Abstract
In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology.
Highlights
Plant genetic engineering is commonly used in plant breeding to improve the productivity and enhance crop fitness of, including yield enhancement, nutritional quality enhancement, herbicide tolerance, drought resistance, pest resistance and viral resistance [1]
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of bovine serum albumin (BSA)-Rhodamine B isothiocyanate (RhB) was performed to confirm the conjugation of RhB to BSA (S1 Fig)
The hydrodynamic diameters, zeta potentials and morphologies of the BSA-RhB conjugate and peptide/protein complexes were characterized by Zetasizer NanoZS (Fig 2) and atomic force microscopy (AFM) (Fig 3)
Summary
Plant genetic engineering is commonly used in plant breeding to improve the productivity and enhance crop fitness of, including yield enhancement, nutritional quality enhancement, herbicide tolerance, drought resistance, pest resistance and viral resistance [1]. Plants are mainly genetically modified by the delivery of exogenous DNA encoding a desired trait via Agrobacterium-mediated transformation or particle bombardment. As compared to protein delivery, the DNA delivery is commonly used to modify the plant due to the ease of preparation, the stability and the smaller size of plasmid DNA. The DNA delivery can cause several potential problems, including the random insertion of exogenous DNA into the plant genome and the uptake of antibiotic resistance genes by pathogenic bacteria in soil via horizontal gene transfer [3]. It is crucial to modify the plant via direct protein delivery strategy. Proteins are more susceptible to the denaturation and loss of functionality due to their fragile tertiary structures [4, 5]
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