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

生物学家可以通过研究将蛋白质从细胞中移除的效果来确定这些蛋白质在生物体中的功能。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基因操作来实现。然而，当这些蛋白质的丢失对生物体来说是致命的时，这种策略是不可行的。此外，一些细胞通路是非常动态的，蛋白质失活的影响可能只能在很短的时间内检测到。该项目将开发一种按需系统，以确定特定蛋白质在植物中降解的目标，并将使植物生物学家能够在短时间内确定从细胞中移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性工具。这项工作将培训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、交流和公共参与方面的最新技术。作为NCSU CAALS-3D项目的一部分，这项研究将包括在实践研究中对未被充分代表的高中生进行推广。这个探索性的、渴望的项目的目标是设计、测试和优化第一个植物兼容的可诱导降解工具，用于灭活植物中的蛋白质靶标。这一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沉默需要几天的孵育时间才能检测到蛋白质水平的变化，这使得它不足以应对动态的细胞事件。一个快速、可诱导的系统来控制植物中的蛋白质丰度，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。所提出的降解子是基于异源E3酶与其目标蛋白之间的特定相互作用。目标蛋白与感兴趣蛋白的融合将与融合到诱导区的E3酶共表达。这种设计将产生一种可诱导的系统，用于按需降解植物中感兴趣的蛋白质。该项目的目标是：1)开发第一个植物可诱导蛋白降解；2)通过靶向两种必需蛋白来展示降解功能。将利用黄金辫子克隆和遗传转化烟草和拟南芥来测试降解的可行性、效率和速度。GFP和两种内源性必需蛋白的定量成像将作为概念验证的测试案例。目标蛋白的诱导降解预计将在30分钟内迅速发生，这将使植物生物学家能够利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。该系统将促进对由必需蛋白质控制的各种细胞过程的分析，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，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.