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bioRxiv · 10.1101/2025.08.22.671880

Extracellular contractile injection systems for high efficiency protein delivery to plants

Legendre, M. G.·Heredia, C. A.·Colee, C. M.·Demirer, G. S.
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Abstract

Protein delivery into plant cells remains a major barrier in plant biotechnology. Extracellular contractile injection systems (eCIS) are phage tail-like nanomachines evolved by bacteria to engage eukaryotic host cells and deliver protein effectors. The Photorhabdus virulence cassette (PVC) is a well-characterized eCIS that naturally targets insect hosts. Here, we retargeted PVCs to plants by engineering their tail fibers to recognize the plant receptor FLAGELLIN SENSITIVE2 (FLS2). We designed and characterized a library of FLS2-targeting PVC variants and evaluated their ability to deliver native and non-native protein cargoes, including a fluorescent protein, Cre recombinase, and R2 retroelement genome editor. Engineered PVCs delivered Cre into wild-type Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf cells with efficiencies of up to 15%. Delivery was FLS2-dependent and scaled with receptor abundance, reaching more than 75% of leaf cells in FLS2-overexpressing lines. We further demonstrated PVC-mediated delivery of R2, resulting in targeted insertion of an mCherry reporter into the plant genome. This work establishes PVCs as receptor-mediated protein-delivery vehicles for plant cells and provides a foundation for targeted, DNA-free genome engineering in plant biotechnology.

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Legendre, M. G., Heredia, C. A., Colee, C. M., Demirer, G. S.. 2025-08-28. Extracellular contractile injection systems for high efficiency protein delivery to plants. https://doi.org/10.1101/2025.08.22.671880

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