Split Nano Luciferase Complementation for Probing Protein-Protein Interactions in Plant Cells.
Split Nano Luciferase Complementation for Probing Protein-Protein Interactions in Plant Cells.
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DOI：
10.1111/jipb.12891
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发表时间：
2019-11
影响因子：
11.4
通讯作者：
Fengzhu Wang;N. Zhang;Yanjun Guo;Ben-Qiang Gong;Jian-Feng Li
中科院分区：
文献类型：
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作者：
Fengzhu Wang;N. Zhang;Yanjun Guo;Ben-Qiang Gong;Jian-Feng Li
蛋白质-蛋白质相互作用（PPIs）是理解植物信号转导途径的基础。裂解萤火虫荧光素酶（Fluc）互补（SLC）试验已广泛用于分析PPI。然而，人们越来越担心Fluc庞大的一半会干扰其融合伴侣的功能。纳米荧光素酶（Nluc）是Fluc的最小替代品，具有更好的稳定性和发光性。在这里，我们开发了一种两用系统，通过基于Nlux的SLC和免疫共沉淀试验检测PPI。这是通过分别与HA或FLAG标记的Nluc半体融合的两种研究蛋白的共表达来实现的。我们通过在原生质体或农杆菌转化的植物中复制多个先前记录的PPI来验证该系统的稳健性。接下来，我们应用该系统来评估拟南芥CERK 1的同源二聚化，真菌激发子几丁质的共受体，以及在几丁质存在或不存在的情况下与其他同源物的异源二聚化。此外，Nluc的分裂片段与拟南芥钙通道CNGC 2和CNGC 4的两个胞质末端融合，以帮助感知细菌诱导子flg 22诱导的变构变化。总的来说，这些结果证明了基于Nlux的SLC测定用于探测植物细胞中的组成型或诱导型PPI和蛋白质变构的有用性。本文受版权保护。All rights reserved.
Deciphering protein-protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual-use system enabling the detection of PPIs through the Nluc-based SLC and co-immunoprecipitation assays. This was realized by co-expression of two proteins under investigation in fusion with the HA- or FLAG-tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium-transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a co-receptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc-based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells. This article is protected by copyright. All rights reserved.