Systemic Delivery of Functional Proteins Into Plants Using an Engineered Membrane Translocation Domain.

Protein-based biopesticides and biostimulants are critical for the future of sustainable agriculture, yet their utility is severely limited by inefficient delivery into plant cells. Traditional cell-penetrating peptides enable protein uptake but lack the efficiency, systemic activity and scalability required for crop production. Here, we present a potential general solution using a novel engineered membrane translocation domain, MTD4, to enable robust, systemic protein delivery into crops and it is suitable for large-scale application. We first demonstrate that MTD4 enables rapid foliar delivery of a model protein (SEP) and, importantly, facilitates its systemic translocation from lower to upper leaves and from roots to shoots, a key requirement for whole-plant protection. To prove the platform's utility, we fused MTD4 to the harpin protein HrpZ, a known defence elicitor. MTD4-HrpZ delivered into tobacco and tomato plants triggered a potent hypersensitive response and systemic acquired resistance, resulting in significant reductions in disease severity from bacterial and fungal pathogens. Remarkably, the MTD4-HrpZ fusion protein was over five times more effective than HrpZ alone, highlighting MTD4's capacity to dramatically enhance protein efficacy. This work introduces MTD4 as a transformative tool for overcoming protein delivery barriers in plants, paving the way for a new generation of high-potency biotherapeutics that can advance sustainable crop protection and reduce dependence on chemical applications.