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.
