(620a) Protein Nanoparticles for Biomolecule Delivery to Plants

Advancements in plant genetic engineering technologies are crucial for addressing food insecurity amid climate change. However, the effectiveness of these technologies is limited by current biomolecule delivery systems. Nanotechnology can circumvent many disadvantages of traditional methods – cargo diversity, species dependence, genomic mutagenesis, etc. – but it remains low in delivery efficiency, particularly for larger biomolecules like proteins, and lack cell-type specific targeting capabilities. To address these challenges, we leverage the protein loading and delivery capabilities of extracellular contractile injection systems (eCISs): natural protein nanoparticles secreted by bacteria to transfer effector proteins into host cells. Recent research has elucidated the mechanism by which eCISs bind host cell receptors and load effector cargoes, presenting the opportunity to re-engineer these particles for plant cell delivery of diverse protein cargo types.

Here, we demonstrate the genetic programming of eCISs to enable the delivery of functional protein cargoes to plant cells. Using a fluorescent reporter assay that detects Cre recombinase activity in plant cells, we show (i) the successful loading of eCISs with a non-native Cre recombinase cargo and (ii) the rational design of eCISs to target specific plant cell membrane receptors. The incorporation of an N-terminal loading tag to the Cre recombinase protein facilitates incorporation of the novel payload, highlighting the cargo modularity of the system. Engineering of the eCIS tail fiber domain to target the ubiquitously expressed plant cell membrane kinase receptor, FLS2, enables efficient delivery of Cre recombinase proteins to plant cells: both protoplast leaf mesophyll cells and intact leaf epithelial cells. Current efforts are focused on expanding our cargo repertoire to include MS2-RNA complexes and CRISPR ribonucleoprotein complexes, aiming to achieve eCIS-mediated RNA interference and genome engineering. Future work will explore an array of cell-type-specific membrane receptors and their epitope ligands to enhance the targeting specificity of the eCIS platform.