Graduation Term

Summer 2025

Degree Name

Master of Science (MS)

Department

School of Biological Sciences

Committee Chair

Viktor Kirik

Committee Member

Kevin Edwards

Committee Member

John Sedbrook

Abstract

All cellular functions depend on protein activity, and the interactions by which proteins perform specific functions. Protein interactions drive cellular processes such as enzymatic activity, complex formation, transcriptional regulation, and signaling, and detecting these interactions allows researchers to elucidate more specific functions and identify complex pathways. Detecting these interactions has long been the goal of many molecular studies resulting in a multitude of available methods which have been optimized depending on the study. Here, we have developed Plant Two-Hybrid, a hybridized protein complementation assay modified from the traditional Yeast Two-Hybrid as an in-planta alternative. The principle of Plant Two-Hybrid is relatively simple, relying on the in vivo reconstitution of the Gal4-VP16 chimeric transcription factor which will turn on the expression of the EGFP-HTB3 reporter gene. By replacing the biosynthetic reporter gene typically used in Yeast Two-Hybrid, Plant Two-Hybrid is easily quantifiable using confocal microscopy. Using Arabidopsis trichome-specific promoters GL2 and MYB5, expression of this system is contained in trichomes limiting whole plant toxicity. These promoters are also active in Nicotiana benthamiana making Plant Two-Hybrid versatile and applicable for quick transient validation of protein-protein interactions. Plant Two-Hybrid was developed to be Gateway compatible so that proteins of interest can be easily expressed within the system. To validate the results of Plant Two-Hybrid, two proteins that have a well-established interaction during trichome development, the interaction between GL3 and MYB23 was quantified and shown to be significantly different from any autoactivation. This method will also be used to evaluate Tonneau1 (TON1) interactions in planta, which will be further validated using FLIM-FRET microscopy.

Access Type

Thesis-Open Access

DOI

https://doi.org/10.30707/ETD.1763755358.724211

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Available for download on Friday, September 10, 2027

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