Simultaneous introduction of multiple biomacromolecules into plant cells using a cell-penetrating peptide nanocarrier

Thagun, Chonprakun; Motoda, Yoko; Kigawa, T.; Kodama, Yutaka; Numata, Keiji

doi:10.1039/d0nr04718j

Cited by 27 publications

(23 citation statements)

References 63 publications

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“…The direct introduction of proteins for trait addition to plants has been studied as an alternative method to gene recombination. , One of these methods is conjugation of a target protein with cell-penetrating peptides (CPPs). CPPs are useful tools for efficiently transporting substances such as nucleic acids and proteins into cells. − Various CPPs have already been developed and are expected to be applied to the medical field. − We have succeeded in introducing proteins such as Citrine, bovine serum albumin, and green fluorescent protein into plant cells by complexing them with CPPs via ionic interactions. , This technique enabled us to impart transient kanamycin resistance to apple leaves by introducing a complex of CPP-fused peptide and neomycin phosphotransferase II (NPTII), a kanamycin resistance protein.…”

Section: Introductionmentioning

confidence: 99%

Peptide-Based Polyion Complex Vesicles That Deliver Enzymes into Intact Plants To Provide Antibiotic Resistance without Genetic Modification

et al. 2020

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Direct delivery of enzymes into intact plants using cell-penetrating peptides (CPPs) is an attractive approach for modifying plant functions without genetic modification. However, by conventional methods, it is difficult to maintain the enzyme activity for a long time because of proteolysis of the enzymes under physiological conditions. Here, we developed a novel enzyme delivery system using polyion complex vesicles (PICsomes) to protect the enzyme from proteases. We created PICsome-bearing reactive groups at the surface by mixing an anionic block copolymer, alkyne-TEG-P(Lys-COOH), and a cationic peptide, P(Lys). The PICsome encapsulated neomycin phosphotransferase II (NPTII), a kanamycin resistance enzyme, and protected NPTII from proteases in vitro. A CPP-modified PICsome delivered NPTII into the root hair cells of Arabidopsis thaliana seedlings and provided kanamycin resistance in the seedlings that lasted for 7 days. Thus, the PICsome-mediated enzyme delivery system is a promising method for imparting long-term transient traits to plants without genetic modification.

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Section: Introductionmentioning

confidence: 99%

Peptide-Based Polyion Complex Vesicles That Deliver Enzymes into Intact Plants To Provide Antibiotic Resistance without Genetic Modification

et al. 2020

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“…One possible improvement of recombination efficiency might be achieved with new CPPs such as BPCH7, which efficiently releases DNA after penetrating inside the cell ( Chuah and Numata, 2018 ); the block copolymer maleimide-conjugated tetra(ethylene glycol) and poly( l -lysine; MAL-TEG-PLL; Miyamoto et al, 2019 ), an endosome-escaping micelle ( Miyamoto et al, 2021 ); and new candidates from a list of 55 peptides ( Numata et al, 2018 ) for efficient gene delivery to chloroplasts. These CPPs may be included into the current cpPD complex as conjugates ( Thagun et al, 2019 , 2020 ), which have raised the transformation efficiency of various types of plastids. Furthermore, based on the behavior of the cpPD complex in the present study, future targets to improve recombination efficiency include changing the properties of the cell wall, harnessing uptake mechanisms such as phagocytosis or pinocytosis, and increasing the colocalization of cp-nucleoids and plasmid DNA.…”

Section: Discussionmentioning

confidence: 99%

Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves

et al. 2021

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Each plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplast genome using peptide–DNA complexes composed of plasmid DNA and a fusion peptide consisting of a cell-penetrating peptide (CPP) and a chloroplast transit peptide (cpPD complex). However, how cpPD complexes are transported into the chloroplast from outside the cell remains unclear. Here, to characterize the route by which these cpPD complexes move into chloroplasts, we tracked their movement from the extracellular space to the chloroplast stroma using a fluorescent label and confocal laser scanning microscopy (CLSM). Upon infiltration of cpPD complexes into the extracellular space of Arabidopsis thaliana leaves, the complexes reached the chloroplast surface within 6h. The cpPD complexes reached were engulfed by the chloroplast outer envelope membrane and gradually integrated into the chloroplast. We detected several cpPD complexes localized around chloroplast nucleoids and observed the release of DNA from the cpPD. Our results thus define the route taken by the cpPD complexes for gene delivery from the extracellular space to the chloroplast stroma.

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“… 17 In addition, functional proteins were successfully internalized into plant cells via functionalized CPPs. 18 , 24 …”

Section: Introductionmentioning

confidence: 99%

“… 25 , 27 The use of these functionalized CPPs and OTPs highlights the advanced development of peptide-based biomolecule delivery systems for plants. Various combinations of cationic CPPs and OTPs have been successfully used to deliver plasmid DNA harboring reporter gene expression cassettes to nuclei and targeted organelles in plant cells using syringe infiltration, 16 − 18 , 24 − 27 vacuum/compression infiltration, 26 , 28 or injection. 27 However, these techniques are impractical for reprogramming plant quality traits under agricultural conditions.…”

Section: Introductionmentioning

confidence: 99%

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts

et al. 2022

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Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.

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Simultaneous introduction of multiple biomacromolecules into plant cells using a cell-penetrating peptide nanocarrier

Abstract: Simple and efficient cell-penetrating peptide-based multiple DNA, RNA and protein codelivery platforms to plant cells have been developed.

Cited by 27 publications

References 63 publications

Peptide-Based Polyion Complex Vesicles That Deliver Enzymes into Intact Plants To Provide Antibiotic Resistance without Genetic Modification

Peptide-Based Polyion Complex Vesicles That Deliver Enzymes into Intact Plants To Provide Antibiotic Resistance without Genetic Modification

Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts

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