Plants

Grooming plants with spray | RIKEN

February 23, 2022

Riken
Kyoto University
Utsunomiya University
University of Tokyo
Science and Technology Promotion Agency

-Development of a simple non-GMO plant modification method-

Researcher Chongparakan Tagun (currently a professor at the Graduate School of Engineering, Kyoto University at the time of the research), Masaki Odawara, visiting researcher Yutaka Kodama (Utsunomiya University), a joint research team composed of professors from the Center for Biological Sciences Education and Research), Keiji Numata Team Leader (Kyoto The vectors and nucleic acids were developed by Misato Otani, associate professor at the Graduate School of Engineering at the University of Tokyo and Associate Professor at the Graduate School of Frontier Science at the University of Tokyo. Develop an easy way to engineer plants.

The results of this study are non-GMO[1]It is a transient modification of crops and is expected to contribute to increased pathogen resistance and metabolite modification.

The collaborative research team this time is “Cell Penetrating Peptide (CPP)”[2]Based on “”, we successfully introduced nucleic acid into plants by spraying nano-scale carriers. In this way, foreign DNA introduced from plant cells or chloroplasts transiently produces proteins, while also producing siRNA.[3]We successfully inhibited the expression of the target protein in plant cells by introducing.

This study is based on scientific journals”ACS NanoPublished ahead of the online edition (23 February: 23 February Japan time).

Illustration of spraying nucleic acid-cell-permeable peptide complexes onto plants

Nucleic acid-cell-permeable peptide complexes are sprayed onto plants by spraying

background

Plant trait modification solves food and environmental problems and brings various wealth to society.However, genetic recombination based on the introduction of foreign DNA into the plant genome[1]Retrofitting factories on an industrial scale is time-consuming and expensive, and there are concerns about human and environmental safety.

Keiji Numata and his team leaders have been developing methods for modifying plants through gene transfer using functional peptides as carriers.Functional peptide modification is simple, low cost, specific organelle[4]This is a method that can be modified by targeting, but only for small-scale introductions, such as small plants or parts of leaves.Note 1-3).

Since spray treatment is a technique that can even be applied on a large scale, such as on farms, the joint research team is now trying to develop a method for plant gene transfer using functional peptides through spray spraying.

Research Methods and Results

The collaborative research team first selected a “cell-penetrating peptide (CPP)” suitable for introducing nucleic acids by spraying. Then, complexes of the selected CPP and the DNA to be introduced are prepared, suspended in a liquid, and sprayed on the leaves of the plants. As a result, in Arabidopsis thaliana as a model plant, tomato and soybean leaves as crops, DNA was efficiently taken up into cells, and a reporter gene was obtained from the taken up DNA.[5]GusExpression of (β-glucuronidase) was confirmed (Fig. 1A, B). Next, RNA interference occurred when a complex of CPP and siRNA was formed and sprayed on the leaves of transgenic tomatoes and Arabidopsis thaliana expressing the fluorescent protein.[6]The expression of fluorescent proteins (GFP and YFP) was successfully inhibited (Fig. 1C).

DNA and RNA uptake profiles in sprayed leaves

Figure 1 DNA and RNA uptake in spray-treated leaves

  • A:Detection of GUS protein in Arabidopsis leaves using cell penetrating peptide (CPP) and DNA. In the left lobe sprayed with DNA and CPP, the activity of the reporter gene-expressed GUS protein produced a blue substance. No GUS protein was produced in the right lobe sprayed with DNA alone.
  • Second:Activity of GUS proteins in Arabidopsis leaves.
  • C:The introduction of siRNA inhibited the expression of green fluorescent protein (GFP) in tomato leaves. Left is when only CPP is sprayed on GFP-expressing leaves, and inside is when only siRNA is sprayed. On the right, when CPP and siRNA were sprayed, RNA interference occurred due to the introduction of siRNA, and GFP was reduced. Magenta is the autofluorescence of chloroplasts.

The collaborating team further developed the technology by using different peptides and successfully delivered nucleic acids into the organelle’s chloroplast. Expression of the reporter gene (luciferase) introduced into the chloroplast was expressed when a complex consisting of KH-AtOEP34, a peptide specifically delivered to the chloroplast, CPP, and DNA was prepared and sprayed on the leaves of white flying birds. .2A). In addition, the siRNA was delivered to the chloroplast by the same method, and the expression of the fluorescent protein expressed in the chloroplast was successfully suppressed by gene recombination (Fig. 2B).

DNA and RNA uptake in spray-treated Arabidopsis chloroplasts

Figure 2 DNA and RNA uptake in spray-treated Arabidopsis chloroplasts

  • A:Luciferase activity in leaf chloroplasts into which a luciferase gene (reporter gene) has been introduced. The horizontal axis represents the time after introduction, and the vertical axis represents the light emission amount. The combination of CPP and KH-AtOEP34 (chloroplast targeting peptide) facilitated the uptake of luciferase gene DNA by the chloroplast.
  • Second:Inhibition of GFP expression by introduction of siRNA. Introduction of peptides (CPP and KH-AtOEP34) and siRNA into plants expressing green fluorescent protein (GFP) in the chloroplast resulted in RNA interference and reduced GFP. Magenta is the autofluorescence of chloroplasts.

From these results, it can be seen that the sprayed DNA and RNA are delivered to a wide range of plant cells and even organelles under the action of CPP and are non-genetically recombined, which can transiently inhibit exogenous gene expression and gene expression. .

future expectations

In this study, we successfully delivered nucleic acids not only to the model plant Arabidopsis, but also to the leaves of crops such as soybean and tomato by spraying the vector with optimized CPP. This method can efficiently and transiently introduce nucleic acids into plants without special equipment, and is a non-transgenic crop that provides transient drug resistance, for example, on a farm-scale planting scale. to modify. On the other hand, risks such as unintentional introduction of nucleic acids into organisms other than target plants will need to be carefully assessed in the future.

In this study, we also observed inhibition of gene expression by introducing siRNA into the chloroplast, which is not thought to cause RNA interference. This is a result that subverts the traditional concept, and it is necessary to analyze the expression inhibition mechanism in detail in the future.

This study is based on the 17 Sustainable Development Goals (SDGs) set by the United Nations in 2016.[7]Among them, we can expect to contribute to “2. Zero Hunger” and “15. Conservation of Abundant Lands”.

Supplementary Instructions

  • 1.Non-GMO, GMO
    In this study, the incorporation and expression of exogenous genes into the DNA of the nucleus or organelles was defined as genetic recombination, while the phenotypic modification without the incorporation of exogenous genes was defined as non-genetic recombination.
  • 2.Cell Penetrating Peptide (CPP)
    A general term for peptides that can penetrate cell membranes and migrate into cells. CPP is an abbreviation for Cell Penetrating Peptide.
  • 3.siRNA
    Double-stranded RNA with 21-25 base pairs. By incorporating synthetic siRNA into cells, the expression of genes with complementary sequences can be inhibited. siRNA is short for Small Interfering RNA.
  • 4.cellular organ
    A general term for membrane-separated organs with special functions and shapes in eukaryotic cells. Including the nucleus, endoplasmic reticulum and Golgi apparatus.
  • 5.reporter gene
    A gene for easy detection and quantification of the expression of the gene to be analyzed. Green fluorescent protein (GFP) quantifies gene expression by its own fluorescence, β-glucuronidase (GUS) quantifies gene expression by staining degraded substrates, and luciferase quantifies gene expression by luminescence of degraded substrates quantify.
  • 6.RNA interference
    A phenomenon in which messenger RNA (mRNA) having a sequence complementary to RNA introduced into cells is degraded and gene expression is inhibited.
  • 7.Sustainable Development Goals (SDGs)
    International goals for the period 2016 to 2030 are set out in the 2030 Agenda for Sustainable Development, adopted at the United Nations summit in September 2015. Composed of 17 goals and 169 goals, the realization of a sustainable world is a common thing, not only developing countries, but also developed countries themselves are working hard, and Japan is also actively working on it. (Reprinted from the website of the Ministry of Foreign Affairs with some modifications).

research support

This research was supported by the Japan Science and Technology Agency (JST) Strategic Innovation Research Promotion Project General Implementation Research ERATO “Numata Organelle Reactor Cluster Project (Research Supervisor: Keiji Numata)”.

Base paper information

  • Chonprakun Thagun, Yoko Horii, Maai Mori, Seiya Fujita, Misato Ohtani, Kousuke Tsuchiya, Yutaka Kodama, Masaki Odahara, Keiji Numata, “Non-transgenic gene regulation by spray delivery of nucleic acid/peptide complexes into plant nucleus and chloroplast”, ACS Nano10.1021/acsnano.1c07723

host

Riken
RIKEN Sustainable Resource Science Center Biopolymer Research Team
Special researcher (at the time of research) チョンパラカン・タグン (Thagun Chonrakun)
(Currently appointed assistant professor at the Graduate School of Engineering, Kyoto University)
Researcher Masaki Odawara
Yutaka Kodama, Visiting Researcher
(Professor, Research Center for Life Science Education, Utsunomiya University)
Numata Keiji
(Professor, Graduate School of Engineering, Kyoto University)

Photo of Researcher Chongparakan Tagung (during research)
Chongparakan Tagong

Department of Frontier Science, The University of Tokyo
Associate Professor Miri Otani

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