TRTech-PGR: Developing an Intracellular Protein Delivery System for In Planta Protein-protein Interaction Studies

Ohio State University, The, Columbus OH

Investigators

Abstract

How proteins interact inside plant cells plays a vital role in how plants grow, adapt, and defend themselves against pests and environmental stresses. However, current tools to study these interactions often rely on non-native systems or invasive methods that can disrupt natural cellular processes. This project will develop a novel, non-invasive protein delivery system to deliver proteins directly into native plant tissues. The new system uses a small, engineered protein called MTD4, which can carry functional proteins into plant cells without disruptive delivery systems and much more efficiently than previous methods. This innovation enables researchers to observe the activity of proteins in real time within live, physiologically intact plant cells, offering a clearer picture of how plant responses are regulated. The approach is fast, flexible, and applicable to a variety of plant species, making it a powerful tool for functional genomics. Development of the system will generate proteomics data from protein-protein interactions that can be used for further functional analysis by the plant community. The project involves strong training and outreach components, providing hands-on research opportunities for graduate students, postdocs, and undergraduates. Through a partnership with Ohio’s Farm Science Review, students will engage with farmers and the public to communicate scientific discoveries and real-world agricultural challenges through storytelling and creative media. This project will enhance our understanding of plant genomes, foster interdisciplinary education, and support the development of resilient, high-performing crops for a sustainable future. This project aims to develop an intracellular protein delivery (ICPD) system to enable study of proteins delivered into plant cells in a physiologically relevant context, by leveraging a highly active membrane translocation domain, MTD4. Current delivery systems, including heterologous expression systems, protoplast transfection, and agroinfiltration are limited by the lack of cellular context, slow protein expression, and/or unintended stress responses. Preliminary studies demonstrate that MTD4 is capable of rapidly delivering a variety of soluble proteins directly into the cytosol of plant cells, with minimal damage to the plant. This project aims to: (1) optimize the ICPD system in tomato and Arabidopsis, and (2) develop ICPD for delivering cargo proteins into various subcellular organelles. This system delivers proteins into plant cells via foliar spray, enabling nuanced analyses of protein function and interactions in real time. By preserving cellular integrity and context, ICPD represents a significant advancement in functional genomics tools for plants. Beyond basic discovery, ICPD has biotechnology potential in high-throughput screening applications and synthetic biology approaches. The project provides robust training and outreach activities designed to foster the next generation of plant scientists and communicators strengthening capacity in plant biology and agricultural biotechnology. Project data resources will be accessible through long-term public repositories. 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.