A centrifugation-assisted peptide-mediated gene transfer method for high-throughput analyses

Kimura, Mitsuhiro; Yoshizumi, Takeshi; Numata, Keiji

doi:10.5511/plantbiotechnology.18.1115a

Cited by 4 publications

(9 citation statements)

References 20 publications

(26 reference statements)

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“…For the introduction of biomolecules complexed with peptides, we describe four methods as reported previously. ,,, In principle, these infiltration methods, except for infiltration by using a needleless syringe, can be applied to various type of plant tissues including callus, but soft tissue is preferable.…”

Section: Methodsmentioning

confidence: 99%

“…The centrifugation-assisted peptide-mediated gene transfer (CAPT) method enables high-throughput infiltration of a large number of leaf discs by centrifugation Soak the plants ( e.g ., leaf discs) in the complex solution prepared in step 2 in a 2 mL round-bottom tube. Centrifuge the tube at 10 000 g for 60 s at 25 °C and keep the plants soaked in the solution for 30 min at 25 °C. Cultivate the plants under the culture conditions. …”

Section: Methodsmentioning

confidence: 99%

“…The centrifugationassisted peptide-mediated gene transfer (CAPT) method enables high-throughput infiltration of a large number of leaf discs by centrifugation. 48 1. Soak the plants (e.g., leaf discs) in the complex solution prepared in step 2 in a 2 mL round-bottom tube.…”

Section: ■ Methodsmentioning

confidence: 99%

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Fusion Peptide-Based Biomacromolecule Delivery System for Plant Cells

Watanabe

Odahara

Miyamoto

et al. 2021

ACS Biomater. Sci. Eng.

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The introduction of DNA, RNA, and proteins into plant cells has become important in plant science with the recent development of innovative technologies such as genome editing. As a new method for the delivery of such biomacromolecules, fusion peptides, which have multiple functional domains, have been developed. The functional domains include cell-penetrating peptides for crossing cell membranes, polycationic peptides for biomacromolecule binding, and organelle-targeting peptides. The fusion peptide-based macromolecule delivery system enables the efficient introduction of DNA, RNA, and proteins, which are much larger in size than the peptide, into plant cells while retaining the activity of the biomacromolecules. Compared to pre-existing delivery methods, this system has advantages in that it does not require any special equipment and can be performed easily and quickly on a wide variety of plants. Furthermore, as a characteristic feature of the fusion peptide system, the application of organelletargeting peptides to fusion peptides allows selective delivery of biomacromolecules to chloroplasts or mitochondria. Here, we provide a representative method of the fusion peptide-based biomacromolecule delivery system and an example of the results of biomacromolecule delivery as promising new tools for plant biology and biotechnology.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Fusion Peptide-Based Biomacromolecule Delivery System for Plant Cells

Watanabe

Odahara

Miyamoto

et al. 2021

ACS Biomater. Sci. Eng.

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show abstract

“…In animal systems, simple co-incubation is enough to internalize the proteins delivered through nanocarriers or CPPs. Due to the presence of the cell wall, mild infiltration force such as vacuum or centrifugal force is applied to deliver protein cargoes in plants without loss of regeneration efficiency ( Kimura et al., 2019 ; Watanabe et al., 2021 ). CPPs have been used in animal cells to deliver genome-editing elements as RNPs non-biolistically, and these short, positively charged peptides were shown to translocate across cell membranes with high delivery efficiencies ( Liu et al., 2014 ; Ramakrishna et al., 2014 ; Alghuthaymi et al., 2021 ; Gustafsson et al., 2021 ).…”

Section: Rnp Delivery: Applications and Limitationsmentioning

confidence: 99%

Transgene-free genome editing and RNAi ectopic application in fruit trees: Potential and limitations

Gouthu

Mandelli²,

Eubanks³

et al. 2022

Front. Plant Sci.

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For the past fifteen years, significant research advances in sequencing technology have led to a substantial increase in fruit tree genomic resources and databases with a massive number of OMICS datasets (transcriptomic, proteomics, metabolomics), helping to find associations between gene(s) and performance traits. Meanwhile, new technology tools have emerged for gain- and loss-of-function studies, specifically in gene silencing and developing tractable plant models for genetic transformation. Additionally, innovative and adapted transformation protocols have optimized genetic engineering in most fruit trees. The recent explosion of new gene-editing tools allows for broadening opportunities for functional studies in fruit trees. Yet, the fruit tree research community has not fully embraced these new technologies to provide large-scale genome characterizations as in cereals and other staple food crops. Instead, recent research efforts in the fruit trees appear to focus on two primary translational tools: transgene-free gene editing via Ribonucleoprotein (RNP) delivery and the ectopic application of RNA-based products in the field for crop protection. The inherent nature of the propagation system and the long juvenile phase of most fruit trees are significant justifications for the first technology. The second approach might have the public favor regarding sustainability and an eco-friendlier environment for a crop production system that could potentially replace the use of chemicals. Regardless of their potential, both technologies still depend on the foundational knowledge of gene-to-trait relationships generated from basic genetic studies. Therefore, we will discuss the status of gene silencing and DNA-based gene editing techniques for functional studies in fruit trees followed by the potential and limitations of their translational tools (RNP delivery and RNA-based products) in the context of crop production.

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“…Compared to canonically mammalian mitochondrial genomes, higher plant mitochondria have larger genomes and greater numbers of mitochondrial genes [2]. In several plant species, the cytoplasmic gene transfer (CAPT) method as a high-throughput means of transferring DNA-peptide complexes to plant organelles [7].…”

Section: Introductionmentioning

confidence: 99%

Particle bombardment-assisted peptide-mediated gene transfer for highly efficient transient assay

et al. 2023

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Objective A centrifugation-assisted peptide-mediated gene transfer (CAPT) method was recently developed as an efficient system for gene delivery into plant cells. However, the gene transfer efficiency of CAPT into plant cells was not entirely satisfactory for detecting transient expression of a transgene driven into mitochondria. Here, we report a new gene delivery system using a method called particle bombardment-assisted peptide-mediated gene transfer (PBPT). Results We investigated various parameters of the PBPT method to increase transient gene expression efficiency in Brassica campestris. The optimal conditions for PBPT were a single bombardment with gold particles coated with a DNA‒peptide complex (6 µg of DNA and 2 µg of peptide) at an acceleration pressure of 5 kg/cm2 and a target distance of 12.5 cm. Moreover, bombardment under the optimal conditions successfully transferred the transgene into the cells of other plant species, namely B. juncea and tomato. Thus, we developed a PBPT method for highly efficient delivery of a DNA‒peptide complex into plant mitochondria.

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A centrifugation-assisted peptide-mediated gene transfer method for high-throughput analyses

Cited by 4 publications

References 20 publications

Fusion Peptide-Based Biomacromolecule Delivery System for Plant Cells

Fusion Peptide-Based Biomacromolecule Delivery System for Plant Cells

Transgene-free genome editing and RNAi ectopic application in fruit trees: Potential and limitations

Particle bombardment-assisted peptide-mediated gene transfer for highly efficient transient assay

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