) Functional assays of NB-ARC variants. Indicated mutations were incorporated into constructs containing the full-length Rx cDNA driven by the Rx genomic promoter. These variants were expressed via agroinfiltration in N. benthamiana leaves with CP and assessed for HR induction over a period of 3 d. The same variants were incorporated into Rx CC-NB-ARC constructs. These variants were transiently expressed via agroinfiltration in N. benthamiana leaves with CP plus the Rx LRR domain, all regulated by P_35S. In this assay, + indicates that an HR was observed within 2 d after infiltration, and – indicates no cell death. –/+ indicates that an HR was observed but was consistently delayed by ∼24 h compared with wild-type Rx. na, not applicable; ne, not expressed (HA-tagged protein was not detectable by protein gel blotting when driven from P_35S). The PVX:GFP resistance assay consisted of agroinfiltrating Rx variants with an infectious PVX:GFP clone and monitoring GFP fluorescence. In this assay, + indicates that no GFP fluorescence was observed 5 d after infiltration, and – indicates no restriction of virus accumulation. –/+ indicates a modest accumulation of PVX:GFP. All HRs were CP-dependent, and all infiltrations were performed at least twice. (B) Coimmunoprecipitation of CC-NB-ARC variants. N. benthamiana leaves were agroinfiltrated with Rx CC-NB-ARC:HA variants and LRR:MYC under P_35S regulation. Two days after infiltration, protein extracts were subjected to immunoprecipitation (IP) with anti-HA (αHA) or anti-Myc (αMyc) antibody–conjugated beads and immunoblotted (IB) with the indicated antibody. This experiment was repeated three times with similar results.

Figure 3.

Interactions between Domains from Different R Proteins. The indicated LRR:HA or GFP:HA proteins were transiently coexpressed in N. benthamiana leaves via agroinfiltration with the indicated CC-NB-ARC:MYC construct together with GFP, all under the control of P_35S. Two days after infiltration, protein extracts were subjected to immunoprecipitation (IP) with either anti-HA (αHA) or anti-Myc (αMyc) antibodies and immunoblotted (IB) with the indicated antibody. This experiment was repeated three times with similar results.

Figure 4.

Rx/GPA2 Chimera Analysis. Chimeric proteins were generated from regions A to E as illustrated in Figure 1. + indicates that a strong HR was observed 2 d after infiltration, and – indicates no HR. –/+ indicates that an HR was observed but was consistently delayed by ∼24 h compared with wild-type Rx plus CP. Each experiment was repeated at least twice with similar results. (A) to (C) Fusion proteins were transiently expressed in N. benthamiana leaves via agroinfiltration under P_35S regulation with either P_35S:GFP or P_35S:CP. (D) In addition to the experiments described above, these constructs were coexpressed with LRR(Y712H) in the presence or absence of CP. Identical results were obtained with wild-type Rx LRR. All constructs were expressed under the regulation of P_35S.

Figure 5.

Effect of Autoactivating Mutations on LRR–ARC Interaction Dynamics. (A) Physical interaction of autoactive variant domains. Wild-type LRR:FH or Rx LRR(Y712H):FH were expressed via agroinfiltration in N. benthamiana leaves with the indicated CC-NB-ARC:HA variants plus either GFP or CP. In combinations that normally would result in an HR, inactivating mutations were introduced into the P-loop (PL: GK175AA) and Kinase 3a motifs (K3: L270P) of Rx and GPA2, respectively. All constructs were regulated by P_35S. Two days after infiltration, protein was extracted and subjected to immunoprecipitation (IP) with either anti-FLAG (αFLAG) or anti-HA (αHA) antibody–conjugated beads and immunoblotted (IB) with the indicated antibody. This experiment was repeated three times with similar results. (B) Functional assays of Rx autoactive variants. The indicated variants were transiently expressed via agroinfiltration in N. benthamiana leaves with either CC-NB-ARC or CC-NB-ARC(D460V) in the presence or absence of CP. + indicates that an HR was observed 2 d after infiltration, and – indicates no HR. (C) Lack of transactivation by the LRRs in some full-length molecules. Inactivating mutations were introduced into the P-loops of RR-GRR (GK175AA) and Bs2 (GK186AA) to create RR-GRR(PL) and Bs2(PL). The indicated constructs were expressed under the regulation of P_35S with or without CP or avrBs2. + indicates that an HR was observed 2 d after infiltration, and – indicates a lack of HR.

Figure 6.

Deletion Analysis of the Rx LRR Domain. (A) Functional assays of LRR variants. The indicated Rx LRR:HA fragments were transiently expressed via agroinfiltration in N. benthamiana leaves with Rx CC-NB-ARC and CP, all under P_35S regulation. + indicates that an HR was observed 2 d after infiltration, and – indicates no HR. (B) Coimmunoprecipitation of CC-NB-ARC by LRR variants. N. benthamiana leaves were agroinfiltrated with Rx CC-NB-ARC:MYC and LRR:HA variants under P_35S regulation. Protein was extracted 2 d after infiltration, immunoprecipitated (I.P.) with anti-Myc (αMyc) or anti-HA (αHA) antibodies, and immunoblotted (I.B.) with the indicated antibody. These experiments were repeated at least three times with similar results.

Figure 7.

Perturbed LRR Model. In this model, the avirulence determinant alters the conformation of, or perturbs, the LRR in a direct or indirect manner. This alteration allows the protein to overcome the autoinhibition conditioned by the ARC2 subdomain and activate downstream signaling (represented as a yellow star). Disruption of the LRR–ARC interaction would proceed simultaneously or subsequent to this activation, allowing the domains of the molecule to reassociate and undergo further rounds of recognition and signaling. In such a model, the combination of the GPA2 ARC2 and Rx LRR domains would lack the structural constraints required to retain the molecule in the autoinhibited state, and the protein would proceed directly to the activated form. However, some additional conformational change induced by CP recognition is required to trigger the dissociation of the Rx LRR from the GPA2 ARC for the process to reiterate.