Rapid depletion of target proteins in plants by an inducible protein degradation system
Rapid depletion of target proteins in plants by an inducible protein degradation system
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DOI：
10.1093/plcell/koae072
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发表时间：
2024-03-06
期刊：
影响因子：
11.6
通讯作者：
Rojas-Pierce，Marcela
中科院分区：
文献类型：
--
作者：
Huang，Linzhou;Rojas-Pierce，Marcela
可诱导蛋白质敲除是在短时间尺度上测试必需蛋白质的功能和捕捉蛋白质在动态事件中的作用的优秀工具。目前的方法通过利用植物生物机制来破坏或隔离蛋白质，例如光遗传学的光感受器的活性或生长素在生长素降解物中介导的泛素化。因此，这些都不适用于植物，因为光和生长素是植物细胞的强烈信号。在这里，我们描述了一种植物中可诱导的蛋白质降解系统，称为E3-DART，用于E3靶向植物蛋白质的降解。E3-DART系统是基于沙门氏菌分泌蛋白H1(SspH1)及其人目标蛋白激酶N1(PKN1)之间的特异性和特征性的相互作用。该系统利用了SspH1的E3催化活性和PKN1的同源区1b(HR1b)结构域的SspH1结合活性。利用烟草和拟南芥(Arabitopsis Thaliana)，我们证明了一种包含SspH1富含亮氨酸重复序列和新的E3连接酶结构域的嵌合蛋白有效地靶向含有HR1b的不同大小的蛋白融合进行降解。通过糖皮质激素反式激活系统对嵌合E3连接酶进行转录调控，可诱导靶蛋白降解，诱导后3 h即可检测到靶蛋白的耗竭。该系统可用于研究任何植物蛋白质的高时间分辨率损失，有可能成为植物细胞生物学的重要工具。
Inducible protein knockdowns are excellent tools to test the function of essential proteins in short time scales and to capture the role of proteins in dynamic events. Current approaches destroy or sequester proteins by exploiting plant biological mechanisms such as the activity of photoreceptors for optogenetics or auxin-mediated ubiquitination in auxin degrons. It follows that these are not applicable for plants as light and auxin are strong signals for plant cells. We describe here an inducible protein degradation system in plants named E3-DART forE3-targetedDegradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between theSalmonella-secreted protein H1 (SspH1) and its human target protein kinase N1 (PKN1). This system harnesses the E3 catalytic activity of SspH1 and the SspH1-binding activity of the homology region 1b (HR1b) domain from PKN1. UsingNicotiana benthamianaandArabidopsis(Arabidopsis thaliana), we show that a chimeric protein containing the leucine-rich repeat and novel E3 ligase domains of SspH1 efficiently targets protein fusions of varying sizes containing HR1b for degradation. Target protein degradation was induced by transcriptional control of the chimeric E3 ligase using a glucocorticoid transactivation system, and target protein depletion was detected as early as 3 h after induction. This system could be used to study the loss of any plant protein with high-temporal resolution and may become an important tool in plant cell biology.