Structural Study Of The Interaction Mechanism Between Potato Disease Resistance Protein Rx With Its Cofactor RanGAP2

Plant genomes encode a large number of NB-LRR receptors, many of them mediate resistance to pathogens in plants, also termed resistance proteins (R proteins). Typical NB-LRR receptors are modular, with a nucleotide binding site domain (NBS) in the center and a C terminal leucine-rich repeat domain (LRR). There are two major classes of NB-LRR receptors that are distinguished by the domains present at their N termini:those that possess a coiled coil (CC) domain and those that possess a Toll-interleukin1receptor like (TIR) domain. In the absence of infection, NB-LRR receptors are maintained in an inactive state through intramolecular interactions between domains. When infection occurs, NB-LRR receptors can initiate a series of resistance signals and prevent infections through recognizing effectors delivered by pathogens. Meanwhile, more and more research data have increasingly revealed that the N terminal domain of NB-LRR receptors, especially CC domains, directly interact with specific intracellular proteins, termed cofactors, enabling the resistance function of NB-LRR receptors. To date, there is limited data about the interaction mechanism and structure of the NB-LRR receptor-cofactor complex.The potato NB-LRR receptors, Rx, which mediates resistance to virus X, has a typical CC-NBS-LRR domain arrangement. Previous studies have demonstrated that the CC domain of Rx plays three roles. First, through the conserved EDVID motif, it mediates intramolecular interactions with the NBS-LRR region. Second, the CC domain of Rx mediates an intermolecular interaction with the cofactor, RanGAP2, which is required for the resistance function of Rx. Third, CC domain of Rx opts to accumulate in the nucleus and plays a role in the intracellular relocation of Rx. Nucleocytoplasmic partitioning may also affect the resistance function of Rx.In this study, the crystal structure of Rx CC domain in complex with RanGAP2WPP domain was resolved. In this complex, the CC domain of Rx is structurally monomeric, and it is strikingly different from the earlier well-characterized structure of barley NB-LRR receptor MLA10CC domain. This difference indicates the structural diversity of CC domains of NB-LRR receptors. The structural diversity may influence the unique and specific recognition of cofactors or effectors.Rx-CC and RanGAP2-WPP bind each other mainly through hydrophobic interactions. Through in vitro and in vivo mutation analysis, two amino acids, Rx Tryptophan-90and RanGAP2Alanine-89were identified as critical for the molecular interaction between Rx-CC and RanGAP2-WPP. Mutation of the two amino acids to aspartate can significantly reduce the interaction between Rx-CC and RanGAP2-WPP, and also severely impair the resistance response of Rx in plants. The surface formed by side chains of EDVID motif, which is involved in intramolecular interactions, is distinct from the surface formed by side chains of amino acids involved in interaction with RanGAP2-WPP. This is consistent with previous speculation.In addition, in this complex, RanGAP2-WPP binding to two a-helices of Rx-CC which are also important for the nuclear accumulation of Rx. By binding the two a-helices, RanGAP2-WPP blocks the transfer of Rx-CC to the nucleus and sequesters it to the cytoplasm.Together, our findings not only reveal the molecular mechanism underlying Rx/RanGAP2interaction, but also interpret the mechanism that RanGAP2mediates nucleocytoplasmic partitioning of Rx.