Deconstructing a molecular switch; structure-function analysis of the CC-NB-LRR proteins RX1 and GPA2

E.J. Slootweg, L.N. Spiridon, O. Caldararu, W. Tameling, H. Pomp, J. Roosien, J. Bakker, G. Smant, M. Joosten, A.J. Petrescu, A. Goverse

Research output: Chapter in Book/Report/Conference proceedingAbstract

Abstract

Many plant immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a coiled coil (CC) and a LRR sensor domain. The cooperation between the CC, switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Through targeted mutagenesis, interaction studies and structural modelling we investigate the way the domains of the potato resistance proteins Rx1 or Gpa2 work together. We demonstrated that the correct cooperation between the CC-NB-ARC and the LRR is focussed on a region in the ARC2 subdomain of the NB-ARC and the N-terminal repeats of the LRR. A mismatch leads either to autoactivation or loss-of-function. Complementary charged surface patches in the ARC2 and LRR are key determinants of the physical interaction between the domains, but do not appear to be involved in signalling. Mutagenesis of specific residues in the ARC2 region 23 affected either elicitor-dependent activation or autoactivation. Furthermore alanine-substitution of several aromatic residues in the CC domain shows that distinct surfaces are required for the interaction of the CC with the NB-ARC-LRR domains or with RanGAP2. The CC domain of Rx1 consists of 4 helices and mutagenesis of the hydrophobic residues required for their interaction revealed that distinct parts of the CC are involved in either the cell death or PVX resistance signaling by Rx1. Currently we are characterising how the binding of RanGAP2 and the recognition of specific elicitors affects the conformation and subcellular localisation of the resistance protein in the cell via fluorescence lifetime imaging.
Original languageEnglish
Title of host publicationProceedings of COST FA 1208: Pathogen-informed strategies for sustainable broad-spectrum crop resistance
Pages22-23
Publication statusPublished - 2014
EventThe second workshop of the COST FA1208 SUSTAIN Action, Bucharest, Romania -
Duration: 10 Sep 201412 Sep 2014

Workshop

WorkshopThe second workshop of the COST FA1208 SUSTAIN Action, Bucharest, Romania
Period10/09/1412/09/14

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