Structure-based mutational analysis of eIF4E in relation to sbm1 resistance to pea seed-borne mosaic virus in pea.
Structure-based mutational analysis of eIF4E in relation to sbm1 resistance to pea seed-borne mosaic virus in pea.
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DOI：
10.1371/journal.pone.0015873
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发表时间：
2011-01-24
期刊：
影响因子：
3.7
通讯作者：
Maule AJ
中科院分区：
文献类型：
--
作者：
Ashby JA;Stevenson CE;Jarvis GE;Lawson DM;Maule AJ
豌豆编码真核翻译起始因子eIF 4 E（eIF 4 ES），该因子支持豌豆种传花叶病毒（PSbMV）的增殖。与其他马铃薯Y病毒的宿主一样，一些豌豆品系含有一个隐性等位基因（sbm 1），编码一个突变体eIF 4 E（eIF 4 ER），该突变体不能与PSbMV无毒蛋白VPg发生功能性相互作用，从而对感染产生遗传抗性。为了研究豌豆eIF 4 E和PSbMV VPg之间的结构-功能关系，我们获得了与m7 GTP结合的eIF 4 ES的X射线结构。晶体学不对称单位包含8个独立的蛋白质拷贝，提供了对eIF 4 E结构保守和灵活区域的见解。为了间接评估关键残基在与VPg和/或m7 GTP结合中的重要性，测试了eIF 4 E中广泛范围的点突变体在抗性豌豆组织中补充PSbMV增殖的能力以及在条件依赖于eIF 4 E异位表达的eIF 4 E缺陷型酵母菌株中补充蛋白质翻译并因此生长的能力。突变体还剖析了eIF 4 ER中多态性的个体贡献，并比较了其他作物物种中抗邻苯二甲酸酯等位基因中单个残基改变的影响。数据显示，eIF 4 E中的基本抗性决定簇对于不同的病毒是不同的，尽管所涉及的关键区域（可能在VPg结合中）是保守的并且与m7 GTP结合区域部分重叠。在大多数情况下，这种重叠导致了病毒增殖和翻译的偶联抑制，尽管存在一些使两个过程解偶联的突变体支持eIF 4 E在马铃薯Y病毒感染中的特定作用可能不限于翻译的观点。这项工作描述了eIF 4 E与马铃薯Y病毒抗性相关的最广泛的结构分析。除了定义eIF 4 E结构内的功能结构域之外，我们还鉴定了具有传递新病毒抗性表型的潜力的eIF 4 E等位基因。
Pea encodes eukaryotic translation initiation factor eIF4E (eIF4ES), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4ER) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection. To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4ES bound to m7GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m7GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4ER and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m7GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation. The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional domains within the eIF4E structure, we identified eIF4E alleles with the potential to convey novel virus resistance phenotypes.
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