Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants

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.

Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance

“…Based on the microscopic analysis, the cells were partially transfected with RhB labeled NPTII. According to our previous study (Ng et al 2016), the peptide-mediated protein delivery was confirmed by several quantitative methods.…”

“…Copyright © 2016 The Japanese Society for Plant Cell and Molecular Biology NPTII protein was labeled with Rhodamine B isothiocyanate (RhB) (Mw: 536.08 g/mol, Sigma-Aldrich. St. Louis, MO), according to a previous report (Ng et al 2016). Fusion peptide 2BP100-K8 (Amino acid sequence: KKLFKKILKYL KKLFKKILKYLKKKKKKKK, Theoretical pI/Mw: 10.75/3851.13 Da) was custom synthesized at the Peptide Synthesis Facility, RIKEN Brain Science Institute, Wako, Japan by using standard 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis (Fields and Noble 1990).…”

“…We recently developed and reported on a direct protein delivery system for intact plants (Ng et al 2016). In this study, target proteins such as bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), and the yellow fluorescent protein (27 kDa) were complexed with fusion peptides and infiltrated into intact A. thaliana leaves.…”

“…The fusion peptide used was designed to consist of a cationic sequence and a cell penetrating peptide, according to previous studies (Lakshmanan et al 2013;Lakshmanan et al 2015;Numata 2015;Numata et al 2014). In the case of protein containing a signal peptide, such as a nuclear localization signal or a peroxisomal targeting signal, the fusion peptide did not disturb the signal peptide; therefore, the target protein could be localized based on the signal peptide (Ng et al 2016). The fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact plants without preventing protein functions (Ng et al 2016).…”

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

“…More importantly, with the use of peptides, genes can be precisely targeted to intracellular organelles such as the mitochondria 21 or plastids (chloroplasts) 22 for expression-a task not achievable by biolistic or Agrobacterium-mediated transformation. Depending on the cargo type, this new plant modification technology can be exploited to deliver proteins 23 and either express (plasmid 21,24 or double-stranded DNA 25 ) or down-regulate (double-stranded RNA 26 ) specific genes within the plant, throughout its cytosolic space [24][25][26] or within a specific organellar compartment 1. Prepare stock solutions of each peptide as follows: (KH) 9 -BP100 (1 mg/ml or 800 nM), Cytcox-(KH) 9 (1 mg/ml), BP100 (1 mg/ml) and (BP100) 2 K 8 (70 µM).…”