Method for fusing plant cells

Abstract

One embodiment of the present invention provides a method for fusing a plant cell A and a plant cell B, the method comprising: step 1 for treating the surfaces of a protoplast of the plant cell A and a protoplast of the plant cell B with a compound represented by formula (I) (each symbol in the formula is as described in the present specification) or a salt thereof, or a solvate or hydrate thereof; and step 2 for mixing the protoplast of the plant cell A and the protoplast of the plant cell B, which have been subjected to the surface treatment in step 1, and culturing the mixture.

Description

Methods for fusing plant cells

[0001] The present invention relates to a method for fusing plant cells, a cell fusion promoter used for the method for fusing plant cells, and the like.

[0002] Cell fusion is the fusion of two or more cells, and can occur not only between cells of the same species but also between cells of different species. In particular, cell fusion between different species has long been used as a method to generate cells with new traits distinct from those of the cells prior to fusion. For example, hybridomas, which are formed by fusing antibody-producing B cells with myeloma cells to prepare monoclonal antibodies, have long been used as replicable cells capable of producing the desired antibodies. In recent years, transplantation of fused cells of mesenchymal stem cells and pancreatic islet cells has been proposed as a new treatment for diabetes (Non-Patent Document 1). Fusion cells of mesenchymal stem cells and pancreatic islet cells maintained islet function even after approximately 20 days of culture. When transplanted into a rat model of type 1 diabetes, they were resistant to immune rejection and demonstrated hypoglycemia for approximately three months. Cell fusion with plant cells can be used to create new plant species, such as the pomato, a new plant species created from the fusion product of potato and tomato.

[0003] As described above, cell fusion is widely used in research, medicine, and the botanical sciences. Known cell-cell fusion methods include the Sendai virus method, the PEG (polyethylene glycol) method, and electrical fusion. In the Sendai virus method, Sendai virus is thought to act as a bridge between cell membranes to promote fusion. This method is difficult to completely avoid viral contamination of the fused cell fraction, making it less suitable for medical use of fused cells. The PEG method does not require specialized equipment and uses inexpensive reagents, making it a commonly used method. However, this method suffers from low cell fusion efficiency and reproducibility, and its cell fusion efficiency varies significantly depending on the cell type. Electrical fusion involves applying an AC voltage to bring cells into contact between electrodes, followed by a DC pulse voltage to open holes in the cell membranes, inducing fusion between the contacting cells. This method can fuse cells more efficiently than the previous two methods. However, this method has drawbacks, such as the need for specialized equipment and the reduced cell viability caused by the application of voltage. Furthermore, cell selectivity is poor, making it difficult to fuse cells of different species. In light of the above, for example, as an efficient cell fusion method that overcomes the problems of conventional methods, a method has been reported in which single-stranded DNA is bound to a PEG-lipid complex, which is then introduced into the cells to be adhered, and the cells are adhered to each other using the complementary sequence of the single-stranded DNA (Non-Patent Documents 1 and 2). Also reported is a method for increasing the efficiency of animal cell fusion by combining the PEG method with a cell fusion promoter (e.g., a peptide-linked PEG-lipid derivative) consisting of a lipid moiety that interacts with the cell membrane, a peptide moiety that holds one of a peptide pair that forms a dimer (a peptide that forms a dimer with another peptide), and a moiety that connects the lipid moiety and the peptide moiety (e.g., PEG) (Patent Document 1).

[0004] International Publication No. 2020 / 262617

[0005] Teramura et al., Biomaterials 31:2229-2235 2010 Teramura et al., Biomaterials 48:119-128 2015

[0006] However, although all of the above methods are excellent, the target of cell fusion is limited to animal cells, and their application to the fusion of plant cells has not been specifically studied, and no useful cell fusion method has yet been provided for plant cells. Furthermore, cell fusion promoters can only increase cell fusion efficiency when used in combination with the PEG method; cell fusion does not occur with the cell fusion promoter alone.

[0007] In view of the above circumstances, the present inventors conducted extensive research and discovered the novel fact that plant cells can be efficiently fused without the use of the PEG method by using a PEG-lipid derivative having a specific structure, and thus completed the present invention. Specific embodiments of the present invention are as follows. However, the present invention is not limited to these embodiments.

[0008] [1] A method for fusing a plant cell A with a plant cell B, comprising the following two steps: (Step 1) reacting a plant cell A with a plant cell B containing a compound represented by the following formula (I):

[0009]

[0010] [wherein X represents a group selected from a peptide which has Cys at its C-terminus and forms a dimer with another peptide, and a phospholipid bilayer-permeable peptide which has Cys at its C-terminus, two m's independently represent an integer of 5 to 18, and n represents an integer of 45 or more and 1250 or less] (also referred to as "compound (I)"), or a salt thereof, or a solvate or hydrate thereof. (Step 2) A step of mixing and culturing the protoplasts of plant cell A and the protoplasts of plant cell B which have been surface-treated in step 1. [2] The fusion method according to the above item [1], wherein steps 1 and 2 are carried out at a pH within the range of 4.6 to 6.6. [3] The fusion method according to [1] or [2] above, wherein in compound (I), two m's are each independently (preferably both) an integer of 5 to 18 (preferably an integer of 7 to 16, more preferably an integer of 7 to 14, and even more preferably an integer of 7 to 12). [4] The fusion method according to any of [1] to [3] above, wherein in compound (I), n is an integer of 80 to 160. [5] The fusion method according to any of [1] to [4] above, wherein in compound (I), X is a peptide having Cys at the C-terminus and forming a dimer with another peptide. [6] The fusion method according to [5] above, wherein X in compound (I) used to treat the surface of protoplasts of plant cell A and X in compound (I) used to treat the surface of protoplasts of plant cell B are different peptides and form a dimer together. [7] The fusion method according to any one of [1] to [6] above, wherein, in compound (I), X is a group selected from EIAALEKEIAALEKEIAALEKGGGC (SEQ ID NO: 1) (also referred to as "fuE3") and KIAALKEKIAALKEKIAALKEGGGC (SEQ ID NO: 2) (also referred to as "fuK3"). [8] The fusion method according to any one of [1] to [4] above, wherein, in compound (I), X is a phospholipid bilayer membrane-permeable peptide having Cys at the C-terminus.[9] The fusion method according to any one of the above items [1] to [4] or [8], wherein in compound (I), X is YGRKKRRQRRRC (SEQ ID NO: 3) (also referred to as "Tat").

[10] The following formula (I):

[0011]

[0012] (wherein X, m, and n are defined as the corresponding symbols defined for compound (I) in [1] above), or a salt thereof, or a solvate or hydrate thereof. *) Here, for preferred embodiments of m, n, and X, reference can be made to the descriptions in [3], [4], [7], and [9] above, respectively.

[0013] In one embodiment, the present invention provides a method for fusing plant cells, and in another embodiment, the present invention provides a cell fusion promoter used for the plant cell fusion method.

[0014] Figures show fluorescence images of Oc cells into which FITC-PEG (5k) lipids (C9, 12, 14, 16, 18) were introduced. Of the figures corresponding to each lipid, the upper left shows a fluorescence image, the upper right shows a bright-field image, and the lower left shows an image obtained by superimposing the fluorescence and bright-field images. Figures show the fluorescence intensity of FITC-PEG (5k) lipids (C9, 12, 14, 16, 18) introduced into Oc cells. Fluorescence images of rice protoplasts into which rhodamine-PEG (5k) lipids (C9, 12, 14, 16, 18) were introduced (top row), and figures show the fluorescence intensity of rhodamine-PEG (5k) lipids (C9, 12, 14, 16, 18) introduced into rice protoplasts (bottom row). This figure shows fluorescent images of Eucalyptus protoplasts transfected with Rhodamine-PEG (5k) lipids (C9, 12, 14, 16, 18). This figure shows fusion of Oc cells (stained green and red) after inducing adhesion by transfection with fuE3 and fuK3-PEG (5k) lipids (C14). As a control experiment, unmodified Oc cells (stained green and red) were used. Images are shown immediately (left column) and 2 hours after (right column) mixing of green and red stained cells. In the figures corresponding to each lipid and incubation time, upper left: CellTracker TM Fluorescent image by Green (green), upper right: CellTracker TMOrange fluorescence image (red), bottom left: bright-field image, bottom right: image obtained by superimposing green and red fluorescence images and bright-field image. When cell fusion occurs, the cells glow yellow in the superimposed image on the bottom right. This shows the fusion rate of Oc cells transfected with fuE3 and fuK3-PEG(5k) lipid (C14). This shows the color of the suspension of rice protoplasts (stained green and red) transfected with Tat-PEG(5k) lipid (C9). This shows a control experiment with rice protoplasts (stained green and red). Left: image obtained by superimposing green and red fluorescence images and bright-field image, right: image obtained by superimposing green and red fluorescence images. This shows the fusion process of rice protoplasts (stained green and red) transfected with Tat-PEG(5k) lipid (C9, 12). After treatment with Tat-PEG(5k) lipid (C9, 12), the cells were observed under a microscope immediately (Ohr), 3 hours, and 6 hours later. (Left: Superimposed image of green and red fluorescent images and bright-field image; Right: Superimposed image of green and red fluorescent images) Fusion rates of rice protoplasts transfected with Tat-PEG(5k) lipid (C9) and Tat-PEG(5k) lipid (C12) are shown. (Right: Superimposed image of green and red fluorescent images) Fusion rates of rice protoplasts transfected with Tat-PEG(5k) lipid (C9) and Tat-PEG(5k) lipid (C12) are shown. (Right: Superimposed image of green and red fluorescent images) Fusion rates of rice protoplasts transfected with Tat-PEG(5k) lipid (C9, 12) are shown. (Right: Superimposed image of green and red fluorescent images) Adhesion and fusion of rice protoplasts transfected with Tat-PEG(5k) lipid (C9, 12) are shown at pH 7.4. Left: Image obtained by superimposing a green and red fluorescent image on a bright-field image. Right: Image obtained by superimposing a green and red fluorescent image. Figure 1 shows the fusion rates of rice protoplasts transfected with Tat-PEG(5k) lipid (C9) and Tat-PEG(5k) lipid (C12) (pH 7.4). Figure 2 shows the results of a significance test for the fusion rates of rice protoplasts at pH 5.6 and 7.4. Figure 3 shows the results of flow cytometry analysis of rice protoplasts (stained green and far-red) transfected with Tat-PEG(5k) lipid (C9, 12).

[0015] The present invention will now be described with reference to specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] [Method for fusing plant cells] One embodiment of the present invention is a method for fusing plant cells as shown below. "A method for fusing plant cell A with plant cell B, comprising the following two steps: (Step 1) reacting a plant cell A with a plant cell B containing a compound represented by the following formula (I):

[0017]

[0018] [wherein X represents a group selected from a peptide which has Cys at its C-terminus and forms a dimer with another peptide, and a phospholipid bilayer-permeable peptide which has Cys at its C-terminus, two m's represent, independently, an integer of 5 to 18, and n represents an integer of 45 or more and 1250 or less] (hereinafter also referred to as "compound (I)"), or a salt thereof, or a solvate or hydrate thereof. (Step 2) A step of mixing and culturing the protoplasts of plant cell A and protoplasts of plant cell B which have been surface-treated in step 1. The above embodiment (also referred to as "the present cell fusion method") will be described in detail below. The present cell fusion method has a technical feature in that the surfaces of protoplasts of plant cell A and protoplasts of plant cell B are treated using compound (I) or a salt thereof, or a solvate or hydrate thereof as a compound that promotes the fusion of plant cells.

[0019] In this specification, the term "fusion" of plant cells refers not only to the adhesion of cell surfaces (cell membranes) to each other, but also to the state in which fusion of cell membranes and mixing of cytoplasm occur. The plant cells (plant cell A and plant cell B) to which the present fusion method is applied are not particularly limited and can be appropriately selected depending on the purpose of fusion, and examples thereof include eucalyptus, pearl millet; Brassicaceae: Arabidopsis thaliana, Arabidopsis lyrata, Brassica rapa, Brassica napus, Brassica campestris; Maple family: sugar maple (Acer saccharum); Euphorbiaceae: castor bean (Ricinus communis); Solanaceae: tobacco (Nicotiana tabacum), eggplant (Solanum melongena), potato (Solanum tuberosum); Fabaceae: soybean (Glycine max), pea (Pisum sativum), broad bean (Vicia faba), wisteria (Wisteria floribunda), peanut (Arachis hypogaea), lotus grass (Lotus japonicus), kidney bean (Phaseolus vulgaris), adzuki bean (Vigna angularis), acacia (Acacia), medicago truncatula), chickpea (Cicer arietinum); Asteraceae: chrysanthemum (Chrysanthemum morifolium), sunflower (Helianthus annuus); Arecaceae: oil palm (Elaeis guineensis, Elaeis oleifera), coconut palm (Cocos nucifera), date palm (Phoenix dactylifera), wax palm (Copernicia);Anacardiaceae: Rhus succedanea, cashew tree (Anacardium occidentale), sumac (Toxicodendron vernicifluum), mango (Mangifera indica), pistachio (Pistacia vera); Cucurbitaceae: pumpkin (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo), cucumber (Cucumis sativus), trichosanthes cucumeroides, bottle gourd (Lagenaria siceraria) var. gourda); Rosaceae: almond (Amygdalus communis), rose (Rosa), strawberry (Fragaria vesca), cherry blossom (Prunus), apple (Malus pumila var. domestica), peach (Prunus persica); Vitaceae: grape (Vitis vinifera), Caryophyllaceae: carnation (Dianthus caryophyllus); Salicaceae: poplar (Populus trichocarpa, Populus nigra, Populus tremula); Poaceae: corn (Zea mays), rice (Oryza sativa) sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), Urartu wheat (Triticum urartu), sorghum (Aegilops tauschii), wheatgrass (Brachypodium distachyon), bamboo (Phyllostachys), sugarcane (Saccharum officinarum), napier grass (Pennisetum pupureum), Erianthus (Erianthus ravenae), Miscanthus virgatum, sorghum (Sorghum bicolor), switchgrass (Panicum); Liliaceae: tulip (Tulipa), lily (Lilium), etc. The plant cells to be fused may be cells of the same species or cells of a different species.

[0020] The present cell fusion method comprises treating the surface of plant cells to be fused using one or two compounds selected from compound (I), which is a PEG-lipid derivative, or a salt thereof, or a solvate or hydrate thereof, as a plant cell fusion promoter.

[0021] Compound (I) will be described below: X in compound (I) is a group selected from a peptide having Cys at the C-terminus and forming a dimer with another peptide, and a phospholipid bilayer-permeable peptide having Cys at the C-terminus.

[0022] As used herein, "a peptide that forms a dimer with another peptide" refers to, for example, peptide A or peptide B when peptide A and peptide B bind to form a dimer. Here, "a peptide that forms a dimer with another peptide" (hereinafter also referred to as "dimer-forming peptide") will be explained using the above-mentioned peptide A and peptide B as an example. The peptide is not particularly limited as long as it has the property of forming a dimer with another peptide. In addition, its length is also not particularly limited as long as it is an easy-to-use length, and for example, it may have 5 to 100 amino acid residues, preferably 10 to 50 residues, and more preferably 20 to 50 residues. An example of a dimer-forming peptide is a peptide represented by the following formula (1). Note that "a" represents the N-terminal side.

[0023]

[0024] In formula (1), a and d are the same or different hydrophobic amino acids, for example, amino acids selected from the group consisting of alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, and tyrosine. e, f, and g are the same or different charged amino acids, for example, amino acids selected from the group consisting of arginine, aspartic acid, glutamic acid, lysine, and histidine. e and f preferably have opposite charges. Furthermore, b is not particularly limited, but is preferably a highly polar uncharged amino acid, for example, an amino acid selected from the group consisting of serine, asparagine, glutamine, and threonine. Furthermore, c is not particularly limited, but examples thereof include alanine. A "peptide having Cys at its C-terminus and forming a dimer with another peptide" may be a peptide having Cys at its C-terminus, including a peptide represented by formula (1), and further including other amino acids in its sequence. Specific examples of suitable peptides include peptides having the amino acid sequences represented by the following: EIAALEKEIAALEKEIAALEKGGGC (SEQ ID NO: 1) (also referred to as "fuE3"), KIAALKEKIAALKEKIAALKEGGGC (SEQ ID NO: 2) (also referred to as "fuK3"), or peptides containing such sequences. Particularly preferred are the above-mentioned "fuE3" and "fuK3". Furthermore, variants thereof are also suitable examples. The above-mentioned "peptide having Cys at the C-terminus and capable of forming a dimer with another peptide" may be commercially available, or may be produced from known raw materials using methods known in the art.

[0025] As used herein, the term "phospholipid bilayer membrane-permeable peptide having Cys at the C-terminus" is not particularly limited as long as it is a peptide having Cys at the C-terminus and permeability through phospholipid bilayer membranes. Furthermore, its length is also not particularly limited as long as it is a convenient length, for example, 5 to 100 amino acid residues, preferably 5 to 50 residues, and more preferably 8 to 20 amino acid residues. Examples of such peptides include transactivator of transcription (Tat) and its mutants that have phospholipid bilayer membrane permeability. Tat is a peptide derived from the transactivator of transcription protein of human immunodeficiency virus, and its amino acid sequence is, for example, YGRKKRRQRRRC (SEQ ID NO: 3). The above-mentioned "phospholipid bilayer membrane-permeable peptide having Cys at the C-terminus" can be a commercially available product, or can be appropriately produced from known raw materials using a method known in the art.

[0026] Examples of "variants of peptides that have Cys at the C-terminus and form dimers with other peptides" and "variants thereof that have Cys at the C-terminus and have phospholipid bilayer permeability" include amino acid sequences that are substantially identical to the above-mentioned SEQ ID NOS: 1 to 3. Examples of amino acid sequences that are substantially identical to the above-mentioned amino acid sequences represented by SEQ ID NOS: 1 to 3 include amino acid sequences that have about 95% or more identity to the amino acid sequences represented by SEQ ID NOS: 1 to 3. Here, "identity" refers to the percentage (%) of identical amino acids out of all overlapping amino acid residues in optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment). Furthermore, "mutants of peptides having Cys at the C-terminus and forming dimers with other peptides" and "mutants thereof having Cys at the C-terminus and permeability through phospholipid bilayer membranes" also include, for example, peptides containing: (1) amino acid sequences in which one or several (1, 2, 3, 4, 5) amino acids are deleted from the amino acid sequences represented by SEQ ID NOs: 1 to 3; (2) amino acid sequences in which one or several (1, 2, 3, 4, 5) amino acids are added to the amino acid sequences represented by SEQ ID NOs: 1 to 3; (3) amino acid sequences in which one or several (1, 2, 3, 4, 5) amino acids are inserted into the amino acid sequences represented by SEQ ID NOs: 1 to 3; (4) amino acid sequences in which one or several (1, 2, 3, 4, 5) amino acids in the amino acid sequences represented by SEQ ID NOs: 1 to 3 are substituted with other amino acids; or (5) amino acid sequences that are a combination thereof. When the amino acid sequence is inserted, deleted, or substituted as described above, the position of the insertion, deletion, or substitution is not particularly limited as long as the phospholipid bilayer membrane permeability is maintained. It is preferable to use similar amino acids for substitution.Here, "similar amino acids" refers to amino acids that are similar in physicochemical properties, and examples thereof include amino acids classified in the same group, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met). Substitution with such similar amino acids is expected to not change the properties of the peptide (i.e., it is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and have been described in various publications (see, for example, Bowie et al., Science, 247:1306-1310 (1990)).

[0027] In compound (I), m defines the length of the carbon chain of the lipid moiety, and two m's are, independently of each other, an integer of 5 to 18. m is preferably an integer of 7 to 16, more preferably an integer of 7 to 14, and even more preferably an integer of 7 to 12. In another preferred embodiment, m is preferably 7, 9, 10, 12, 14, 16, or 18, more preferably 7, 9, 10, 12, 14, or 16, and even more preferably 7, 10, or 12.

[0028] In compound (I), n defines the degree of polymerization of PEG, and represents an integer of 45 or more and 1250 or less. n is preferably an integer of 80 or more and 160 or less, and more preferably an integer of 100 or more and 120 or less.

[0029] Compound (I) can be used in its free form, but can also be used in the form of a salt, solvate, or hydrate for carrying out the present fusion method (hereinafter collectively referred to as "the compound of the present invention"). Suitable salts can be selected appropriately depending on the purpose of the present fusion method, and examples include salts with bases such as alkali metal salts (e.g., sodium salt, potassium salt), alkaline earth metal salts (e.g., calcium salt), etc.; and salts with amino acids (e.g., glycine salt, lysine salt, arginine salt, histidine salt, ornithine salt). When carrying out the present fusion method in the food or pharmaceutical fields, it is preferable to select a pharmaceutically acceptable salt from these. Solvates can be selected appropriately depending on the purpose of the present fusion method, and examples include alcohol solvates such as ethanol solvates.

[0030] (Method for producing compound (I)) Compound (I) can be produced by introducing an X-S group into a compound represented by the following formula (II) (also referred to as "compound (II)") or a salt thereof, which corresponds to the structural portion of compound (I) other than the X-S group.

[0031]

[0032] (In the formula, each symbol has the same meaning as the corresponding symbol defined for compound (I) in embodiment [1] of the section [Summary of the Invention].) Compound (II) can be obtained by appropriate synthesis by a person skilled in the art using raw materials and methods known in the art (see, for example, Patent Document 1). Suitable salts of compound (II) include the salts mentioned for compound (I). Introduction of the X-S group can be appropriately performed by a person skilled in the art using raw materials and methods known in the art (see, for example, Patent Document 1). For example, this can be performed by Michael addition of the side chain thiol group of the Cys residue to the maleimide group of compound (II) using a "peptide that forms a dimer with another peptide" or a "phospholipid bilayer-permeable peptide," each of which has a Cys residue at its C-terminus. This reaction can be performed by a method known in the art, the method described in the Examples section below, or a method equivalent thereto.

[0033] (Steps 1 and 2) Step 1 is a step of treating the surfaces of protoplasts of plant cell A and protoplasts of plant cell B with the compound of the present invention. Step 2 is a step of mixing and culturing the protoplasts of plant cell A and protoplasts of plant cell B surface-treated in step 1. In the above steps, as described above, plant cell A and plant cell B may be of the same species or different species. Here, the determination of whether they are of the same species or different species follows the definition of species classification based on scientific names. In the above steps, the surfaces of protoplasts of plant cell A and plant cell B are treated with the compound of the present invention. Protoplasts are plant cells whose cell walls have been dissolved, resulting in spherical cells surrounded by a cell membrane. Protoplasts can be prepared by dissolving plant tissue with a plant tissue-disintegrating enzyme and treating the resulting cells with a cell wall-degrading enzyme (e.g., pectinase, cellulase, etc.) using a method commonly used in the art. For more specific details, see the description in the Examples section below.

[0034] The mixing ratio of the protoplasts of plant cells A and B to the compound of the present invention is not particularly limited, but for example, ４ ~10 ６ A cell suspension of about 100 μg may be centrifuged (100 g, 5 minutes) to form a pellet, and after removing as much of the supernatant as possible, the compound of the present invention may be added and mixed to a final concentration of 0.05 mg / mL or more, preferably 0.1 mg / mL or more. Before seeding the protoplasts to be fused, they may be washed with an appropriate solution (e.g., PBS) to remove excess compound of the present invention. The density at which the protoplasts are seeded can be freely selected as appropriate depending on the intended use and properties of the protoplasts.

[0035] More specific embodiments of Step 1 and Step 2 will be explained separately for a case where X in compound (I) is a peptide having Cys at the C-terminus and forming a dimer with another peptide, and a case where X is a phospholipid bilayer-permeable peptide having Cys at the C-terminus. However, both embodiments share the common technical feature of treating the surfaces of protoplasts of plant cell A and protoplasts of plant cell B using compound (I) or a salt thereof, or a solvate or hydrate thereof as a compound that promotes plant cell fusion.

[0036] (Aspect 1) When X is a peptide having Cys at the C-terminus and forming a dimer with another peptide (Step 1) In this aspect, the surface of protoplasts of plant cell A is treated with one of two types of compound (I) or a salt thereof, or a solvate or hydrate thereof (also referred to as "compound (Ia)"), and the surface of protoplasts of plant cell B is treated with the other (also referred to as "compound (Ib)"), characterized in that X (X1) of compound (Ia) and X (X2) of compound (Ib) are different peptides that form a dimer together. In this aspect, the prepared protoplasts of plant cell A are subjected to surface treatment with compound (Ia), and the prepared protoplasts of plant cell B are subjected to surface treatment with compound (Ib), respectively. Specifically, for example, protoplasts of plant cell A are suspended in a solution of compound (Ia), while protoplasts of plant cell B are suspended in a solution of compound (Ib), and the respective suspensions are incubated at a temperature that does not adversely affect the cells (e.g., about 4°C to 37°C) for an appropriate period of time (e.g., about 15 minutes to 1 hour). (Step 2) Step 2 is a step in which the protoplasts of plant cell A and the protoplasts of plant cell B that have been surface-treated in step 1 are mixed and cultured. In this step, a suspension of protoplasts of plant cell A after the surface treatment obtained in step 1 and a suspension of protoplasts of plant cell B after the surface treatment are combined, and the resulting suspensions are further incubated at a temperature that does not adversely affect the cells (e.g., about 4°C to 37°C) for an appropriate period of time (e.g., about 1 hour to 10 hours). The progress of cell fusion can be confirmed, for example, by carrying out the cell fusion method of the present invention using compound (I) that has been fluorescently labeled or the like, and observing the progress over time during the incubation in step (2) using an appropriate observation means (e.g., a confocal microscope).

[0037] The pH of the system during steps (1) and (2) can be determined appropriately by those skilled in the art. From the viewpoint of efficient plant cell fusion, the pH is preferably within the range of 4.6 to 6.6, and more preferably within the range of 5.0 to 6.0.

[0038] In embodiment 1, protoplasts of plant cell A are surface-treated with compound (Ia) having X1, and protoplasts of plant cell B are surface-treated with compound (Ib) having X2, whereby compound (Ia) having X1 is introduced onto the cell surface of plant cell A, and compound (Ib) having X2 is introduced onto the cell surface of plant cell B (Step 1). When the two protoplasts are then mixed, the interaction between X1 and X2 brings the two protoplasts close to each other, facilitating the exchange reaction of lipids in the cell membranes of the close protoplasts, thereby promoting their fusion (Step 2).

[0039] (Aspect 2) When X is a phospholipid bilayer membrane-permeable peptide having Cys at the C-terminus (Step 1) In this aspect, one compound selected as compound (I) (X is a phospholipid bilayer membrane-permeable peptide having Cys at the C-terminus) or a salt thereof, or a solvate or hydrate thereof (also referred to as "compound (Ic)") is used for the surface treatment of both protoplasts of plant cell A and protoplasts of plant cell B. In this aspect, as in the above-mentioned "(1) When X is a peptide having Cys at the C-terminus and forming a dimer with another peptide," the protoplasts of plant cell A and the protoplasts of plant cell B can be subjected to the surface treatment with compound (Ic) separately, or steps (1) and (2) can be carried out consecutively. For example, fused cells can also be obtained by preparing protoplasts of plant cell A and plant cell B, preparing a mixture containing each protoplast and compound (Ic), and incubating the mixture at a temperature that does not adversely affect the cells (e.g., about 4°C to 37°C) for an appropriate period of time (e.g., about 1 hour to 10 hours). The above mixture may be obtained, for example, by mixing suspensions containing protoplasts of both plant cell A and plant cell B to remove impurities, centrifuging the mixture, removing the supernatant, and then adding a solution of compound (I) to the cell pellet.

[0040] In this embodiment, the pH in the system during steps (1) and (2) can be determined as appropriate by those skilled in the art. From the viewpoint of efficient plant cell fusion, the pH is preferably within the range of 4.6 to 6.6, and more preferably within the range of 5.0 to 6.0.

[0041] In aspect 2, compound (Ic) is introduced into protoplasts of plant cell A, and the protoplasts aggregate with protoplasts of plant cell B, to the surface of which compound (Ic) has also been introduced, via the phospholipid bilayer-permeable peptide X having Cys at the C-terminus exposed on the surface. This facilitates lipid exchange reactions in the cell membranes of the adjacent protoplasts, and the protoplasts of plant cell A and plant cell B fuse together without using any other fusion method (including the PEG method).

[0042] The mixing ratio of the cells and the cell fusion promoter is not particularly limited, but for example, ４ ~10 ６ The cell fusion-promoting compound or the like may be added to and mixed with cells of about 1000 μg / ml so that the final concentration of the cell fusion-promoting compound or the like is 0.05 mg / mL or more, preferably 0.1 mg / mL or more. Before seeding the cells to be fused, they may be washed with an appropriate solution (e.g., PBS) to remove excess cell fusion promoter. The cell density when seeding the cells can be freely selected as appropriate depending on the intended use and properties of the cells.

[0043] The present cell fusion method has been described in detail above. As will be described in the Examples section below, the present cell fusion method can efficiently achieve plant cell fusion without the need for the conventional PEG method. This is one of the notable advantages of the present cell fusion method.

[0044] [Cell fusion promoter for plant cell fusion] Another embodiment of the present invention is a cell fusion promoter for plant cell fusion (hereinafter also referred to as "plant cell fusion promoter") containing compound (I) or a salt thereof, or a solvate or hydrate thereof. The plant cell fusion promoter may be compound (I) or a salt thereof, or a solvate or hydrate thereof itself, or may be formulated by dissolving it in an appropriate solvent, for example. The form of the formulation and formulation conditions can be appropriately determined by one skilled in the art depending on the intended use. The plant cell fusion promoter can be suitably used for plant cell fusion by the method described above for the "cell fusion method of the present invention." The plant cell fusion promoters share the technical feature of containing, as an ingredient, compound (I), which is a PEG-lipid derivative having a specific structure, or a salt thereof, or a solvate or hydrate thereof. Specific modes of use will be described below, dividing them into cases where X in compound (I) has Cys at the C-terminus and forms a dimer with another peptide, and cases where X has Cys at the C-terminus and is a phospholipid bilayer-permeable peptide.

[0045] (Use Mode 1) When X is a Peptide Having Cys at the C-Terminus and Forming a Dimer with Another Peptide In this mode, two compounds are selected from compound (I), each of which has as X a "peptide having Cys at the C-terminus and forming a dimer with another peptide" that is different from the other but which together form a dimer, and the surface of protoplasts of plant cell A is treated with one of the compounds or a salt thereof, or a solvate or hydrate thereof, and the surface of protoplasts of plant cell B is treated with the other compound or a salt thereof, or a solvate or hydrate thereof. Therefore, when promoting cell fusion with the present plant cell fusion promoter, a pair of two cell fusion promoters are used, each containing the two mutually binding compounds selected from compound (I).

[0046] (Use Mode 2) When X is a phospholipid bilayer membrane-permeable peptide having Cys at the C-terminus In this mode, one compound in which X is a phospholipid bilayer membrane-permeable peptide having Cys at the C-terminus is selected from compound (I), and the surfaces of protoplasts of plant cell A and protoplasts of plant cell B are treated with the compound or a salt thereof, or a solvate or hydrate thereof. Therefore, when cell fusion is promoted with the present plant cell fusion promoter, one cell fusion promoter containing the one compound selected from compound (I) is used.

[0047] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art may modify the embodiments of the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also included within the scope of the present invention.

[0048] (Materials) The materials used in the examples below were obtained as follows: Rice Oc cultured cells were purchased from the RIKEN BioResource Research Center. TM Green and CellTracker TMOrange was purchased from Invitrogen. Millex-GV low protein binding durapore (PVDF) Membrane 0.22 μm was purchased from Merck Millipore Ltd. As a phospholipid, 1,2-Dinonanoyl-sn-glycero-3-phosphocholine (DC9PC) was purchased from Avanti Polar. Lipids, 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), was purchased from Bachem, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1,2-Distaroyl-sn-glycero-3-phosphoethanolamine (DSPE), α-3-[(3-maleimido-1-oxopropyl)aminopropyl-ω-(succinimidyloxycarboxy)]polyethylene glycol (Mal-PEG (1kDa)-NHS, MW1000Da), α-3-[(3-maleimido-1-oxopropyl)aminopropyl-ω-(succinimidyloxycarboxy)]polyethylene glycol (Mal-PEG (5kDa)-NHS, MW5000Da), and α-3-[(3-maleimido-1-oxopropyl)aminopropyl-ω-(succinimidyloxycarboxy)]polyethylene Glycol (Mal-PEG (40 kDa)-NHS, MW 40,000 Da) was purchased from NOF Corporation. Synthetic peptides (FITC-GC; Rhodamine-GC; EIAALEKEIAALEKEIAALEKGGGC (SEQ ID NO: 1) (fuE3); KIAALKEKIAALKEKIAALKEGGGC (SEQ ID NO: 2) (fuK3); and YGRKKRRQRRRC (SEQ ID NO: 3) (Tat)) were purchased from GenScript. Diethyl ether, dichloromethane, and triethylamine were purchased from Kanto Chemical Co., Ltd.Spin columns (for desalting) were purchased from Thermo Fisher Scientific. Calcium chloride, sodium chloride, L-cysteine, cholesterol, sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), ethanol, Dulbecco's phosphate-buffered saline (PBS; pH 7.4), and mixed salts for Murashige-Skoog medium were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Mannitol, KCl, MES, and β-mercaptoethanol were purchased from Nacalai Tesque. Cellulase "onozuka" R-10, Cellulase "onozuka" RS, and Macerozyme R-10 were purchased from Yakult Pharmaceutical Co., Ltd. 0.1% Pectolyase Y-23 was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. 2,4-D sodium monohydrate (2,4-Dichlorophenoxy) acetic acid sodium salt monohydrate was purchased from Sigma-Aldrich Co. CellTracker. TM Green, CellTracker TM Orange, CellTrace TM Oregon Green 488 carboxylic Acid Diacetate, Succinimidyl Ester and CellTracker TM Deep Red was purchased from Invitrogen.

[0049] (Synthesis of FITC-PEG (5k) lipid (C9, 12, 14, 16, 18)) Here, C9 corresponds to compound (I) where m is 7, and C12, C14, C16, and C18 correspond to compound (I) where m is 10, 12, 14, and 16, respectively. The same applies below. (1) DC9PC (28 mg) was dissolved in 10 mL of diethyl ether, and 2 mL of 250 mM ethanolamine-HCl (pH 4.2) and 1 mL of a 30 U / mL aqueous solution of phospholipase D (from Streptomyces sp., Asahi Kasei Pharma, T-138) were added. The mixture was stirred at 35°C for 90 minutes with a stirrer (1000 rpm). After the reaction, the diethyl ether was distilled off with a nitrogen stream. 0.2 mL of ultrapure water was added, followed by 4 mL of chloroform and 8 mL of methanol to obtain a homogeneous solution. Then, 1 mL of chloroform and 1 mL of ultrapure water were added, and the mixture was centrifuged (2500 rpm) to separate into two phases. The chloroform phase was collected and evaporated under reduced pressure to obtain DC9PE. (2) 1,2-Dinonanoyl-sn-glycero-3-phosphoethanolamine (DC9PE (also referred to as "C9"), 20 mg, 1 Eq), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE (also referred to as "C12"), 20 mg, 1 Eq), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE (also referred to as "C14"), 20 mg, 1 Eq), 1 Either 2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE (also referred to as "C16"), 20 mg, 1 Eq) or 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE (also referred to as "C18"), 20 mg, 1 Eq) was added to Mal-PEG (5 kDa)-NHS (180 mg, 1.1 Eq) and triethylamine (5.0 μL) in dichloromethane, and the mixture was stirred at room temperature for 2 days. The reaction solution was reprecipitated with diethyl ether (3.0 L) and dried under vacuum to obtain Mal-PEG (5 kDa) lipid (DC9PE, DLPE, DMPE, DPPE, DSPE) (yield 80%) as a white powder.(3) A fluorescent peptide (FITC-GC) was conjugated to Mal-PEG(5k) lipid. Specifically, Mal-PEG(5k) lipid (100 μL, 10 mg / mL in PBS, 1 Eq) and a fluorescent peptide solution (FITC-GC: 11 μL, 10 mg / mL in DMSO, 1.1 Eq) were mixed and stirred at room temperature for one day. FITC-conjugated PEG lipids (FITC-PEG(5k) lipids(C9, 12, 14, 16, 18)) composed of the fluorescent peptide (FITC-GC), PEG with a molecular weight of 5 kDa, and lipids (C9, 12, 14, 16, 18) were obtained.

[0050] (Synthesis of Rhodamine-PEG(5k) lipid (C9, 12, 14, 16, 18)) A fluorescent peptide (Rhodamine-GC) was conjugated to Mal-PEG(5k) lipid. Specifically, Mal-PEG(5k) lipid (100 μL, 10 mg / mL in PBS, 1 Eq) and a fluorescent peptide solution (Rhodamine-GC: 13 μL, 10 mg / mL in DMSO, 1.1 Eq) were mixed and stirred at room temperature for 1 day. A Rhodamine-conjugated PEG lipid (Rhodamine-PEG(5k) lipid(C9, 12, 14, 16, 18)) composed of a fluorescent peptide (Rhodamine-GC), PEG with a molecular weight of 5 kDa, and lipid (C9, 12, 14, 16, 18) was obtained.

[0051] (Synthesis of fuE3-PEG(5k) lipid (C14) and fuK3-PEG(5k) lipid (C14)) Synthetic peptides (fuE3 and fuK3) were each conjugated to Mal-PEG(5k) lipid. Specifically, Mal-PEG(5k) lipid (100 μL, 10 mg / mL in PBS, 1 Eq) was mixed with synthetic peptide solution (fuE3: 51 μL, fuK3: 51 μL, 10 mg / mL in DMSO, 1.1 Eq) and stirred at room temperature for 1 day. Peptide-PEG-lipids (fuE3-PEG(5k)lipid(C9, 12, 14, 16, 18), fuK3-PEG(5k)lipid(C9, 12, 14, 16, 18)) composed of synthetic peptides (fuE3, fuK3), PEG with a molecular weight of 5 kDa, and lipids (C9, 12, 14, 16, 18) were obtained.

[0052] (Synthesis of Tat-PEG(5k) lipid (C9, 12, 14, 16, 18)) The membrane-permeable peptide (Tat) was conjugated to Mal-PEG(5k) lipid. Specifically, Mal-PEG(5k) lipid (100 μL, 10 mg / mL in PBS, 1 Eq) and membrane-permeable peptide solution (Tat: 30 μL, 10 mg / mL in DMSO, 1.1 Eq) were mixed and stirred at room temperature for one day. This resulted in the formation of membrane-permeable peptide-PEG-lipid (Tat-PEG(5k) lipid (C9, 12, 14, 16, 18) composed of the membrane-permeable peptide (Tat), PEG with a molecular weight of 5 kDa, and lipid (C9, 12, 14, 16, 18).

[0053] (Isolation of Oc cell protoplasts) (1) Buffer used: Mixed salts for Murashige-Skoog (MS) medium: MS_VT: 0.5 mg / mL nicotinic acid, 0.5 mg / mL pyridoxine·HCl, 0.1 mg / mL thiamine·HCl, 2 mg / mL glycine; MS_inositol (40 mg / mL myo-inositol): 2,4-D (0.2 mg / mL 2,4-D sodium monohydrate ((2,4-Dichlorophenoxy) acetic acid sodium salt monohydrate, Sigma-Aldrich); KOH (1N); MS Medium (1 L MS salt mix 1 bag, 30 g Sucrose, 1 mL MS_VT, 2.5 mL MS_inositol, 5 mL 2,4-D) Enzyme solution: 1% Cellulase "Onozuka" RS, 0.1% Pectolyase Y-23, MS medium) (2) Method Oc cell suspension (500 μL) was suspended in enzyme-containing medium (10 mL) (Cellulase "Onozuka" RS (final 1%), Pectolyase Y-23 (final 0.1%)) and incubated at room temperature for 2 hours on a rotating rotor (9 rpm). Then, 100 μL of the suspension was suspended in PBS (900 μL).

[0054] (Protoplast isolation from rice plants) Rice protoplasts were isolated from rice (Oryza sativa, Nipponbare) plants grown at 28°C, 50-70% humidity, and in the dark. (1) Digestion buffer used: Digestion buffer (600 mM mannitol, 20 mM KCl, 10 mM CaCl ２ , 20mM MES (pH 5.7)) Enzyme solution (1% Cellulase "onozuka" RS, 0.5% Macerozyme R-10, 0.1% BSA, 10mM β-mercaptoethanol) W5 buffer (150mM NaCl, 125mM CaCl ２ , 5mM KCl, 2mM MES (pH 5.7)) (2) Method Thirty-five rice culms (excluding expanded parts) approximately 5 days after sowing were sampled and finely chopped with a scalpel so that the cut surface was oblique. The samples were placed in enzyme solution so that all samples were immersed, and degassing and normal pressure were repeated three times. The tubes were shielded from light with aluminum foil and incubated in an incubator at 26.5°C with up-and-down stirring (40 rpm) for 3 hours. The entire suspension was filtered through a 40μm cell strainer, collected in a new 50mL tube, and gently pushed out from the top of the cell strainer with a tip. W5 buffer (20mL) was added to the tube containing the suspension to wash the remaining sample, and the process of adding 3mL at a time to the cell strainer and gently pushing out with a tip was repeated. The collected 40 mL was centrifuged (200 g, 10 minutes) and the supernatant was removed. W5 buffer (20 mL) was added and gently suspended, and the mixture was centrifuged again (200 g, 5 minutes) and the supernatant was removed. This process was repeated twice. The pellet was suspended in W5 buffer and used as a protoplast solution for experiments.

[0055] (Protoplast isolation from eucalyptus plants) Eucalyptus protoplasts were isolated at 22°C and 60 μmol / s / m only on the day of germination. ２The enzyme was isolated from eucalyptus (Eucalyptus globulus) plants grown under conditions of 1000 nm of light and 1000 nm of dark. (1) Buffer used: Digestion buffer (600 mM mannitol, 20 mM KCl, 10 mM CaCl ２ , 20mM MES (pH 5.7)) Enzyme solution (1% Cellulase "onozuka" RS, 0.5% Macerozyme R-10, 0.1% BSA, 10mM β-mercaptoethanol) W5 buffer (150mM NaCl, 125mM CaCl ２ , 5mM KCl, 2mM MES (pH 5.7)) MMg buffer (400mM Mannitol, 15mM MgCl2.6H ２ 0, 4 mM MES (pH 5.7)) (2) Method Cotyledons from 40 eucalyptus plants were collected approximately 15 days after sowing and divided into two tubes. The abaxial epidermis was peeled using the tape sandwich method and immediately placed in digestion buffer (40 mL / tube). This procedure of incubating for 30 minutes and replacing the digestion buffer was repeated three times. Enzyme solution was added, and the mixture was subjected to degassing and atmospheric pressure three times. After that, the mixture was incubated for 90 minutes in an incubator at 26.5°C with up-and-down stirring (40 rpm). The entire suspension was filtered through a 70 μm cell strainer and collected in a new 50 mL tube. W5 buffer (20 mL) was added to the tube containing the suspension, and the suspension was filtered again while washing. The collected 40 mL was centrifuged (100 g, 5 minutes) and the supernatant was removed. W5 buffer (20 mL) was added and gently suspended, and the suspension was centrifuged again (200 g, 5 minutes) and the supernatant was removed. This procedure was repeated twice. The pellet was suspended in W5 buffer and used as a protoplast solution in experiments.

[0056] [Surface Modification of Cultured Plant Cells] Example A: Surface Modification of Oc Cell Protoplasts (1) Differences in the carbon chains of fluorescent molecule-conjugated PEG lipids modified on the surface membrane of Oc cell protoplasts were evaluated. FITC-PEG (5k) lipid (C9, 12, 14, 16, 18) solution (50 μL, 1 mg / mL in MS medium) was added to the Oc cell protoplast pellet (1.0 × 10 ５ (50 μL; 5, 2, 1, 0.5 mg / mL in MS medium) was added to the cells and incubated at room temperature for 30 minutes. After centrifugation and rinsing with MS medium, the cells were observed under a confocal microscope. When cells were treated with FITC-conjugated PEG lipid, fluorescence was observed on the cell surface at the time of treatment, confirming that the cell surface had been modified with FITC-conjugated PEG lipid (Figure 1). The number of cells in which fluorescence was observed on the cell surface was particularly high for lipid C14. (2) Quantitative analysis of fluorescent molecule-conjugated PEG lipid on the Oc cell surface. The amount of modification due to differences in the carbon chain of fluorescent molecule-conjugated PEG lipid modified on the surface membrane of Oc cell protoplasts was evaluated using fluorescent molecules. FITC-PEG (5k) lipid (C9, 12, 14, 16, 18) solution (50 μL; 5, 2, 1, 0.5 mg / mL in MS medium) was added to the Oc cell protoplast pellet (1.0 × 10 ５ The cells were then added to a 1000 ml sample of FITC-conjugated PEG-lipid (C9, 12, 14, 16, 18) and incubated at room temperature for 30 minutes. After rinsing with MS medium by centrifugation, 0.1 mL of pure water was added to the cell pellet to induce hypotonic hemolysis. The fluorescence intensity of the disrupted cell solution was measured using a fluorometer, and the difference in the amount of modification due to lipid length (lipids C9, 12, 14, 16, 18) was quantified from the fluorescence intensity obtained by measuring the FITC fluorescent peptide. Stronger fluorescence was detected at all lipid lengths compared to the control, confirming that the cell surface had been modified with FITC-conjugated PEG-lipid (Figure 2). High fluorescence was particularly detected on the surface of Oc cell protoplasts with lipid C14.

[0057] Example B: Surface modification of rice protoplasts (1) The amount of modification depending on the carbon chain of the fluorescent molecule-bound PEG lipid modified on the surface membrane of rice protoplasts was evaluated. Rhodamine-PEG (5k) lipid (C9, 12, 14, 16, 18) solution (50 μL, 1 mg / mL in W5 buffer) was added to rice protoplast pellets (1.0 × 10５ (50 μL; 5, 2, 1, 0.5 mg / mL in W5 buffer) was added to the rice protoplast pellet (1.0 × 10) and incubated for 30 minutes. After centrifugation and rinsing with W5 buffer, the cells were observed under a confocal microscope. When the cells were treated with rhodamine-conjugated PEG lipid, fluorescence was observed on the cell surface at the time of treatment, confirming that the cell surface had been modified with rhodamine-conjugated PEG lipid (Figure 3, upper panel). (2) Quantitative analysis of fluorescent molecule-conjugated PEG lipid on the surface of rice protoplasts. The amount of modification due to differences in the carbon chain of the fluorescent molecule-conjugated PEG lipid modified on the surface membrane of rice protoplasts was evaluated using fluorescent molecules. Rhodamine-PEG (5k) lipid (C9, 12, 14, 16, 18) solution (50 μL; 5, 2, 1, 0.5 mg / mL in W5 buffer) was added to rice protoplast pellet (1.0 × 10) ５ The cells were added to a 1000 ml (1000 ml) of PEG-lipid-binding solution and incubated at room temperature for 30 minutes. After rinsing with W5 buffer by centrifugation, 0.1 mL of pure water was added to the cell pellet to lyse the cells. The fluorescence intensity of the lysed cell solution was measured using a fluorometer, and the difference in the amount of modification due to lipid length (C9, 12, 14, 16, 18) was quantified from the fluorescence intensity obtained by measuring the rhodamine fluorescent peptide. Stronger fluorescence was detected at all lipid lengths compared to the control, confirming that the cell surface had been modified with rhodamine-conjugated PEG lipid (Figure 3, bottom panel). Lipid C14 in particular exhibited high fluorescence intensity on the rice protoplast surface.

[0058] Example C: PEG-lipid modification of eucalyptus protoplasts (1) Buffers used for surface modification of eucalyptus protoplasts PBS: (pH 7.4) W5 buffer: (pH 5.6) PBS (W5): (pH 5.6) adjusted to the same osmotic pressure as W5 buffer MMg buffer: (pH 5.6) PBS (MMg): (pH 5.6) adjusted to the same osmotic pressure as MMg buffer (2) The effects of differences in buffer (pH, osmotic pressure) on modification were investigated using PBS adjusted to the same osmotic pressure as W5 buffer (PBS (W5)) and PBS adjusted to the same osmotic pressure as MMg buffer (PBS (MMg)). Rhodamine-PEG (5k) lipid (C14, 16) solution (50 μL, 1 mg / mL in W5 buffer, PBS (W5), MMg, PBS (MMg)) was added to the protoplast pellets (1.0 × 10 ５ The cells were added to a 500 ml buffer (5kJ / ml) and incubated at room temperature for 30 minutes. After rinsing with each buffer by centrifugation, the cells were observed under a confocal microscope. When the cells were treated with the rhodamine-conjugated PEG lipid, ring-shaped fluorescence was observed on the cell surface in the W5 buffer and MMg buffer used for the rhodamine-PEG (5k) lipid (C14), confirming that the cell surface had been modified with the rhodamine-conjugated PEG lipid (Figure 4).

[0059] From the above studies in Examples A to C, it was found that for FITC-PEG (5k) lipid and Rhodamine-PEG (5k) lipid, C14 is the optimal lipid, and that surface modification at pH 5.6 is optimal for plant cell fusion. Based on this new finding, cell fusion studies (Example 1) were carried out for fuE3-PEG lipid and fuK3-PEG lipid. [Cell Fusion] Example 1: Oc cell fusion experiment using fuE3-PEG lipid and fuK3-PEG lipid (pH 5.6) Oc cell protoplasts were cultured using CellTracker. TM Green or CellTracker TM Stained with Orange (red). CellTracker TMGreen-labeled Oc cell protoplasts (2.0 × 10 ６ ) were suspended in fuE3-PEG(5k) lipid (C14) solution (50 μL, 1 mg / mL in MS medium) and analyzed by CellTracker. TM Orange (red)-labeled Oc cell protoplasts (2.0 × 10 ６ 2.0 × 10 cells) were suspended in fuK3-PEG lipid (C14) solution (50 μL, 1 mg / mL in MS medium) and incubated at room temperature for 30 minutes. Each was rinsed with MS medium, centrifuged, and resuspended in MS medium. ６ CellTracker TM Green-labeled fuE3-PEG (5k) lipid (C14) solution-modified Oc cell protoplasts and 2.0 × 10 ６ CellTracker TM A suspension (50 μL) of Oc cell protoplasts modified with orange (red)-labeled fuK3-PEG lipid (C14) solution was incubated in a 15 mL Falcon tube. The cells were observed immediately and 2 hours later using a confocal microscope (Figure 5). The Oc cell protoplasts modified with fuE3-PEG lipid (C14) and fuK3-PEG lipid (C14) showed a 35% cell-cell fusion rate, a significant increase compared to the control unmodified Oc cells (1%) (Figure 6). These results demonstrate that fuE3-PEG lipid (C14) and fuK3-PEG lipid (C14) induce cell-cell fusion.

[0060] In addition to the above, cell fusion using Tat-PEG lipid was also investigated (Examples 2 to 4). [Cell fusion] Example 2: Rice protoplast cell fusion using Tat-PEG lipid (pH 5.6) Rice protoplast suspension (1 mL, 8.0 × 10 ６ ) was prepared and CellTracker TM Green or CellTracker TM The cells were stained with orange (red). The stained cells were mixed, centrifuged, and the supernatant was removed. The cell pellet (4.0 × 10６ Tat-PEG(5k) lipid (C9) or Tat-PEG(5k) lipid (C12) solutions (1 mg / mL, 50 μL) were added to rice protoplasts and incubated at room temperature for 6 hours. Cell adhesion and cell fusion were confirmed compared with unmodified control rice protoplasts (Figures 7 and 8). Cells were also observed under a confocal microscope immediately, 3 hours, and 6 hours after incubation (Figure 9). Most of the cells in the Tat-PEG lipid-treated rice protoplasts were observed to be yellow, confirming cell fusion compared with unmodified control rice protoplasts. The cell fusion rates in the Tat-PEG(5k) lipid-modified rice protoplasts (C9 and C12) were 34% (C9) and 33% (C12), respectively, significantly higher than those in unmodified control rice protoplasts (0.5%) (Figure 10). This indicates that cell-cell fusion is induced by Tat-PEG(5k) lipid (C9, 12).

[0061] [Cell fusion] Example 3: Rice protoplast cell fusion using Tat-PEG lipid (pH 7.4) Rice protoplast suspension (1 mL, 2.0 × 10 ６ ) was prepared and CellTracker TM Green or CellTracker TM The cells were stained with orange (red). The stained cells were mixed, centrifuged, and the supernatant was removed. The cell pellet (1.0 x 10 ６ Tat-PEG(5k) lipid (C9) or Tat-PEG(5k) lipid (C12) solution (50 μL, 1 mg / mL in PBS) was added to the rice protoplasts and incubated at room temperature for 6 hours. Cell adhesion and cell fusion were confirmed compared to unmodified control rice protoplasts (Figure 11). Cells were also observed under a confocal microscope immediately, 3 hours, and 6 hours after treatment (Figure 12). Most of the cells in the Tat-PEG lipid-treated rice protoplasts were observed to be yellow, confirming cell fusion compared to unmodified control rice protoplasts (Figure 13).

[0062] The results of the significance test showed that the fusion efficiency of rice protoplasts mediated by Tat-PEG(5k) lipid (C9, 12) was significantly higher at pH 5.6 than at pH 7.4 (Figure 14).

[0063] [Cell fusion] Example 4: Flow cytometric analysis of rice protoplast cell fusion by Tat-PEG lipid (pH 5.6) Rice protoplast suspension (1 mL, 1.0 × 10 ７ ) was prepared and TM Oregon Green 488 Carboxylic Acid Diacetate, Succinimidyl Ester (green) or CellTracker TM The cells were stained with Deep Red (far red). The stained cells were mixed, centrifuged, and the supernatant was removed. The cell pellet (2.0 × 10 ６ To the rice protoplasts (50 μL), Tat-PEG(5k) lipid (C9) or Tat-PEG(5k) lipid (C12) solution (50 μL, 1 mg / mL in W5 buffer) was added and incubated at room temperature for 0, 3, and 6 hours. Immediately after incubation, and after 0, 3, and 6 hours, the cells were analyzed by flow cytometry. Most of the rice protoplasts treated with Tat-PEG lipid exhibited both green and far-red coloring, confirming cell aggregation and fusion compared to unmodified control rice protoplasts (Figure 15).

[0064] The present invention relates to a method for fusing plant cells, a cell fusion promoter used for the plant cell fusion method, etc. Therefore, the present invention is expected to be used in fields related to plant cell engineering, etc., and in the fields of food and pharmaceuticals.

[0065] This application is based on patent application No. 2023-192501 filed in Japan (filing date: November 10, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for fusing plant cell A and plant cell B, comprising the following two steps. (Process 1) The following equation (I): 【Chemistry 1】 [In the formula, X represents a group selected from peptides that form dimers with other peptides and have Cys at the C-terminus, and phospholipid bilayer-permeable peptides that have Cys at the C-terminus.] The two values ​​of m represent integers between 5 and 18, independently of each other. n represents an integer between 45 and 1250. A step of treating the surface of the protoplasts of plant cell A and plant cell B with one or two compounds selected from the compounds represented by ("Compound (I)"), or salts thereof, or solvates or hydrates thereof. (Step 2) A step in which the protoplasts of plant cell A and plant cell B, which were surface-treated in step 1, are mixed and cultured.

2. Steps 1 and 2 are performed within a pH range of 4.6 to 6.

6. The fusion method according to claim 1.

3. In compound (I), the two values ​​of m are independently integers between 7 and 12. The fusion method according to claim 1 or 2.

4. In compound (I), n is an integer between 80 and 160. The fusion method according to claim 1 or 2.

5. In compound (I), X is a peptide that forms a dimer with another peptide having Cys at its C-terminus. The fusion method according to claim 1 or 2.

6. X of compound (I) obtained by treating the surface of a protoplast of plant cell A, X of compound (I), obtained by treating the surface of a protoplast of plant cell B, These are peptides that are distinct from each other but combine to form a dimer. The fusion method according to claim 5.

7. In compound (I), X is A group selected from EIAALKEKEIAALKEKEIAALKEKGGGC (SEQ ID NO: 1) ("fuE3") and KIAALKEKIAALKEKIAALKEGGGC (SEQ ID NO: 2) ("fuK3") The fusion method according to claim 5.

8. In compound (I), X is a phospholipid bilayer-permeable peptide having Cys at its C-terminus. The fusion method according to claim 1 or 2.

9. In compound (I), X is YGRKKRRQRRRC (Sequence ID 3) ("Tat") That is, The fusion method according to claim 8.

10. The following equation (I): 【Transformation 6】 [In the formula, X represents a group selected from peptides that form dimers with other peptides and have Cys at the C-terminus, and phospholipid bilayer-permeable peptides that have Cys at the C-terminus.] The two values ​​of m represent integers between 5 and 18, independently of each other. n represents an integer between 45 and 1250. A cell fusion promoter for plant cell fusion containing a compound represented by or a salt thereof, or a solvate or hydrate thereof.

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