Extracellular contractile injection systems for high efficiency protein delivery to plants

Efficient delivery of functional proteins into plant cells remains a major barrier in plant biotechnology. Extracellular contractile injection systems (eCISs) are phage tail-like nanomachines evolved by bacteria to interface with eukaryotic host cells and deliver protein effectors. The Photorhabdus virulence cassette (PVC), a well-characterized eCIS, naturally targets insect hosts but can be reprogrammed for protein cargo delivery in mammalian systems. Here, we adapted PVCs for targeted delivery to plants by engineering their tail fibers to recognize a natural plant immune receptor, FLAGELLIN SENSITIVE2 (FLS2). We designed a library of FLS2-binding PVC variants and demonstrated efficient loading and delivery of non-native cargoes, including a fluorescent reporter protein and the Cre recombinase. We showed that engineered PVCs can deliver these proteins to Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf cells with efficiencies up to 40%. We elucidated that the delivery efficiency is correlated with receptor surface density, demonstrating that receptor selection and expression level are key parameters for optimization. This work establishes PVCs as novel, programmable protein delivery nanoparticles for plants, capable of targeting plant membrane receptors and effectively delivering diverse functional proteins. By enabling precise, DNA-free delivery of gene editing proteins, plant-targeted PVCs provide the framework for next-generation genome engineering strategies with broad potential in agricultural nanobiotechnology.