EAGER: An inducible degron system for control of protein degradation in plants.

生物学家可以通过研究从细胞中去除蛋白质的效果来确定蛋白质在活生物体中的功能。在大多数情况下，这可以通过导致蛋白质表达减少或蛋白质失活的遗传操作来实现。然而，当这些蛋白质的丢失对生物体是致命的时，这种策略是不可行的。此外，一些细胞途径是非常动态的，并且蛋白质失活的影响可能仅在短时间内可检测到。该项目将开发一种按需系统，以靶向植物中降解的特定蛋白质，并使植物生物学家能够在短时间内确定从细胞中去除这些蛋白质的效果。这种蛋白质降解系统适用于任何植物途径，因此将成为植物生物学的变革工具。这项工作将培养两名本科生和一名博士后研究助理，掌握分子生物学、活细胞成像、沟通和公众参与方面的最先进技术。该研究将包括外展代表性不足的高中学生在动手研究的一部分NCSU的CAALS-3D program.The的目标是设计，测试和优化第一个植物兼容的诱导降解决定子工具的蛋白质目标在植物中的失活。该系统还将能够表征必需蛋白质，目前唯一可用的遗传工具是地塞米松诱导的RNA沉默。RNA沉默需要数天的孵育才能检测到蛋白质水平的变化，这使得它不适合动态细胞事件。一个快速、可诱导的系统来控制植物中蛋白质的丰度将有助于研究高度动态的过程，如内膜运输和细胞周期控制。所提出的降解决定子是基于异源E3酶与其靶蛋白之间的特异性相互作用。靶蛋白与感兴趣的蛋白的融合物将与融合至诱导型结构域的E3酶共表达。该设计将导致用于植物中感兴趣的蛋白质的按需降解的诱导系统。该项目的目标是：1）开发第一个植物诱导蛋白降解决定子; 2）通过靶向两个必需蛋白来证明降解决定子的功能。将利用金辫基因克隆和转化烟草和拟南芥的方法来验证该降解决定子的可行性、效率和转化速度。GFP和两种内源性必需蛋白质的定量成像将用作概念验证的测试案例。预期靶蛋白的诱导降解在30分钟内迅速发生，这将允许植物生物学家使用稳定转化的植物以无与伦比的时间分辨率查询任何感兴趣的途径。该系统将有助于分析由必需蛋白质控制的各种细胞过程，并将提高蛋白质敲除实验的时间分辨率。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估。

Biologists can identify the functions of proteins in living organisms by studying the effect of removing such proteins from the cell. In most cases, this can be accomplished by genetic manipulations that result in reduced protein expression or protein inactivation. This strategy, however, is not feasible when the loss of such proteins is lethal to the organism. Moreover, some cellular pathways are very dynamic, and the effect of protein inactivation may be detectable only within short time frames. This project will develop an on-demand system to target specific proteins for degradation in plants and will allow plant biologists to determine the effect of removing such proteins from the cell within a short time. This protein degradation system is applicable to any plant pathway and therefore will become a transformative tool for plant biology. This work will train two undergraduates and a postdoctoral research associate in state-of-the-art techniques in molecular biology, live-cell imaging, communication and public engagement. The research will include outreach to underrepresented high-school students in hands-on research as part of NCSU's CAALS-3D program.The goal of this exploratory EAGER project is to design, test and optimize the first plant-compatible inducible degron tool for inactivation of protein targets in plants. This system will also enable the characterization of essential proteins, for which the only genetic tool available today is dexamethasone-induced RNA silencing. RNA silencing requires days of incubation before changes in protein levels are detected, which makes it inadequate for dynamic cellular events. A fast, inducible system to control protein abundance in plants will be useful for studies of highly dynamic processes such as endomembrane trafficking and cell cycle control. The proposed degron is based on the specific interaction between a heterologous E3 enzyme and its target protein. A fusion of the target protein with a protein of interest will be co-expressed with the E3 enzyme fused to an inducible domain. This design will result in an inducible system for on-demand degradation of proteins of interest in plants. The aims of this project are: 1) To develop the first plant inducible protein degron; and 2) To demonstrate degron functionality by targeting two essential proteins. GoldenBraid cloning and genetic transformation into Nicotiana and Arabidopsis will be used to test the feasibility, efficiency and speed of the degron. Quantitative imaging of GFP and two endogenous essential proteins will be used as test cases for proof of concept. The induced degradation of targeted protein is expected to occur rapidly, within 30 min, which will allow plant biologists to query any pathway of interest with unmatched time resolution using stably transformed plants. This system will facilitate analyses of a wide variety of cellular processes that are controlled by essential proteins and will improve temporal resolution in protein knockdown experiments.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.