Guo Liwei, Zhang Yikun, Ma Mengqi, Liu Qiang, Zhang Yanan, Peng Youliang, Liu Junfeng
Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, No. 2 Yunamingyuanxilu, Beijing 100193, People's Republic of China.
Acta Crystallogr F Struct Biol Commun. 2018 Apr 1;74(Pt 4):262-267. doi: 10.1107/S2053230X18003618. Epub 2018 Mar 28.
RGA5 is a component of the Pia resistance-protein pair (RGA4/RGA5) from Oryza sativa L. japonica. It acts as an immune receptor that directly recognizes the effector AVR1-CO39 from Magnaporthe oryzae via a C-terminal non-LRR domain (RGA5A_S). The interaction between RGA5A_S and AVR1-CO39 relieves the repression of RGA4, leading to effector-independent cell death. To determine the structure of the complex of RGA5A_S and AVR1-CO39 and to understand the details of this interaction, the complex was prepared by fusing the proteins together, by mixing them in vitro or by co-expressing them in one host cell. Samples purified via the first two strategies were crystallized under two different conditions. A mixture of AVR1-CO39 and RGA5A_S (complex I) crystallized in 1.1 M ammonium tartrate dibasic, 0.1 M sodium acetate-HCl pH 4.6, while crystals of the fusion complex RGA5A_S-TEV-AVR1-CO39 (complex II) were grown in 2 M NaCl. The crystal of complex I belonged to space group P321, with unit-cell parameters a = b = 66.2, c = 108.8 Å, α = β = 90, γ = 120°. The crystals diffracted to a Bragg spacing of 2.4 Å, and one molecule each of RGA5A_S and AVR1-CO39 were present in the asymmetric unit of the initial model. The crystal of complex II belonged to space group I4, with unit-cell parameters a = b = 137.4, c = 66.2 Å, α = β = γ = 90°. The crystals diffracted to a Bragg spacing of 2.72 Å, and there were two molecules of RGA5A_S and two molecules of AVR1-CO39 in the asymmetric unit of the initial model. Further structural characterization of the interaction between RGA5A_S and AVR1-CO39 will lead to a better understanding of the mechanism underlying effector recognition by R proteins.
RGA5是来自粳稻的稻瘟病抗性蛋白对（RGA4/RGA5）的一个组分。它作为一种免疫受体，通过C端非亮氨酸重复序列结构域（RGA5A_S）直接识别稻瘟病菌的效应子AVR1-CO39。RGA5A_S与AVR1-CO39之间的相互作用解除了对RGA4的抑制，导致效应子非依赖型细胞死亡。为了确定RGA5A_S与AVR1-CO39复合物的结构并了解这种相互作用的细节，通过将蛋白质融合在一起、在体外混合或在一个宿主细胞中共表达来制备复合物。通过前两种策略纯化的样品在两种不同条件下结晶。AVR1-CO39与RGA5A_S的混合物（复合物I）在1.1 M酒石酸氢二铵、0.1 M乙酸钠-HCl pH 4.6中结晶，而融合复合物RGA5A_S-TEV-AVR1-CO39（复合物II）的晶体在2 M NaCl中生长。复合物I的晶体属于空间群P321，晶胞参数a = b = 66.2，c = 108.8 Å，α = β = 90，γ = 120°。晶体衍射到2.4 Å的布拉格间距，初始模型的不对称单元中每个含有一个RGA5A_S分子和一个AVR1-CO39分子。复合物II的晶体属于空间群I4，晶胞参数a = b = 137.4，c = 66.2 Å，α = β = γ = 90°。晶体衍射到2.72 Å的布拉格间距，初始模型的不对称单元中有两个RGA5A_S分子和两个AVR1-CO39分子。对RGA5A_S与AVR1-CO39之间相互作用的进一步结构表征将有助于更好地理解R蛋白识别效应子的潜在机制。
