Arginine-rich intracellular delivery peptides synchronously deliver covalently and noncovalently linked proteins into plant cells.

Protein transduction domains (PTDs) are small peptides with a high content of basic amino acids, and they are responsible for cellular uptake. Many PTDs, including arginine-rich intracellular delivery (AID) peptides, have been shown to transport macromolecules across membranes and into cells. In this study, we demonstrated for the first time that AID peptides could rapidly and efficiently deliver proteins into plant cells in both covalent and noncovalent protein transductions (CNPT) simultaneously. The optimal molecular ratio between an AID peptide carrier and cargo in CNPT was about 3:1. Fluorescence resonance energy transfer (FRET) analysis revealed protein-protein interactions between AID peptide carriers and cargos after CNPT in cells. The possible mechanisms of AID peptides-mediated cellular entry might involve a combination of multiple internalization pathways. Therefore, applications by AID peptide-mediated CNPT may provide a simple and direct transport strategy for delivering two proteins in agricultural systems.