Crystallization of the rice immune receptor RGA5A_S with the rice blast fungus effector AVR1-CO39 prepared via mixture and tandem strategies
Crystallization of the rice immune receptor RGA5A_S with the rice blast fungus effector AVR1-CO39 prepared via mixture and tandem strategies
复制标题
通过混合和串联策略制备水稻免疫受体 RGA5A_S 和稻瘟菌效应子 AVR1-CO39 的结晶。
DOI：
10.1107/s2053230x18003618
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发表时间：
2018-04-01
影响因子：
0.9
通讯作者：
Liu, Junfeng
中科院分区：
文献类型：
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作者：
Guo, Liwei;Zhang, Yikun;Liu, Junfeng
RGA5是Oryza sativa L. japonica抗性蛋白对（RGA4/RGA5）的一个组成部分。它作为一种免疫受体，通过c端非lrr结构域（RGA5A_S）直接识别来自Magnaporthe oryzae的效应物AVR1-CO39。RGA5A_S和AVR1-CO39之间的相互作用解除了RGA4的抑制，导致不依赖于效应器的细胞死亡。为了确定RGA5A_S和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的条件下结晶。AVR1-CO39和RGA5A_S（配合物I）在1.1 M酒石酸铵双碱、0.1 M醋酸钠- hcl pH 4.6中结晶，而融合配合物RGA5A_S- tev -AVR1-CO39（配合物II）在2 M NaCl中结晶。配合物I晶体属于空间群P3121，其晶胞参数为a = b = 66.2, c = 108.8 Å， α = β = 90, γ = 120°。晶体衍射到2.4 Å的Bragg间距，RGA5A_S和AVR1-CO39各有一个分子存在于初始模型的不对称单元。配合物II晶体属于空间群I4，其晶胞参数a = b = 137.4, c = 66.2 Å， α = β = γ = 90°。晶体衍射到Bragg间距为2.72 Å，在初始模型的不对称单元中存在两个RGA5A_S分子和两个AVR1-CO39分子。RGA5A_S与AVR1-CO39相互作用的进一步结构表征将有助于更好地理解R蛋白识别效应物的机制。
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 P3121, 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.