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

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.