title: Structure and Function of the TIR Domain from the Grape NLR Protein RPV1.
abstract: The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax andArabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevineMuscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices ("AE" interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from theArabidopsisRPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling.
authors.affiliation: School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, BrisbaneQLD, Australia; Research School of Biology, The Australian National University, CanberraACT, Australia.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Williams
authors.affiliation: Guangxi Crop Genetic Improvement and Biotechnology Key Lab, Guangxi Academy of Agricultural SciencesNanning, China; Commonwealth Scientific and Industrial Research Organisation, UrrbraeSA, Australia; College of Food Science and Nutritional Engineering, China Agricultural UniversityBeijing, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Yin
authors.affiliation: School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane QLD, Australia.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Foley
authors.affiliation: School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane QLD, Australia.
authors.forename: Lachlan W
authors.initials: LW
authors.lastname: Casey
authors.affiliation: School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane QLD, Australia.
authors.forename: Megan A
authors.initials: MA
authors.lastname: Outram
authors.affiliation: Australian Synchrotron, Clayton VIC, Australia.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Ericsson
authors.affiliation: College of Food Science and Nutritional Engineering, China Agricultural UniversityBeijing, China; Department of Plant Science, Shanghai Jiao Tong UniversityShanghai, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Lu
authors.affiliation: School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane QLD, Australia.
authors.forename: Mikael
authors.initials: M
authors.lastname: Boden
authors.affiliation: Guangxi Crop Genetic Improvement and Biotechnology Key Lab, Guangxi Academy of Agricultural SciencesNanning, China; Commonwealth Scientific and Industrial Research Organisation, UrrbraeSA, Australia.
authors.forename: Ian B
authors.initials: IB
authors.lastname: Dry
authors.affiliation: School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane QLD, Australia.
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
keywords: Muscadinia rotundifolia; Plasmopara viticola; X-ray crystallography; grapevine downy mildew; nucleotide-binding oligomerisation domain (NOD)-like receptor (NLR); plant disease resistance; toll/interleukin-1 receptor (TIR)