Nuclear Delivery of Exogenous Gene in Mature Plants Using Nuclear Location Signal and Cell-Penetrating Peptide Nanocomplex

Abstract

Delivery and expression of exogenous plasmid DNA (pDNA) into mature plants for plant genetic engineering mainly rely on Agrobacterium-mediated transformation and biolistic bombardment. Meanwhile, the process of pDNA entering the nucleus via traditional methods is random and there is no nuclear targeting effect. Here, we reported a dual-peptide-based gene delivery system to achieve nuclear delivery of exogenous pDNA in mature plants. This system is a combination of engineered peptides composed of nuclear location signal (NLS) fusion with the DNA binding domain (KH) 9 and cell-penetrating peptides (CPPs), which could bind pDNA to form a sphere-like CPP-NLS (KH) -pDNA nanocomplex. We found that this system could overcome barriers of the nuclear pore complexes (NPCs) and deliver exogenous pDNA entering the nucleus efficiently. Besides, we demonstrated that the CPP-NLS (KH) -pDNA nanocomplex could efficiently cross the rigid plant cell wall into the nucleus at 24 h after treatment and bring about a transient expression in plant suspension cells and living plants 3 days after treatment. This strategy has obvious advantages of good biocompatibility, nongenomic integration, and nonspecies limitation and shows the potential of NLS peptides as gene carriers, which would provide a delivery tool to complement preset methods serving plant genetic engineering.

DNA Delivery by Virus-Like Nanocarriers in Plant Cells

“…Recent advancements in nanotechnology have revealed the potential of nanomaterials in facilitating the delivery of genetic materials, such as plasmid DNA 23−25 and siRNA, 26,27 as well as biomacromolecules like functional proteins, 28 active ingredients, 29,30 nutrients, 31 and therapeutics 32 in plants. Singlewalled carbon nanotubes (SWCNTs), 23,24,33 mesoporous silica nanoparticles (MSNs), 34,35 layered double hydroxide (LDH) clay nanosheets, 26 and functional peptide−DNA complexes 25,36 Several studies have demonstrated the possibility of carbon nanotube-mediated gene delivery in plant nucleus, 23,36 chloroplast, 24,33 and mitochondrial 25 genomes. However, there is a need to develop high aspect ratio nanomaterials for plant transformation that are degradable, biocompatible, and manufactured with controlled aspect ratios on a large scale.…”

“…Recent advancements in nanotechnology have revealed the potential of nanomaterials in facilitating the delivery of genetic materials, such as plasmid DNA − and siRNA, , as well as biomacromolecules like functional proteins, active ingredients, , nutrients, and therapeutics in plants. Single-walled carbon nanotubes (SWCNTs), ,, mesoporous silica nanoparticles (MSNs), , layered double hydroxide (LDH) clay nanosheets, and functional peptide–DNA complexes , have demonstrated delivery of functional DNA/RNA cargoes into plant cells without mechanical assistance. Several studies have demonstrated the possibility of carbon nanotube-mediated gene delivery in plant nucleus, , chloroplast, , and mitochondrial genomes.…”

“…Single-walled carbon nanotubes (SWCNTs), ,, mesoporous silica nanoparticles (MSNs), , layered double hydroxide (LDH) clay nanosheets, and functional peptide–DNA complexes , have demonstrated delivery of functional DNA/RNA cargoes into plant cells without mechanical assistance. Several studies have demonstrated the possibility of carbon nanotube-mediated gene delivery in plant nucleus, , chloroplast, , and mitochondrial genomes. However, there is a need to develop high aspect ratio nanomaterials for plant transformation that are degradable, biocompatible, and manufactured with controlled aspect ratios on a large scale.…”