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 for E3-targeted Degradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between the Salmonella 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. Using Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana), we show that a chimeric protein containing the Leucine-Rich Repeat (LRR) and novel E3 ligase (NEL) 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.

中文翻译：

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通过诱导蛋白降解系统快速消耗植物中的靶蛋白

诱导性蛋白质敲低是在短时间内测试必需蛋白质功能并捕获蛋白质在动态事件中的作用的绝佳工具。目前的方法通过利用植物生物学机制来破坏或隔离蛋白质，例如用于光遗传学的光感受器的活性或生长素降解决定子中生长素介导的泛素化。由此可见，这些不适用于植物，因为光和生长素对植物细胞来说是强信号。我们在这里描述了一种名为 E3-DART 的植物诱导蛋白降解系统，用于 E3 靶向植物蛋白降解。E3-DART 系统基于沙门氏菌分泌蛋白 H1 (SspH1) 与其人类靶蛋白激酶 N1 (PKN1) 之间的特定且充分表征的相互作用。该系统利用 SspH1 的 E3 催化活性和 PKN1 的同源区域 1b (HR1b) 结构域的 SspH1 结合活性。利用本塞姆氏烟草和拟南芥 (Arabidopsis thaliana)，我们发现含有富含亮氨酸重复序列 (LRR) 和 SspH1 的新型 E3 连接酶 (NEL) 结构域的嵌合蛋白可有效靶向含有 HR1b 的不同大小的蛋白质融合物进行降解。使用糖皮质激素反式激活系统通过嵌合 E3 连接酶的转录控制来诱导靶蛋白降解，并且早在诱导后 3 小时就检测到靶蛋白的消耗。该系统可用于以高时间分辨率研究任何植物蛋白的损失，并可能成为植物细胞生物学的重要工具。