Williams Simon J, Yin Ling, Foley Gabriel, Casey Lachlan W, Outram Megan A, Ericsson Daniel J, Lu Jiang, Boden Mikael, Dry Ian B, Kobe Bostjan
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.
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.
Front Plant Sci. 2016 Dec 8;7:1850. doi: 10.3389/fpls.2016.01850. eCollection 2016.
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 and . 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 grapevine . 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 the RPS4/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.
N端Toll/白细胞介素-1受体/抗性蛋白（TIR）结构域已被证明在模式植物亚麻和[未提及的植物]中对于防御信号传导既是必需的也是充分的。在这些生物体的实例中，TIR结构域的自缔合对于信号传导功能是必需的，尽管是通过不同的界面。在这里，我们研究了来自野生葡萄[未提及具体品种]的含有抗性蛋白RPV1的TIR结构域的这些特性。在没有额外侧翼序列的情况下，RPV1 TIR结构域在烟草中瞬时表达时具有自活性，这表明单独的TIR结构域就能够进行细胞死亡信号传导。我们以2.3埃的分辨率确定了RPV1 TIR结构域的晶体结构。在晶体中，RPV1 TIR结构域形成二聚体，主要通过αA和αE螺旋中的残基（“AE”界面）介导。这个界面与在RPS4/RRS1抗性蛋白对的TIR结构域的二聚体复合物中发现的界面相同。我们表明，在晶体中介导二聚体相互作用的AE界面上暴露于表面的残基在含植物TIR结构域的蛋白质中高度保守。虽然我们无法证明RPV1 TIR结构域在溶液中或使用酵母双杂交时的自缔合，但AE界面上暴露于表面的残基的突变会阻止细胞死亡自活性表型。此外，已知在亚麻L6 TIR结构域的细胞死亡信号传导功能中起重要作用的残基的突变也被证明是RPV1 TIR结构域介导的细胞死亡所必需的。我们的数据表明，多个TIR结构域表面控制着RPV1 TIR结构域的细胞死亡功能，并且我们认为保守的AE界面可能在TIR-NLR信号传导中具有普遍功能。
