Physical Association of the NB-LRR Resistance Protein Rx with a Ran GTPase–Activating Protein Is Required for Extreme Resistance to Potato virus X[W][OA]

Nucleotide binding leucine-rich repeat (NB-LRR) proteins play an important role in plant and mammalian innate immunity. In plants, these resistance proteins recognize specific pathogen-derived effector proteins. Recognition subsequently triggers a rapid and efficient defense response often associated with the hypersensitive response and other poorly understood processes that suppress the pathogen. To investigate mechanisms associated with the activation of disease resistance responses, we investigated proteins binding to the potato (Solanum tuberosum) NB-LRR protein Rx that confers extreme resistance to Potato virus X (PVX) in potato and Nicotiana benthamiana. By affinity purification experiments, we identified an endogenous N. benthamiana Ran GTPase–Activating Protein2 (RanGAP2) as an Rx-associated protein in vivo. Further characterization confirmed the specificity of this interaction and showed that the association occurs through their N-terminal domains. By specific virus-induced gene silencing of RanGAP2 in N. benthamiana carrying Rx, we demonstrated that this interaction is required for extreme resistance to PVX and suggest that RanGAP2 is part of the Rx signaling complex. These results implicate RanGAP-mediated cellular mechanisms, including nucleocytoplasmic trafficking, in the activation of disease resistance

Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation

Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP)

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

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