Collaborative Research: Tools4Cells: An Inducible Degron Tool for Plant Cell Biology

Proteins are the catalytic agents in the cell and carry out most cellular reactions. Control of the abundance or activity of cellular proteins can be used to modify cellular pathways and, by extension, control the health and viability of an organism. While systems for targeted destruction of proteins of interest are available for research and therapeutics in animals, very few tools exist to control protein abundance in plants.

Instead, plant scientists are currently reliant on methods that regulate protein abundance at the level of mRNA expression, which are inherently slow. This research will enhance the capabilities of a recently developed tool to control protein degradation in plants called E3 DART, thus providing the opportunity to control the abundance of a protein target at the protein level.

Specifically, this work will expand the mode of activation of E3 DART and widen its applicability to multiple plant species of research and commercial importance. The Broader Impacts of this work include its intrinsic merit as the optimized tool will enhance fundamental plant biology research and may be deployed in the future for applied agronomic innovations.

Agronomic industry innovations that could benefit include developing novel herbicide resistance traits, engineering pathogen resistance by degrading pathogen effectors or design of new haploid technologies for faster breeding. Additional activities include outreach and development of teaching tools for museum activities, high school and/or undergraduate courses, and plasmid designs and plant lines deposited in public repositories. The research team will continue to mentor young scientists to develop a strong workforce in STEM.

Inducible protein degradation systems are an important, but untapped resource for the study of protein function in plant cells. The recently developed E3-targeted Degradation of Plant Proteins (E3-DART) is a protein degradation system based on the activity of a Novel E3 Ligase (NEL) from Salmonella. The goals of this work are to optimize the E3 DART system such that it can be chemically controlled and, combined with other recombinant strategies, used in proof-of concept experiments to test the function of specific endomembrane proteins.

This complementary set of tools, which are lacking in model plant systems, will provide deeper insights than previously possible into the highly dynamic, temporal, and spatial molecular mechanisms of organelle biogenesis and endomembrane trafficking. The specific aims of this research are to: 1) Develop a ligand-inducible E3-DART system; 2) Control E3-DART activity with novel recombinant tools; and 3) Develop proof-of-concept methodology with E3-DART to study endomembrane protein function and synchronized secretory protein trafficking.

A robust system to control protein degradation will have a significant impact on plant biology. Key for the development of such systems is to engineer plant lines in which the degron-tagged protein of interest functionally complements a mutant, and the E3 DART activity and target protein degradation are controlled in a tunable and reversible manner.

Such capability will allow for future characterization of the function of essential proteins involved in dynamic cellular processes in plants in ways not achievable with existing tools.

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.