Quadruple mutant of solanaceous plant capable of mass production of heterologous protein

Abstract

To provide a cell or a plant body that allows a large amount of heterologous proteins to be produced efficiently by one-time transformation, relative to conventional methods.SOLUTION: A Solanaceous plant cell carries loss-of-function type mutations in the DCL2 gene, the DCL3 gene, and the DCL4 gene, which exhibits an improved ability to express foreign genes.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a mutant of a Solanaceous plant capable of mass-producing a heterologous protein. More specifically, the present invention relates to a Solanaceous plant cell or plant body lacking the function of a Dicer-like protein (DCL), and a method for producing a heterologous protein using the cell or plant body.

Background Art

[0002] A system using plants for the production of heterologous proteins has attracted attention as an alternative to systems using bacteria, yeast, and animal cells. Certain plants such as Nicotiana benthamiana can be cultivated on a large scale and enable the mass production of heterologous proteins. As methods for producing heterologous proteins in plants, there are two methods: a method using stable transformation and a method of transient overexpression. Although stable transformation has advantages in terms of reproducibility and large-scale production, it takes time to establish transformants and is not suitable for the rapid expression of a wide variety of constructs. On the other hand, transient expression in plants can produce high-titer heterologous proteins in a short period of time. Transient expression can be achieved by a method using a modified plant virus or a method using Agrobacterium tumefaciens that introduces a gene into somatic tissue through infiltration. In order to efficiently produce a heterologous protein by the latter method, it is necessary to improve transcriptional activity and increase the mRNA level, or to improve translational activity and maximize the translation of mRNA (for example, Non-Patent Document 1).

[0003] By the way, animals prevent the growth of viruses mainly through the immune mechanism represented by the antibody-antigen reaction. Since plants do not have an acquired immune mechanism, RNA interference (referred to as RNA silencing in plants) plays an important role in virus resistance. Through this mechanism, gene expression regulation and transposon expression suppression are also carried out. In RNA silencing in plants, there are two pathways: the RNAi pathway in which small-interfering RNA (siRNA) derived from exogenous long double-stranded RNA such as viruses acts as a defense system, and the miRNA pathway in which microRNA (miRNA), which is encoded in the genome as a precursor RNA with a hairpin structure and controls life phenomena such as development, differentiation, and environmental response, functions.

[0004] The inventors of the present invention previously focused on two types of pathways in the RNAi pathway: the Post-transcriptional gene silencing (PTGS) pathway that cleaves and suppresses translation of mRNA complementary to siRNA, and the Transcriptional gene silencing (TGS) pathway that suppresses complementary genes through DNA methylation and performs transcriptional regulation. Then, in order to create a plant that does not cause PTGS, using N. benthamiana suitable for the Agrobacterium method, a transformant in which the DCL genes (dcl2, dcl4a, and dcl4b) involved in the PTGS pathway were disrupted was prepared. As a result, it was found that in this transformant, the expression of the introduced gene increased compared to the wild-type plant (Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] In the transient expression of heterologous proteins in plants using plant viruses, viral growth is inhibited by RNA silencing, and suppression of the expression of the transgene may also occur. Therefore, in the conventional methods for expressing heterologous proteins, there has been a problem that it takes time and cost to obtain a sufficient amount of protein. Accordingly, an object of the present invention is to provide a cell or a plant body that can efficiently produce a large amount of heterologous proteins by a single transformation as compared with the conventional methods.

Means for Solving the Problems

[0008] The present inventors attempted to develop a method for further highly expressing exogenous genes by improving the invention described in Patent Document 1. As a result of intensive research, mutants in which not only the PTGS pathway but also the TGS pathway were inhibited were prepared, and by using these mutants, the inventors succeeded in significantly improving the gene expression efficiency. In Arabidopsis thaliana, a model plant of the Brassicaceae family, mutants of the PTGS pathway and the TGS pathway have been analyzed in detail. However, since immune induction by Agrobacterium occurs (for example, Tsuda K. et al., Plant J, 69(4):713-719 (2012), etc.), the relationship between transient heterologous protein expression and RNA silencing has not been studied. On the other hand, many examples have been reported in which the transient heterologous protein expression efficiency in N. benthamiana was increased by expressing an RNA silencing suppressor of a plant virus that only suppresses the PTGS pathway (such as p19 of Tomato bushy stunt virus) (for example, Takeda A. et al., FEBS Lett., 532(1-2):75-79 (2002), etc.). However, no example has been reported in which the transient heterologous protein expression efficiency in N. benthamiana was increased by suppressing the TGS pathway. That is, it is difficult to predict that the transient gene expression efficiency will be significantly increased by inhibiting the TGS pathway in addition to the PTGS pathway in N. benthamiana. The remarkable improvement in the expression efficiency of exogenous genes in the Solanaceae plants according to the present invention was surprising. In addition, among the mutants of the above Solanaceae plants, some were found in which the expression of the introduced gene was increased by about 430 times compared to the wild-type plant body. As a result of further research based on these findings, the present invention was completed.

[0009] That is, the present invention is as follows. [1] A Solanaceae plant cell having a loss-of-function mutation in the DCL2 gene, the DCL3 gene, and the DCL4 gene, and having an improved ability to express a foreign gene. [2-1] The cell according to [1], having a loss-of-function mutation in the DCL4a and DCL4b genes. [2-2] The cell according to [1] or [2-1], which has loss-of-function mutations in the DCL2 gene, DCL3 gene, DCL4a gene, and DCL4b gene in all alleles. [3-1] The cell according to any one of [1] to [2-2], wherein the solanaceous plant belongs to the genus Nicotiana. [3-2] The cell according to [3-1], wherein the plant belonging to the genus Nicotiana is Nicotiana benthamiana. [4-1] The cell according to any one of [1] to [3-2], which is characterized by not having a foreign gene. [4-2] The cell according to [4-1], which is a null mutant. [4-3] The cell according to any one of [1] to [3-2], wherein the plant cell is a recombinant. [4-4] The cell according to [4-3], which is characterized by not having the transgene used for gene recombination. [5-1] A plant body having the cell according to any one of [1] to [4-4]. [5-2] The plant body according to [5-1], which is characterized by not having a foreign gene. [5-3] The plant body according to [5-2], which is a null mutant. [6] (1) A step of introducing a nucleic acid encoding a target protein into the cell according to any one of [1] to [4-4] or the plant body according to any one of [5-1] to [5-3], and (2) A step of expressing the protein in the cell or plant body into which the nucleic acid has been introduced in step (1). A method for producing a target protein, comprising the above steps. [7] (3) The method according to [6], further comprising a step of isolating the protein from the cell or plant body expressing the target protein obtained in step (2). [8] The method according to [6] or [7], wherein the step (1) includes infecting a cell or a plant with an Agrobacterium or a plant virus vector having a nucleic acid encoding a target protein. [9-1] The method according to any one of [6] to [8], wherein the nucleic acid has a sequence encoding a virus-derived replication system or a plant virus vector. [9-2] The method according to any one of [6] to [9-1], wherein the target protein is at least one selected from the group consisting of an enzyme, an antibody, an interferon, and an antigen.

[10] (1) Infecting a cell according to any one of [1] to [4-4] or a plant according to any one of [5-1] to [5-3] with a virus and propagating the virus, and (2) Recovering the virus propagated by the step (1) A method for propagating a virus, comprising:

[11] The method according to

[10] , wherein the virus is an attenuated plant virus. [Advantages of the Invention]

[0010] According to the present invention, there is provided a mutant of a Solanaceae plant capable of mass-producing a heterologous protein. In one embodiment, by transient expression in the leaves of this mutant, the foreign protein (heterologous protein) can be expressed 400 times or more higher than in the case of wild-type plants, and thus it exhibits particular strength especially when overexpressing multiple genes simultaneously such as an antibody. Further, in this mutant, expression suppression by RNA silencing does not occur, so that it is possible to highly express the protein for a longer period. Furthermore, since this mutant can be a null segregant from which a foreign gene has been removed, it also has an advantageous effect that it can be excluded from the scope of the Cartagena method, and like wild-type plants, it can be cultivated in an open system. [Brief Description of the Drawings]

[0011]

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Modes for Carrying Out the Invention

[0012] 1. A plant cell having a loss-of-function mutation in the gene of the dicer-like (DCL) protein or a plant body containing the cell The present invention provides a plant cell having a loss-of-function mutation in a gene of all Dicer-like proteins (also referred to as "DCL gene") selected from the group consisting of DCL2 gene, DCL3 gene, and DCL4 gene, or a plant body containing the cell (hereinafter sometimes referred to as "the mutant of the present invention").

[0013] The mutant of the present invention can improve the expression ability of a foreign gene by suppressing RNA silencing in plants. RNA silencing in plants is roughly classified into a Transcriptional gene silencing (TGS) pathway that methylates the promoter region of a gene to be suppressed and performs suppression at the transcriptional level (the outline is shown in FIG. 1), and a Post-transcriptional gene silencing (PTGS) pathway that degrades mRNA derived from the gene to be suppressed and performs suppression at the post-transcriptional level (the outline is shown in FIG. 2). These are carried out via three types of siRNAs having lengths of 21, 22, and 24 nt.

[0014] The TGS pathway that methylates DNA via 24 nt siRNA is also known as RNA directed DNA methylation (RdDM) and was discovered in 1994 as a phenomenon in which only PSTVd-specific sequences were methylated upon Potato spindle tuber viroid (PSTVd) infection (Wassenegger et al., Cell, 76(3):567-576 (1994)). It is now known to be involved in transposon suppression, virus resistance, DNA methylation in germ cells, etc. (Sigman M.J. et al., Nature Plants 7(11):1461-1474 (2021); Long J. et al., Science, 373(6550):eabh0556 (2021)). RdDM begins with RNA polymerase IV (Pol IV), which moves around the genome, recognizing and transcribing abnormal DNA sequences such as repetitive sequences. The transcribed Pol IV-RNA is double-stranded by RNA dependent RNA polymerase 2 (RDR2), and this long dsRNA is cleaved by Dicer like 3 (DCL3). After cleavage, the 3'-OH group of the DCL3 product is methylated by Hua Enhancer 1 (HEN1), generating 24 nt siRNA. The 24 nt siRNA is incorporated into AGO4 and forms a DNA methyltransferase group including methyltransferase 1 (MET1 or DMT1), domains rearranged methyltransferase 2 (DRM2), and RNA induced silencing complex (RISC).This RISC binds to the RNA polymerase V transcript as a scaffold, methylates the promoter region of the recognized DNA sequence, and suppresses transcription (Matzke M.A. and Mosher R.A., Nat Rev Genet, 15(6):394-408 (2014); Zilberman D. Cao X. and Jacobsen C.S., Science, 299(5607):716-719 (2003); Havecker E.R. et al., Plant Cell, 22(2):321-334 (2010)).

[0015] The PTGS pathway that degrades mRNA via 21- and 22-nt siRNAs is known to be involved in virus resistance and the production of trans-acting siRNAs (tasiRNAs), which are endogenous siRNAs derived from the TAS locus. PTGS can be subdivided into sense-post-transcriptional gene silencing (S-PTGS), which is induced from sense-strand mRNA, and inverted repeat-post-transcriptional gene silencing (IR-PTGS), which is induced from hairpin-shaped dsRNA consisting of inverted repeats. The S-PTGS pathway begins with the double-stranded formation of ssRNA derived from DNA polymerase II (Pol II) or ssRNA viruses by the SGS3 / RDR6-body composed of RNA-dependent RNA polymerase 6 (RDR6) and suppressor of gene silencing 3 (SGS3) (Kumakura N. et al., FEBS Lett, 583(8):1261-1266 (2009)). This dsRNA is cleaved by Dicer-like 4 (DCL4) or Dicer-like 2 (DCL2), and 21-nt siRNAs are generated from the DCL4 products and 22-nt siRNAs are generated from the DCL2 products via methylation of the 3'-OH group by HEN1 (Fukudome A. and Fukuhara T., J Plant Res, 130(1):33-44 (2017); Bouche N. et al., EMBO J, 25(14):3347-3356 (2006)). The resulting RNA-induced silencing complex (RISC), formed by the incorporation of these into AGO1 with RNase activity, binds to and degrades mRNA with a sequence homologous to the siRNA. Since the IR-PTGS pathway is induced from dsRNA, it does not require double-stranded formation by RDR6 and SGS3 (Bouche N. et al., EMBO J, 25(14):3347-3356 (2006)).

[0016] Dicer is a double-stranded RNA (dsRNA)-specific endoribonuclease. In eukaryotes, it plays an important role in inducing transcriptional and post-transcriptional gene silencing by cleaving single-stranded RNAs with stem-loop structures, such as dsRNA and microRNA precursor transcripts, into 21-24 nucleotide (nt) small RNAs. In plants, proteins with similar functions to the above-mentioned Dicer are known as Dicer-like (DCL) proteins. DCL proteins, together with AGO and RDR proteins, play important roles in RNA silencing as a defense mechanism against viruses. In Arabidopsis thaliana, four DCL proteins (DCL1-4) that specifically produce siRNAs of different sizes are known (Bologna N.G. and Voinnet O., Annu Rev Plant Biol, 65:473-503 (2014)).

[0017] DCL is classified as a class 4 RNaseIII with dsRNA as a substrate and is known as a family protein with an ATPase / DExD / H-box helicase domain, an RNaseIII domain, a PAZ (Piwi / Argonaute / Zwille) domain, and a dsRNA-binding domain from the N-terminal side (Bouche N. et al., EMBO J, 25(14):3347-3356 (2006)). From studies on Arabidopsis thaliana, DCL2, DCL3, and DCL4 are known to generate siRNAs. DCL3, which generates 24 nt siRNAs and is involved in TGS, has 1-2 nt 3' overhangs and prefers short dsRNAs of 30-37 nt as substrates. Also, it preferentially cleaves dsRNAs with adenine and uridine on the 5' side. It has ATP-independent activity and shows stronger activity in the presence of physiological concentrations of inorganic phosphate, 150-200 mM KCl, or NaCl. DCL4, which generates 21 nt siRNAs and is involved in PTGS, acts together with DRB4. Also, it prefers dsRNAs longer than DCL3 as substrates, and the efficiency of generating 21 nt siRNAs decreases when the length is shorter than 50 nt.

[0018] When the genomic sequence of the DCL of the target plant cell is not known, the genomic sequence of the DCL may be identified by a method known per se based on information of a plant cell whose DCL genomic sequence is known. For example, in the case of the genomic sequences of DCL2, DCL3, and DCL4 of Nicotiana benthamiana (also known as: Nicotiana tabacum cv. Xanthi), they can be identified by the following method, for example. Using the CDS sequences of DCL2, DCL3, and DCL4 reported in a known document (Nakasugi et al., PLoS ONE, 9, e91776 (2014)) as reference sequences, perform a BLAST search against the Scaffold of Nicotiana benthamiana genome v1.0.1 to identify the genomic DNA sequences of DCL2 (Niben101Scf08272), DCL3 (Niben101Scf06666), DCL4a (Niben101Scf11936), and DCL4b (Niben101Scf01789). The CDS sequence (base sequence) of DCL2 of Nicotiana benthamiana identified by the above method is represented by SEQ ID NO: 1, the amino acid sequence is represented by SEQ ID NO: 2, the CDS sequence (base sequence) of DCL3 is represented by SEQ ID NO: 3, the amino acid sequence is represented by SEQ ID NO: 4, the CDS sequence (base sequence) of DCL4a is represented by SEQ ID NO: 5, the amino acid sequence is represented by SEQ ID NO: 6, the CDS sequence (base sequence) of DCL4b is represented by SEQ ID NO: 7, and the amino acid sequence is represented by SEQ ID NO: 8.

[0019] When there are multiple DCL genes that are considered to have the same function on the same genome, such as DCL4a and DCL4b of Nicotiana benthamiana described above, the mutant of the present invention may have a loss-of-function mutation in only one of the genes, but from the viewpoint of the expression ability of a foreign gene, it is preferable to have loss-of-function mutations in both genes.

[0020] In the present invention, the "loss-of-function mutation of a gene" means a mutation in which a nonsense mutation or a frameshift mutation is introduced into the gene, or a part or all of the sequence of the gene is deleted, added, or replaced with another sequence, etc., resulting in the loss of the function of the protein encoded by the gene. The mutant of the present invention preferably has a loss-of-function mutation introduced homozygously (that is, has a loss-of-function mutation in the genes of all alleles of the DCL protein). Due to the above loss-of-function mutation, a shorter and incomplete DCL protein is produced compared to the wild-type protein, or the expression of the DCL protein is no longer observed. Since the incomplete DCL gene product can be degraded at both the mRNA level and the protein level, the mutation position causing the loss-of-function mutation is not particularly limited. Examples of such mutation positions include the middle of the double-stranded RNA binding domain (dsrm a or dsrm b) present on the C-terminal side or upstream of the start point of the domain, but it is preferably in the region on the N-terminal side, such as the region consisting of the DEAD domain, the non-coding region, and the helicase C domain, or in the vicinity thereof. In one embodiment, the mutant of the present invention has a mutation that causes a loss-of-function mutation at exon 6 of DCL2 or a site upstream thereof. In one embodiment, the mutant of the present invention has a mutation that causes a loss-of-function mutation at exon 11 of DCL3 or a site upstream thereof. In one embodiment, the mutant of the present invention has a mutation that causes a loss-of-function mutation at exon 9 of DCL4a or a site upstream thereof. In one embodiment, the mutant of the present invention has a mutation that causes a loss-of-function mutation at exon 9 of DCL4b or a site upstream thereof. In one embodiment, the mutant of the present invention has mutations that cause loss-of-function mutations at exon 6 of DCL2 or a site upstream thereof, exon 11 of DCL3 or a site upstream thereof, exon 9 of DCL4a or a site upstream thereof, and exon 9 of DCL4b or a site upstream thereof.

[0021] As shown in the following examples, the mutant of the present invention has an improved ability to express a foreign gene due to having a loss-of-function mutation in the gene of the DCL protein as compared with the wild-type cell or plant. In one embodiment, when a foreign gene is introduced into the tissue (e.g., leaf) of the mutant of the present invention in which mutations have been introduced into the DCL2 gene, DCL3 gene, DCL4a gene, and DCL4b gene, in the tissue, compared with the tissue of a plant (control plant) that does not have the above loss-of-function mutation in all the genes of the DCL protein, it expresses a protein 400 times or more (e.g., about 430 times). Although the above expression is a comparison of the expression level in the tissue of the plant, it is presumed that a similar improvement in the expression ability is also recognized when compared at the cell level. Therefore, in one aspect of the present invention, the mutant (cell) of the present invention has an ability to express a foreign gene 50 times or more (e.g., 100 times, 150 times, 200 times, 300 times, 400 times, or more (e.g., about 430 times)) as compared with a cell that does not have a loss-of-function mutation. The expression ability of such a foreign gene itself can be evaluated by comparison with the amount of protein by Western blotting, enzyme activity, or the amount of mRNA measured by RT-PCR. However, when using specific numerical values such as the above 50 times or more as an index, like the method described in the examples, the enzyme activity of firefly luciferase encoded by the foreign gene introduced into the cell using the Agrobacterium method is used as a reference based on the value measured 6 days after inoculation with Agrobacterium. Of course, it is not essential for the mutant of the present invention to have the above firefly luciferase gene as a foreign gene.

[0022] Examples of the Solanaceae plant from which the cells used in the present invention are derived include, but are not limited to, plants belonging to the genus Nicotiana, plants belonging to the genus Solanum, plants belonging to the genus Capsicum, plants belonging to the genus Datura, plants belonging to the genus Brugmansia, and the like. A preferred Solanaceae plant is a plant belonging to the genus Nicotiana (also referred to as a "Nicotiana plant").

[0023] Examples of plants belonging to the genus Nicotiana include, for example, Nicotiana acaulis, Nicotiana acuminata, Nicotiana africana, Nicotiana alata, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana attenuata, Nicotiana benavidesii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana bonariensis, Nicotiana cavicola, Nicotiana clevelandii, Nicotiana cordifolia, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana forgetiana, Nicotiana fragrans, Nicotiana glauca, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana ingulba, Nicotiana kawakamii, Nicotiana knightiana, Nicotiana langsdorfi, Nicotiana linearis, Nicotiana longiflora, Nicotianalongiflora), Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana obtusifolia, Nicotiana occidentalis, Nicotiana otophora, Nicotiana paniculata, Nicotiana pauczjlora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotiana quadrivalvis, Nicotiana raimondii, Nicotiana repanda, Nicotiana rosulata, Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifolia, Nicotiana spegauinii, Nicotiana stocktonii, Nicotiana suaveolens, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana thyrsiflora, Nicotiana tomentosa (NicotianaNicotiana tomentosa, Nicotiana tomentosifomis, Nicotiana trigonophylla, Nicotiana umbratica, Nicotiana undulata, Nicotiana velutina, Nicotiana wigandioides, and hybrids of tobacco plants, etc. Among them, Nicotiana benthamiana is preferred.

[0024] Examples of plants belonging to the genus Solanum include Solanum aethiopicum, Solanum americanum, Solanum carolinense, Solanum betaceum, Solanum lycopersicum, Solanum lyratum, Solanum mammosum, Solanum melongena, Solanum muricatum, Solanum nigrum, Solanum pseudocapsicum, Solanum tuberosum, etc.

[0025] Examples of plants belonging to the genus Capsicum include Capsicum annuum, Capsicum baccatum, Capsicum cardenasii, Capsicum chinense, Capsicum frutescens, Capsicum pubescens, etc.

[0026] Examples of plants belonging to the genus Datura include, for example, Datura metel, Datura inoxia, Datura stramonium, etc. Examples of plants belonging to the genus Brugmansia include, for example, Brugmansia arborea, Brugmansia suaveolens, etc.

[0027] As used herein, the "plant body" includes any of a plant individual, a plant organ, a plant tissue, a plant cell, and a seed. Examples of plant organs include roots, leaves, stems, and flowers. In addition, plant cells include not only cultured cells but also cells in a plant body. Furthermore, various forms of plant cells, such as suspension-cultured cells, protoplasts, leaf sections, root sections, callus, immature embryos, pollen, etc. are included. The cells constituting the plant body are partially or entirely the mutants of the present invention (i.e., having a loss-of-function mutation in the DCL gene), but it is preferable that all the cells constituting the plant body are the mutants of the present invention.

[0028] As used herein, "not having a foreign gene" means not having at least the introduced gene (e.g., a nucleic acid encoding a CRISPR-Cas system, etc.) used in producing the recombinant. Here, the base insertions into the target site resulting from genome editing using non-homologous end joining (NHEJ) repair (typically several tens of base pairs or less), or the unintended base insertions other than the sequences derived from the donor DNA into the target site resulting from genome editing using homologous recombination repair (HDR) are not regarded as foreign genes. "Cells or plants not having a foreign gene" include not only cells and plants from which the above-introduced gene has been removed (e.g., null-segregant, cells and plants from which the introduced gene has been removed using a transposon system, etc.), but also cells and plants having spontaneous mutations. In addition, "null mutant" means an individual among the progeny individuals obtained by crossing recombinants with each other or a recombinant with a non-recombinant that does not have the introduced foreign gene.

[0029] The loss-of-function mutation of the DCL protein gene possessed by the variant of the present invention may be, for example, a natural mutation or an artificially introduced mutation (mutation caused by mutagenesis, homologous recombination of genes, or a mutation resulting from genome editing). A cell or a plant having a gene into which such an artificial mutation has been introduced is also referred to as a "recombinant gene" or a "transformant". The natural mutation of the above gene can occur naturally due to replication errors and gene damage. Examples of the above mutagenesis include exposure of cells or plants to radiation, neutron rays, ultraviolet rays, or mutagenic substances (e.g., EMS, etc.). Homologous recombination of genes can be carried out by homologously recombining part or all of the target gene with a recombinant sequence containing a sequence homologous to such a region according to known gene recombination techniques. Genome editing can be carried out by known techniques (e.g., zinc-finger nucleases: ZFN, transcription activator-like effector nucleases: TALEN, and CRISPR / Cas9 system, etc.). In addition, plants having a loss-of-function mutation in one or more DCL genes are crossed (e.g., dcl3 - / - and dcl2 - / - / 4a - / - / 4b - / - are crossed) to obtain a variant of the present invention having a loss-of-function mutation in multiple types of DCL genes.

[0030] From the viewpoint of being able to easily introduce a loss-of-function mutation of a desired gene, it is preferable to introduce it by genome editing. Therefore, the mutant of the present invention can be produced by introducing a loss-of-function mutation into the DCL protein gene of solanaceous plant cells by genome editing. Thus, in another aspect of the present invention, there is provided a method for producing the mutant of the present invention, which includes a step of introducing into solanaceous plant cells a complex of a nucleic acid sequence recognition module targeting at least one gene selected from the group consisting of DCL2 gene, DCL3 gene and DCL4 gene and a nuclease or deaminase (hereinafter sometimes referred to as "nucleic acid-modifying enzyme complex") or a nucleic acid encoding the complex. In the present specification, the nucleic acid encoding the nucleic acid-modifying enzyme complex includes a base sequence encoding the nucleic acid sequence recognition module and a base sequence encoding a nuclease or deaminase, and when using the CRISPR-Cas system, the complex further includes a sequence encoding a guide RNA.

[0031] In the present specification, the "nucleic acid sequence recognition module" means a molecule or molecular complex having the ability to specifically recognize and bind to a target sequence on a DNA strand. By binding of the nucleic acid sequence recognition module to the target sequence, it becomes possible for a nuclease or deaminase linked to the module to act specifically on the sequence or its vicinity.

[0032] Examples of the nucleic acid sequence recognition module include, in addition to the CRISPR-Cas system, zinc finger motif, TAL effector, and PPR motif, a fragment containing a DNA-binding domain of a protein capable of specifically binding to DNA, such as a restriction enzyme, a transcription factor, and an RNA polymerase. Preferably, it is the CRISPR-Cas system, zinc finger motif, TAL effector or PPR motif.

[0033] The zinc finger motif is formed by linking 3 to 6 different Cys2His2-type zinc finger units (each finger recognizes approximately 3 bases), and can recognize a target sequence of 9 to 18 bases. The zinc finger motif can be prepared by known methods such as the Modular assembly method (Nat Biotechnol (2002) 20: 135-141), the OPEN method (Mol Cell (2008) 31: 294-301), the CoDA method (Nat Methods (2011) 8: 67-69), and the Escherichia coli one-hybrid method (Nat Biotechnol (2008) 26:695-701). For details on the preparation of the zinc finger motif, reference can be made to, for example, Japanese Patent No. 4968498.

[0034] The TAL effector has a repeating structure of modules with approximately 34 amino acids as a unit. The binding stability and base specificity are determined by the 12th and 13th amino acid residues (referred to as RVDs) of one module. Since each module has a high degree of independence, it is possible to prepare a TAL effector specific to a target sequence simply by connecting the modules. Preparation methods using open resources have been established for TAL effectors (such as the REAL method (Curr Protoc Mol Biol (2012) Chapter 12: Unit 12.15), the FLASH method (Nat Biotechnol(2012) 30: 460-465), the Golden Gate method (Nucleic Acids Res (2011) 39: e82), etc.), and it is possible to design a TAL effector for a target sequence relatively easily. For details on the preparation of the TAL effector, reference can be made to, for example, Japanese Patent Publication No. 2013-513389.

[0035] The PPR motif consists of 35 amino acids and is configured to recognize a specific nucleotide sequence by a series of consecutive PPR motifs that recognize one nucleobase. Only the 1st, 4th, and ii(-2)th amino acids of each motif recognize the target base. There is no dependence on the motif composition and no interference from the motifs on both sides. Therefore, similar to TAL effectors, it is possible to create a PPR protein specific to the target sequence simply by linking PPR motifs. For details on the production of PPR motifs, reference can be made to, for example, Japanese Patent Application Laid-Open No. 2013-128413.

[0036] When using the CRISPR-Cas system, it is provided as a complex with a CRISPR-RNA (crRNA) containing a sequence complementary to the target sequence and, if necessary, a trans-activating RNA (tracrRNA) required for recruitment of the Cas protein (when tracrRNA is required, it can be provided as a chimeric RNA with crRNA). An RNA molecule consisting of crRNA alone or a chimeric RNA linking crRNA and tracrRNA, which constitutes a nucleic acid sequence recognition module in combination with the Cas protein, is collectively referred to as "guide RNA".

[0037] The Cas protein used in the present invention is not particularly limited as long as it can form a complex with a guide RNA and recognize and bind to a target sequence in a target gene and a protospacer adjacent motif (PAM) adjacent thereto, but is preferably Cas9 or Cpf1 (Cas12a). Examples of Cas9 include Cas9 derived from Streptococcus pyogenes (SpCas9; PAM sequence NGG (N is A, G, T, or C; the same applies hereinafter)), Cas9 derived from Streptococcus thermophilus (StCas9; PAM sequence NNAGAAW), Cas9 derived from Neisseria meningitidis (NmCas9; PAM sequence NNNNGATT), Cas9 derived from Staphylococcus aureus (SaCas9; PAM sequence: NNGRRT), Cas9 derived from Campylobacter jejuni (CjCas9; PAM sequence NNNVRYM (V is A, G, or C; R is A or G; Y is T or C; M is A or C)), and a group of modified Cas9 with modified PAM recognition specificity, but is not limited thereto. Preferably, it is SpCas9 with less restriction by PAM (substantially two bases). Examples of Cpf1 include Cpf1 derived from Francisella novicida (FnCpf1; PAM sequence NTT), Cpf1 derived from Acidaminococcus sp. (AsCpf1; PAM sequence NTTT), Cpf1 derived from Lachnospiraceae bacterium (LbCpf1; PAM sequence NTTT), but is not limited thereto. As the above Cas protein, those having the ability to cleave both strands of a double-stranded DNA strand, those having nickase activity in which only the ability to cleave one strand is inactivated, and those in which the ability to cleave both strands is inactivated can all be used.For example, in the case of SpCas9, a D10A mutant lacking the cleavage ability of the strand complementary to the guide RNA (thus having nickase activity against the strand forming a complementary strand with the guide RNA), in which the 10th Asp residue is converted to an Ala residue, or an H840A mutant lacking the cleavage ability of the strand forming a complementary strand with the guide RNA (thus having nickase activity against the strand opposite to the strand forming a complementary strand with the guide RNA), in which the 840th His residue is converted to an Ala residue, and furthermore, its double mutant (dCas9) can be used. Also, in the case of FnCpf1, mutants lacking the cleavage ability of both strands, in which the 917th Asp residue is converted to an Ala residue (D917A) or the 1006th Glu residue is converted to an Ala residue (E1006A), can be used.

[0038] By binding any of the above nucleic acid sequence recognition modules to a nuclease or a deaminase, a nucleic acid modifying enzyme complex can be prepared. Also, when using the CRISPR-Cas system, a guide RNA that recognizes a target sequence and a Cas protein having nuclease activity recruited by the guide RNA form a nucleic acid modifying enzyme complex. Alternatively, a guide RNA, a Cas protein in which at least one of the DNA cleavage abilities is inactivated (also referred to as a "mutant Cas protein"), and a deaminase form a nucleic acid modifying enzyme complex. For details on the preparation of a nucleic acid modifying enzyme complex having a deaminase, reference can be made to, for example, International Publication No. WO 2015 / 133554.

[0039] Examples of the nuclease used in the present invention include Cas proteins (e.g., Cas9, Cpf1), endonucleases (e.g., restriction enzymes such as FokI), and exonucleases. Examples of the deaminase include cytidine deaminase that can convert cytosine or 5-methylcytosine into uracil or thymine, respectively, adenosine deaminase that can convert adenine into hypoxanthine, guanosine deaminase that can convert guanine into xanthine, and the like. More preferably, as the cytidine deaminase, activation-induced cytidine deaminase (hereinafter also referred to as AID), which is an enzyme that introduces mutations into immunoglobulin genes in the acquired immunity of vertebrates, and the like can be mentioned. The origin of the deaminase is not particularly limited, and for example, PmCDA1 (Petromyzon marinus cytosine deaminase 1) derived from lamprey, AID (Activation-induced cytidine deaminase; AICDA) or APOBEC protein derived from mammals (e.g., human, pig, cow, horse, monkey, etc.) can be used.

[0040] The nucleic acid-modifying enzyme complex is typically introduced into cells in the form of the nucleic acid encoding the complex, which may be DNA or RNA, but is preferably DNA. In the case of DNA, it is preferably double-stranded DNA and is provided in the form of an expression vector placed under the control of a promoter functional in the host cell. Such nucleic acids may be prepared using genetic engineering techniques or chemically synthesized. When the nucleic acid is DNA, the full-length DNA encoding the protein can be constructed by chemically synthesizing the DNA strands or by connecting partially overlapping short oligonucleotide DNAs using methods such as the PCR method or the Gibson Assembly method. The advantage of constructing the full-length DNA by chemical synthesis or in combination with the PCR method or the Gibson Assembly method is that the codons used can be designed over the entire length of the CDS according to the host into which the DNA is introduced. When expressing heterologous DNA, increasing the protein expression level can be expected by converting the DNA sequence into codons frequently used in the host organism. Data on codon usage frequencies in the host to be used can be obtained, for example, by using the Genetic Code Usage Frequency Database published on the homepage of the Kazusa DNA Research Institute, or by referring to literature that describes the codon usage frequencies in each host.

[0041] An expression vector containing DNA encoding a nucleic acid sequence recognition module and / or a nuclease or deaminase can be produced, for example, by ligating the DNA downstream of a promoter in a vector containing a promoter that can function in plant cells. The vector replicable in plant cells is not particularly limited as long as it has an origin of replication that functions in plant cells (e.g., ori of Ti plasmid, Ri plasmid, etc.), but it is preferably also equipped with an origin of replication of Escherichia coli (e.g., ColE1 ori, etc.). When using the Agrobacterium method as a gene introduction method, it is necessary to further contain a T-DNA fragment (including border sequences RB and LB) excluding the pathogenic genes of Ti plasmid and Ri plasmid. Examples include pBI101 and pBI121 (Clontech) derived from pBIN19 and improved vectors using these as backbones (e.g., pRI909, pRI910, pRI101, pRI201 (Takara Bio), etc.), but are not limited thereto. Also, a method using a plant virus vector (e.g., Potato virus X (PVX) vector, Turnip mosaic virus (TuMV) vector, Tobacco mosaic virus (TMV) vector, Cowpea mosaic virus (CPMV) vector, etc.) (e.g., Geneware system, etc.) may be used.

[0042] From the perspective of expression level, when using the Agrobacterium method, the above plasmid may be a replication system derived from a virus. In other words, the above plasmid may be one in which only the region necessary for the replication of the virus is incorporated from the viral genome. Examples of such virus-derived replication systems include, but are not limited to, the replication system derived from Turnip mosaic virus (TuMV), the replication system derived from Tobacco mosaic virus (TMV) (e.g., the magnICON system), the replication system derived from Cowpea mosaic virus (CPMV) (e.g., the Profica system), the replication system derived from Bean yellow dwarf virus (BeYDV) (e.g., the Tsukuba system (WO 2018 / 220929)). In addition to the region necessary for virus replication, the above plasmid may also have other regions, such as a region encoding a viral coat protein, and may also have a sequence encoding a plant virus vector. Alternatively, a plasmid without the above virus-derived replication system may also be used.

[0043] Any promoter that can function in plant cells may be used. By using an inducible promoter (e.g., the PR1α gene promoter induced by injury or salicylic acid treatment, the rd29A gene promoter induced by drought, low temperature, or abscisic acid treatment, the GST-27 gene promoter induced by dichloromid treatment, etc.) as the promoter, the number of cells may be increased until the induction starts. Also, a constitutive promoter can be used as the promoter. Examples of the constitutive promoter include the cauliflower mosaic virus (CaMV) 35S promoter, the CaMV 19S promoter, the nopaline synthase (NOS) promoter, the parsley-derived ubiquitin promoter (Pcubi4-2), etc. Those in which these promoters or fragments thereof are connected in tandem (e.g., 2x35S) can also be used. Also, the present inventors have previously found that the Flt (Full length transcriptional) promoters of plant DNA viruses belonging to the genus Carlavirus, such as Peanut chlorotic streak virus (PClSV), which is a virus in the same family as CaMV, Figwort mosaic virus (FMV), which is a plant DNA virus belonging to the genus Soymovirus, Mirabilis mosaic virus (MMV), which is a plant virus belonging to the genus Carlavirus, Cassava vein mosaic virus (CsVMV), which is a plant virus belonging to the genus Cavemovirus, and Dahlia mosaic virus (DMV), which is a plant virus belonging to the genus Carlavirus, can also be used as a plant gene expression system in the same manner as the 35S promoter (in particular, the effect can be enhanced with the Flt promoter derived from MMV). Also, when expressing a foreign gene, instead of using a single type of promoter, the expression level can be improved by using a plurality of types of promoters to express the foreign gene. Therefore, even when only one type of foreign gene is to be expressed, it is also preferable to use a plurality of promoters.Although not wishing to be bound by any theory, it is speculated that the improvement in the expression level is because when a construct having the same promoter is introduced into a plant cell, the number of the promoters on the genome of the plant cell becomes larger than that in the case of using a plurality of promoters, and thus TGS is relatively likely to occur. Further, the above Flt promoter may have the 5'UTR derived from the virus from which each promoter is derived removed.

[0044] The expression vector can contain, if desired, a terminator (e.g., NOS terminator, pea rbcS3A terminator, heat shock protein (HSP) 18.3 terminator, etc.), a translation enhancer (e.g., rice-derived alcohol dehydrogenase 5'untranslated region (OsADH-5'UTR), tobacco mosaic virus (TMV)-derived Ω sequence, etc.), a 3'regulatory region (e.g., rice-derived actin gene (Act1) 3'UTR, etc.), a polyA addition signal, a selection marker such as a drug resistance gene (e.g., G418 resistance gene (nPtII), hygromycin resistance gene (hpt), etc.).

[0045] On the other hand, the DNA encoding the guide RNA can be chemically synthesized, for example, using a DNA / RNA synthesizer. The length of the target sequence is, for example, 15 to 30 nucleotides, preferably 18 to 25 nucleotides. Also, the DNA encoding the guide RNA can be inserted into the same expression vector as described above, but as the promoter, it is preferable to use a pol III-based promoter (e.g., SNR6, SNR52, SCR1, RPR1, U3, U6, H1 promoter, etc.) and a terminator (e.g., polyT sequence (T6 sequence, etc.)).

[0046] By introducing an expression vector containing DNA encoding a nucleic acid sequence recognition module and / or a nuclease or deaminase into a host plant cell and culturing the host cell, a nucleic acid modifying enzyme complex can be expressed intracellularly. The introduction of the nucleic acid modifying enzyme complex into the cell can be carried out by methods known per se. For example, it can be introduced in the form of DNA encoding a nucleic acid sequence recognition module and / or a nuclease or deaminase (e.g., nucleic acid in the form of an expression vector). The introduction of the expression vector can be carried out according to known methods (e.g., Agrobacterium method, PEG method, electroporation method, particle gun method, whisker direct introduction method, etc.) for appropriate tissues (e.g., callus, root, leaf, seed, meristem, etc.) depending on the type of plant. Usually, the Agrobacterium method is used. For example, when applying the Agrobacterium method to plants of the genus Nicotiana, for example, a part of a tobacco tissue (e.g., tobacco leaf) or hypocotyl is excised and infected with Agrobacterium (in other words, Agrobacterium is seeded). After selecting recombinants, callus is induced, and then rooting is induced to obtain a genetically modified plant. Alternatively, callus may be infected with Agrobacterium. As such Agrobacterium, typically, one into which DNA encoding a nucleic acid sequence recognition module and / or a nuclease or deaminase (when using the CRISPR-Cas system, an expression cassette containing DNA encoding a Cas protein and / or a complex of a mutant Cas protein and a deaminase and DNA encoding a guide RNA) is introduced into the T-DNA fragment of the Agrobacterium expression vector is used. When using the PEG method or electroporation method, protoplasts are prepared from appropriate cells or tissues according to a conventional method, and an expression vector is introduced into the protoplasts. In the case of the particle gun method, the expression vector adsorbed on gold microparticles can be introduced into callus, immature embryos, meristems present in shoot apices or axillary buds, etc. using a particle gun. In the particle gun method and the Agrobacterium method, since gene transfer often results in chimeras, it is necessary to use sample cells in which the above nucleic acid is introduced into germ line cells at a high frequency for transformation.For example, embryos, hypocotyl sections, embryogenic callus, isolated growing points, etc. can be mentioned.

[0047] The culture of the plant cells into which the vector has been introduced can be carried out according to known methods depending on the type thereof. As the medium used for the culture, a solid medium (e.g., an agar medium, an agarose medium, a gellan gum medium, etc.) is preferable. Further, the medium preferably contains a carbon source, a nitrogen source, inorganic substances, etc. necessary for the growth of the transformant. For example, N6 medium, MS medium, MSR medium, LS medium, B5 medium, etc. are used as the basal medium. Plant growth substances (e.g., auxins, cytokinins, etc.) and the like may be appropriately added to the medium. The pH of the medium is preferably about 5 to about 8. The culture temperature can be appropriately selected usually within the range of about 20 to about 35°C depending on the type of the plant cells. For example, in the case of plant cells of the genus Nicotiana, they can be usually cultured at 28 to 33°C, preferably 30 to 33°C.

[0048] The selection of the plant cells or plants of the present invention can be appropriately carried out by a known method according to the type of the marker gene used by those skilled in the art. For example, when a drug resistance gene (e.g., a drug resistance gene such as a kanamycin resistance gene (nptII), a hygromycin resistance gene (hpt), etc.) is used, the plant cells into which the loss-of-function mutation of the present invention has been introduced are cultured in the presence of the corresponding drug, whereby the plant cells into which a mutation or the like has been introduced into the target region can be selected.

[0049] In the present invention, the transformant clone in which mutagenesis has been confirmed can be redifferentiated into a plant by a redifferentiation method known per se. If a plant containing plant cells into which a mutation has been introduced into the target region is obtained, it is possible to obtain progeny from the plant by sexual reproduction or asexual reproduction. Further, it is also possible to obtain propagation materials (for example, seeds, fruits, cuttings, strains, calli, protoplasts, etc.) from the plant, its progeny or clones, and mass-produce the plant based on them. Therefore, the present invention includes plants containing the plant cells of the present invention, progeny and clones of the plants, and propagation materials of the plants, their progeny, and clones. When a mutation has been introduced into heterozygosity, by self-pollinating the obtained plant to obtain a T1 plant and further self-pollinating the T1 plant to obtain a T2 plant, a plant into which a mutation has been introduced into homozygosity can be obtained. Further, for a plant into which a mutation has been introduced into homozygosity, by selecting an individual in which the foreign gene used at the time of mutagenesis, that is, the gene encoding the above nucleic acid-modifying enzyme complex, does not exist, or by further advancing generations, a null-segregant can be produced.

[0050] 2. A method for producing a protein in a plant cell having a loss-of-function mutation in the DCL gene or a plant body containing the cell In another embodiment, the present invention provides a method for producing a target protein using the mutant of the present invention (hereinafter sometimes referred to as "the method for producing the protein of the present invention"). The method includes, for example, (1) a step of introducing a nucleic acid encoding a target protein (hereinafter sometimes referred to as "target protein-encoding nucleic acid") into the mutant of the present invention, and (2) a step of expressing the protein in the mutant of the present invention into which the target protein-encoding nucleic acid has been introduced in step (1), and includes.

[0051] The target protein-encoding nucleic acid contains a gene encoding the target protein, and the gene may be a gene that the cell into which the nucleic acid is introduced originally has (in this case, the protein encoded by the gene will be overexpressed), but is preferably a gene that the cell does not originally have (that is, a gene encoding a heterologous protein).

[0052] The target protein-coding nucleic acid may be DNA or RNA, but is preferably DNA. In the case of DNA, it is preferably double-stranded DNA and is provided in the form of an expression vector placed under the control of a promoter functional in the host cell.

[0053] The expression vector is not particularly limited as long as it has an origin of replication that functions in plant cells (e.g., ori of Ti plasmid, Ri plasmid, etc.), but it is preferably also equipped with an origin of replication of Escherichia coli (e.g., ColE1 ori, etc.). When the Agrobacterium method is used as the gene introduction method, it is necessary to further include a T-DNA fragment (including the border sequences RB and LB) excluding the pathogenic genes of the Ti plasmid and Ri plasmid. Examples include pBI101 and pBI121 (Clontech) derived from pBIN19, and improved vectors using this as a backbone (e.g., pRI909, pRI910, pRI101, pRI201 (Takara Bio), etc.), but are not limited thereto. Also, a method using a plant virus vector (e.g., Potato virus X (PVX) vector, Turnip mosaic virus (TuMV) vector, Tobacco mosaic virus (TMV) vector, Cowpea mosaic virus (CPMV) vector, etc.) (e.g., Geneware system, etc.) may be used.

[0054] From the perspective of the expression level, when using the Agrobacterium method, the plasmid may be a replication system derived from a virus. In other words, the plasmid may contain only the region necessary for the replication of the virus integrated from the viral genome. Examples of such virus-derived replication systems include, but are not limited to, the replication system derived from Turnip mosaic virus (TuMV), the replication system derived from Tobacco mosaic virus (TMV) (e.g., magnICON system), the replication system derived from Cowpea mosaic virus (CPMV) (e.g., Profica system), the replication system derived from Bean yellow dwarf virus (BeYDV) (e.g., Tsukuba system (WO 2018 / 220929)). In addition to the region necessary for virus replication, the plasmid may have other regions, such as a region encoding a viral coat protein, and may also have a sequence encoding a plant virus vector. Alternatively, a plasmid without the above virus-derived replication system may be used.

[0055] Regarding the method for preparing the nucleic acid encoding the target protein, the type of expression vector, other elements such as the promoter and terminator contained in the expression vector, or the method for introducing the expression vector into cells and the method for culturing cells, etc., the description for the nucleic acid encoding the above nucleic acid-modifying enzyme complex can be cited. In this case, "encoding the nucleic acid-modifying enzyme complex" and "encoding the nucleic acid sequence recognition module and / or nuclease or deaminase" shall be read as "encoding the target protein".

[0056] The target protein is not particularly limited, and examples include high-value-added proteins such as antibodies and interferons (e.g., IFN-α, IFN-β, IFN-ω, IFN-ε, IFN-κ, IFN-γ, IFN-λ, etc.), antigens, and enzyme proteins that are difficult to express (e.g., horseradish peroxidase, etc.). As an antigen, for example, by using a part of a viral protein, it becomes possible to mass-produce useful antigens for the development of vaccines and antibodies against the virus. Alternatively, it is also possible to mass-produce cancer antigens useful for the development of anticancer agents.

[0057] Examples of such viruses include viruses of the family Herpesviridae such as those of the genus Varicellovirus (e.g., Varicella Zoster virus, etc.), those of the genus Simplexvirus (e.g., Herpes simplex virus 2, etc.), those of the genus Roseolovirus (e.g., human herpesvirus 6, 7, etc.); viruses of the family Flaviviridae such as those of the genus Hepacivirus (e.g., hepatitis C virus, etc.), those of the genus Flaviviridae (e.g., Japanese encephalitis virus, Zika virus, etc.); viruses of the family Coronaviridae such as those of the genus Betacoronavirus (e.g., Severe acute respiratory syndrome coronavirus:SARS-CoV, SARS-CoV-2, Middle East respiratory syndrome coronavirus, etc.); viruses of the family Orthomyxoviridae such as Influenzavirus A, Influenzavirus B, Influenzavirus C;Viruses of the family Paramyxoviridae, such as viruses of the genus Morbillivirus (e.g., Measles morbillivirus, etc.), viruses of the genus Orthopneumovirus (e.g., respiratory syncytial virus, etc.); Examples of retroviruses include viruses of the family Retroviridae, such as viruses of the genus Lentivirus (e.g., Human Immunodeficiency Virus, etc.).;

[0058] In the above step (2), the target protein can be expressed by culturing the cells into which the nucleic acid encoding the target protein has been introduced. Alternatively, the target protein can also be expressed by infecting a part of the plant body (e.g., leaf) with Agrobacterium or a plant virus vector having the nucleic acid encoding the target protein and then growing the plant body.;

[0059] In addition, the target protein can be isolated by extracting and purifying the useful component from the cells obtained in step (2) by a method known per se, or by secreting the target protein into the culture medium and recovering the protein. Alternatively, the cells can be subjected to freezing and drying treatments and used as they are. Similarly, the target protein of the plant body can be isolated by extracting and purifying the useful component from the plant body by a method known per se. Alternatively, the plant body can be used as it is. Therefore, the method for producing the protein of the present invention may include a step of isolating the protein from the cells or the plant body expressing the target protein obtained in (3) step (2). Examples of the above-mentioned method for isolation known per se include a method of crushing the plant body with a mixer or a mortar and then immersing it in an appropriate buffer such as physiological saline to remove the crushed residue by centrifugation or filtration.;

[0060] 3. A method for virus propagation in a plant cell having a loss-of-function mutation in the DCL gene or a plant body containing the cell In the present invention, a plant cell having a loss-of-function mutation in the DCL gene produced therein or a plant body containing such a cell lacks virus resistance, and thus can efficiently propagate even a plant virus lacking immunosuppressive ability. Therefore, in still another aspect, the present invention provides a method for propagating a virus, comprising: (A) a step of infecting a mutant of the present invention with a virus and propagating the virus; and (B) a step of recovering the virus propagated by the step (A). Examples of such a virus include attenuated plant viruses used as biopesticides. Both the above steps (A) and (B) can be carried out by methods known per se.

[0061] "Attenuated plant virus" means a virus with reduced pathogenicity to a host. Since it retains the ability to grow in the host, it can be used as a biopesticide because it can suppress the onset of diseases caused by viruses that infect later by pre-treating plants. Examples of attenuated plant viruses include attenuated viruses of tomato mosaic virus (e.g., TMV-L11A), cucumber mosaic virus, attenuated viruses of capsicum mild mottle virus (e.g., TMV-P), attenuated viruses of watermelon green mottle mosaic virus, attenuated viruses of zucchini yellow mosaic virus, attenuated viruses of soybean mosaic virus, attenuated viruses of citrus tristeza virus, etc., but are not limited thereto. For example, the above-mentioned attenuated plant virus can be infected and propagated in large quantities in a plant cell having a loss-of-function mutation in the Dicer-like (DCL) protein produced in the present invention or a plant body containing such a cell.

[0062] The present invention will be described more specifically with reference to the following examples, which are merely illustrative and do not limit the scope of the present invention in any way.

Examples

[0063] <Materials and Methods> Nicotiana benthamiana Supermix A (Sakata Seeds): Seeds were sown in plastic pots filled with soil mixed at a ratio of vermiculite = 1:1. Growth was carried out under long-day conditions with a temperature of 24°C, a 16-hour light period and an 8-hour dark period by fluorescent lamps. For agroinfiltration, plants at about 4 to 5 weeks after sowing were used. Also, N. benthamiana grown in a sterile state was used for the production of transformed individuals and seedlings for sRNA-seq.

[0064] Generation of dcl4a / 4b mutants and dcl2 / 4a / 4b mutants by genome editing The present inventors previously created dcl4a / 4b mutants (#3-2-6, #3-2-11, #3-2-12) and dcl2 / 4a / 4b mutants (#3-4-7) by genome editing in which the plasmid pWAT135-FLAG-hcoCas9-3002 for genome editing was introduced into wild-type N. benthamiana (Patent Document 1). This plasmid expresses hcoCas9 under the control of the RPS5A promoter and HSP18.2 terminator derived from A. thaliana. In addition, a cassette expressing DCL2-gRNA3 from the U6-26 promoter, DCL3-gRNA1 from the U6-1 promoter, and DCL4-gRNA1 from the U6-29 promoter is linked. It is suggested that in the dcl4a / 4b mutants, a single-base insertion of A occurs at the 5th base upstream of the PAM sequence in the sequences targeted by DCL4-gRNA1 in the DCL4a and b genes on the genome, resulting in a premature stop codon due to the induced frameshift. In the dcl2 / 4a / 4b mutants, a 5-base deletion of AACCT occurs at the 5th to 9th bases upstream of the PAM sequence in the sequence targeted by DCL2-gRNA3 in the DCL2 gene on the genome, and a single-base insertion of A occurs at the 5th base upstream of the PAM sequence in the sequences targeted by DCL4-gRNA1 in the DCL4a and b genes on the genome, resulting in a premature stop codon due to the induced frameshift (Figs. 4 to 8).

[0065] Bacteria Escherichia coli For the amplification of plasmids and the selection of colonies into which the plasmids were introduced, Escherichia coli DH5α (genotype: F-, Φ80lacZΔM15, Δ(lacZYA-argF), U169, recA1, endA1, hsdR17(rK-, mK+), phoA, supE44, λ-thi-1, gyrA96, relA1) was used.

[0066] Agobacterium tumefaciens For transient gene expression experiments by agroinfiltration, the GV3101(pMP90) strain of A. tumefaciens was used. The GV3101 strain has a rifampicin (Rif) resistance gene on the genome, Vir genes necessary for transformation on the helper plasmid, and a gentamicin (Gen) resistance gene. For the generation of transformants of N. benthamiana, the GV3101 strain (GV3101 pSoup-Spec) into which the conjugative plasmid pSoup-Spec (Kumakura, N., et al., PLoS One, 8(11):e79219 (2013)) having a spectinomycin (Spe) resistance gene was introduced into the GV3101 strain was used.

[0067] Plasmids used pWAT735-FLAG-hcoCas9-NbDCL3-1,2 A plasmid derived from pWAT135, having an RPS5A promoter and an HSP18.2 terminator derived from Arabidopsis thaliana, and expressing the hcoCas9 gene under the control of this RPS5A promoter. It also has a cassette for expressing a gRNA targeting the DCL3 gene (NbDCL3) of N. benthamiana under the control of the U6-26 promoter. In this study, it was used to induce a frameshift for the purpose of knocking out the DCL3 gene of N. benthamiana.

[0068] pBIC-p35 It is a plasmid with a 35S promoter and terminator derived from cauliflower mosaic virus (CaMV). The T-DNA region contains a kanamycin resistance gene, and its expression is controlled by the Nos promoter. In this study, it was used as an Empty vector (EV) when confirming the functional deficiency of dcl3 and dcl2 / 4a / 4b.

[0069] pBIC-GFP It has a 35S promoter and terminator derived from CaMV. GFP is expressed under the control of the 35S promoter (Takeda A. et al., FEBS Lett, 532(1-2):75-79 (2002)). In this study, it was used as a transgene when confirming the functional deficiency of dcl3 and dcl2 / 4a / 4b.

[0070] pBIC-dsGFP It has a 35S promoter and terminator derived from CaMV. After the inverted repeat sequence of GFP is transcribed, double-stranded GFP RNA is formed, thus inducing the IR-PTGS pathway (Takeda A. et al., FEBS Lett, 532(1-2):75-79 (2002)). In this study, it was used as an inducer of IR-PTGS when confirming the functional deficiency of dcl3 and dcl2 / 4a / 4b.

[0071] pAT006 It is a plasmid derived from pMDC123 and has a 35S promoter and terminator derived from CaMV. It has Km resistance in transformed Escherichia coli. In this study, it was used as an EV when analyzing RNA silencing induced during agroinfiltration.

[0072] pAT006-Fluc-iFAD2 It has the 35S promoter and terminator derived from CaMV. The firefly luciferase (Fluc) gene is expressed under the control of these promoters. Also, an intron of the Arabidopsis thaliana-derived AtFAD2 gene is inserted into the Fluc gene to prevent the accidental translation of Fluc in Agrobacterium cells. In this study, it was used as the transgene when analyzing RNA silencing induced during agroinfiltration.

[0073] The reagents used are shown in Table 1.

[0074]

Table 1-1

[0075]

Table 1-2

[0076]

Table 1-3

[0077]

Table 1-4

[0078]

Table 1-5

[0079]

Table 1-6

[0080]

Table 1-7

[0081]

Table 1-8

[0082]

Table 1-9

[0083] Experimental methods Agrobacterium transformation 300 - 500 ng of plasmid was added to a 1.5 mL tube containing 40 μL of Agrobacterium GV3101 strain or G3101 pSoup-Spec. After freezing in liquid nitrogen, it was left standing at 37°C for 30 minutes. After standing, 360 μL of SOC was added and it was left standing at 28°C for 2 hours. Then, in the case of the GV3101 strain, 200 μL of the Agrobacterium bacterial solution was spread on an LB agar medium containing Rif, Gen, Km (50 μg / mL each), and in the case of the G3101 pSoup-Spec strain, 200 μL of the Agrobacterium bacterial solution was spread on an LB agar medium containing Rif, Gen, Spe, Km (50 μg / mL each), and cultured at 28°C for 48 hours.

[0084] Sterilization of N. benthamiana seeds Approximately 30 N. benthamiana seeds were placed in a 1.5 mL tube, and 1 mL of DW and 2 μL of PPM TM (Nacalai Tesque) were added and mixed by inverting. This tube was left standing at 4°C overnight for sterilization and then evenly spread on an MS8 solid medium. Excess moisture on the MS8 solid medium was removed, the petri dish was sealed with surgical tape, and grown at 24°C for 3 weeks.

[0085] N. benthamiana transformation A colony of Agrobacterium G3101 pSoup-Spec strain carrying pWAT-735-FLAG-hcoCas9-NbDCL3-gRNA1,2 with the gene to be introduced into N. benthamiana was picked up with a 10-μL tip and inoculated into 10 mL of LB liquid medium containing Rif, Gen, Km, and Spe (50 μg / mL each), and cultured with shaking at 28°C for 24 hours. The culture solution of this Agrobacterium was centrifuged at 4,000 rpm at 4°C for 15 minutes to remove the supernatant, 22.5 mL of MS1 liquid medium was added, and the pellet was suspended. The subsequent operations were carried out in a clean bench. Leaves of N. benthamiana aseptically cultivated on MS8 solid medium (2.4.8) for 3 weeks were cut into pieces of about 1 cm2 with a scalpel and immersed in the Agrobacterium solution transferred to a petri dish for 2 minutes. After 2 minutes, the leaf sections were transferred onto a sterilized Kim towel and dried, and then transferred onto MS1 solid medium (2.4.6) containing Km (50 μg / mL) and cefotaxime (100 μg / mL), and left standing under dark conditions at 22°C for 48 hours. After 48 hours, they were immersed in 22.5 mL of MS1 liquid medium containing cefotaxime (250 μg / mL) in a petri dish and left standing under dark conditions at 22°C for 24 hours. After 24 hours, the leaf sections were transferred onto a sterilized Kim towel and dried, and then transferred onto MS4 solid medium (2.5.16) containing Km (50 μg / mL) and cefotaxime (100 μg / mL). This medium was placed in an incubator (Nippon Medical Chemicals, LPH-411S) set at 22°C with a 16-hour light period and an 8-hour dark period to induce callus. When shoots were induced after callus induction on MS4 solid medium, the growing shoots were aseptically cut with a scalpel in a clean bench and transferred onto MSR solid medium (2.4.9) containing Km (50 μg / mL) and cefotaxime (100 μg / mL). This medium was placed in an incubator (Nippon Medical Chemicals, LPH-411S) set at 22°C with a 16-hour light period and an 8-hour dark period to induce rooting. After confirmation of rooting, the plants were taken out of the MSR solid medium, the roots were washed with deionized water, and the attached MSR solid medium was removed. The period until the plants were transferred to soil and acclimated was covered with a dome for heat and humidity retention (Sakata Seed, half tray (a tray that makes watering easy)) and cultivated while maintaining the humidity.

[0086] DNA extraction Genomic DNA was extracted from each plant and used to confirm the genotype. One leaf disk was collected in a 2 mL tube containing 1 stainless steel bead, and immediately frozen with liquid nitrogen. Then it was disrupted with a paint shaker (product number and conditions) for 2 minutes. After disruption, 100 μL of DNA extraction buffer was added and vortexed, followed by centrifugation at 15,000 rpm, room temperature for 2 minutes. After centrifugation, 70 μL of the supernatant was added to a 1.5 mL tube containing 70 μL of isopropanol and vortexed. Then it was centrifuged at 15,000 rpm, room temperature for 5 minutes. After centrifugation, the supernatant was removed and air-dried for 5 minutes. After air-drying, 50 μL of 1x TE was added and vortexed to obtain the DNA sample.

[0087] PCR This was performed to confirm the mutations of each NbDCL gene. Using the genomic DNA extracted from the generated transgenic plants as a template, amplification was carried out by PCR using sequence-specific primers for the sequence to be confirmed. The following shows each condition and primer sequences. The sequences in Table 3 are shown as SEQ ID NOs: 9 - 30 in order from the top.

[0088]

Table 2

[0089]

Table 3

[0090] After PCR, agarose gel electrophoresis or DNA-PAGE was performed. If necessary, the gel of the target size was recovered and sequence analysis was carried out.

[0091] DNA-PAGE This was done to confirm PCR products of less than 200 bp. A gel with a thickness of 1 mm was used, and an electrophoresis apparatus and the gel were prepared and left standing for 1 hour. After gel preparation, 0.5x TBE Buffer was added to the electrophoresis tank. After cleaning the wells by pipetting, the samples were applied. The same amount of 1x Loading Buffer as the samples was applied to both ends and unused wells to reduce smiling. Then, electrophoresis was performed at 100 V and 50 mA using the migration rate of BPB as a guide (for about 60 - 80 minutes). Thereafter, the band of the target size was confirmed.

[0092] Agroinfiltration It was carried out to induce RNA silencing in dcl mutants. A colony of Agrobacterium harboring the plasmid with the gene to be introduced was picked up with a 10 μL tip and put into 5 mL of LB liquid medium containing Rif, Gen, and Km (50 μg / mL each), and cultured with shaking at 28°C for 24 hours. The next day, the culture solution was centrifuged at 4,000 rpm at 4°C for 15 minutes to remove the supernatant, 5 mL of Vir Induction Medium was added, and the container was shaken to suspend the pellet. After suspension, it was shaken at 28°C for 24 hours. The next day, the suspension was centrifuged at 4,000 rpm at 4°C for 15 minutes to remove the supernatant, 5 mL of Infiltration Solution was added, and the pellet was suspended in the same manner as the previous day. 200 μL of the prepared Agrobacterium solution was mixed with 800 μL of Infiltration Solution, placed in a Disposable Semi Micro Cuvette, 800 μL - 1.5 mL, UV Plastic 10 mm (WPA), and the OD600 was measured using a Biowave CO8000 Cell Density meter (WPA). After measurement, it was diluted with Infiltration Solution so that the Agrobacterium solution had an OD600 = 0.8. The prepared Agrobacterium solutions were mixed in a 1:1 ratio depending on the use and used as an infiltration solution. Holes were made in the leaves of N. benthamiana cultivated for 4 - 5 weeks with a syringe needle, and the prepared infiltration solution was injected from the back side of the leaf using a 1 mL syringe without a needle (NIPRO). Thereafter, the plants were cultivated under the conditions of 22°C, a 16-hour light period, and an 8-hour dark period for 48 hours.

[0093] Luc assay Luciferase activity was measured to evaluate the inducibility of RNA silencing under transient overexpression. One leaf disk was collected with the lid of a 2 mL tube containing one disruption bead (Strain beads, SUS-304, 5 / 32 inch) and frozen in liquid nitrogen. The collected sample was disrupted with a paint shaker (FAST&FLUID, SK550) for 2 minutes. While being careful not to let the leaves dissolve, it was gently centrifuged to let the leaves sink to the bottom and then cooled again. 300 μL of a solution obtained by diluting 5-fold concentrated Picagene cultured cell lysate (Toyobo Ink, PGC-50) 5-fold was added thereto, and then this sample tube was vortexed with a Micro Mixer E-36 (TAITEC) for 2 minutes to mix. Then, it was centrifuged at 4°C, 15,000 rpm for 3 minutes to precipitate the leaf pieces. Next, 4 μL of the supernatant was added to a luminescent substrate solution PGL (Toyobo Ink, PGL-1500) containing 40 μL of luciferin and gently pipetted. One minute after adding the supernatant, the FLuc enzyme activity was measured with a GloMax® Navigator Microplate Luminometer (Promega, GM2000). The measurement was performed with quick read and an exposure time of 10 seconds.

[0094] Protein extraction from leaves of N. benthamiana Performed for SDS-PAGE sample preparation. Four leaf disks were collected with the lid of a 2 mL tube containing one disruption bead and cooled in liquid nitrogen. After disrupting the leaves using a paint shaker, centrifugation was performed at 3,000 rpm for 1 minute at 4°C to let the leaves sink to the bottom of the tube. 80 μL of 1x SDS sample buffer was added thereto and gently mixed by pipetting. Then, heat block treatment was performed at 95°C for 5 minutes, allowed to stand on ice for 5 minutes, and then centrifuged at 15,000 rpm for 3 minutes at 4°C. The supernatant after centrifugation was used as the protein sample solution.

[0095] Protein concentration measurement and adjustment This was done to align the proteins in the extracted samples. Referring to the instruction manual of XL-Bradford [SDS-PAGE compatible] (Farmers), the sample volume to be measured was changed from 20 μL to 3 μL.

[0096] SDS-PAGE A gel with a thickness of 1 mm was used, and the electrophoresis apparatus and gel preparation were carried out. After gel preparation, 1x SDS running buffer (2.4.24) was added to the electrophoresis tank. After cleaning the wells by pipetting, a sample equivalent to 3 μg of protein was applied. To both ends and unused wells, the same amount of 1x SDS sample buffer (2.4.17) as the sample was applied for smile reduction, and 5 μL of CLEARLY Staind Protein Ladder (Takara Bio) was used for the marker. Then, electrophoresis was performed at a constant current of 25 mA until passing through the stacking gel and 50 mA after passing through. The electrophoresis time was set to about 60 - 90 minutes after changing to 50 mA, with the mobility of BPB as a reference. The part containing RbcL was stained with CBB staining solution for loading control, and the part containing GFP was used for measuring the amount of GFP by Western blotting.

[0097] Transfer, blocking, antibody reaction This was performed to measure the amount of GFP transiently expressed in N. benthamiana leaves. A portion near the molecular weight of GFP (about 27 kDa) was excised from the gel on which SDS-PAGE was performed. Also, one Immobilon-P Transfer membrane (Millipore) larger than the excised gel and four pieces of filter paper larger than the membrane were cut out. The membrane was shaken in 100% MtOH for 10 seconds to increase protein adsorption. Then, the gel, filter paper, and membrane were each shaken in 1x Towbin buffer (2.4.26) for 5 minutes. After shaking, transfer was performed at 25 V for 90 minutes using 1x Towbin buffer as the transfer buffer, a large transfer apparatus (Nippon Eido, NA-1518), and a power supply for transfer (Bio Craft, BP-T8). After transfer, the membrane was washed with 1x TBS.T for 5 minutes and then shaken with Western Blocking buffer at room temperature for 60 minutes or more to block the membrane. After blocking, the membrane was washed with 1x TBS.T for 5 minutes. After washing, the membrane and 15 mL of GFP antibody dilution were placed in a Hybri-Bag Soft (Cosmo Bio), sealed with a Polysheeter (Fuji Impulse), and then shaken at 4°C overnight. The next day, 1 mL of Luminata TM Classico Western HRP Substrate (Millipore) was dropped onto the entire membrane and signal detection was performed with an ImageQuant LAS 4000.

[0098] Total RNA extraction (for siRNA Northern blotting) 150 mg of inoculated leaves of N. benthamiana cultivated for 4 - 5 weeks were collected into a 2 mL tube containing 2 stainless steel beads (SUS304 5 / 32 inch). The tube was placed in liquid nitrogen to freeze the leaves and crushed for 3 minutes with a shaker (FAST&FLUID, SK550). After crushing, 1.5 mL of TRI Reagent (SIGMA) was added and vortexed well. After vortexing, it was allowed to stand at room temperature for 10 minutes, 200 μL of chloroform was added, and it was vortexed again. After allowing it to stand at room temperature for 2 minutes, it was confirmed that the suspension was separated into two layers and centrifuged at 13,000 rpm at 4°C for 15 minutes. 500 μL of the supernatant was added to a 1.5 mL tube containing 500 μL of isopropanol and inverted and mixed. After allowing it to stand at room temperature for 10 minutes, it was centrifuged at 13,000 rpm at 4°C for 10 minutes. The supernatant was removed, and 400 μL of UltraPure TM Distilled Water (invitrogen) was added to dissolve the RNA pellet. Then, 200 μL of RNA Precipitation Solution and 200 μL of isopropanol were added and inverted and mixed. After allowing it to stand at room temperature for 10 minutes, it was centrifuged at 13,000 rpm at 4°C for 10 minutes. The supernatant was removed, and 800 μL of 75% ethanol was added to wash the RNA pellet. After washing, it was centrifuged at 13,000 rpm at room temperature for 2 minutes, and the supernatant was removed. The remaining pellet was air-dried until it became transparent, and then dissolved in 11 μL of UltraPure TM Distilled Water to obtain a Total RNA sample. The concentration, A260 / 230, and A260 / 280 of the extracted Total RNA were measured with Thermo Scientific TM NanoDrop 2000 (Thermo Fisher Scientific), dissolved in LMV buffer, frozen with liquid nitrogen, and stored at -80°C.

[0099] Total RNA Extraction (for RNA Comprehensive Analysis) Five N. benthamiana seedlings cultivated aseptically in the form of MS8 solid medium for 10 - 11 days were used after completely removing the medium adhering to the roots with deionized water. Then, they were crushed in the same manner as in 2.5.14, 1 mL of TRI Reagent was added, and after suspension by vortexing, they were left standing at room temperature for 10 minutes. After standing, 200 μL of chloroform was added, and they were suspended by vortexing and left standing at room temperature for 3 minutes. After standing, they were centrifuged at 13,000 rpm at 4°C for 10 minutes. After centrifugation, 500 μL of the supernatant was transferred to a 1.5 mL tube containing 500 μL of isopropanol, mixed by inverting, and left standing at room temperature for 10 minutes. After standing, they were centrifuged at 13,000 rpm at 4°C for 10 minutes. After centrifugation, the supernatant was removed, and the pellet was washed with 80% ethanol. After washing, they were centrifuged at 13,000 rpm at room temperature for 3 minutes. After centrifugation, the ethanol was removed, and the RNA pellet was air-dried until it became transparent. Then, 20 μL of UltraPure TM Distilled Water was added to dissolve the pellet. The concentration, A260 / 230, and A260 / 280 of the extracted total RNA were measured with a Thermo Scientific TM NanoDrop 2000. After confirming that it was not degraded by agarose gel electrophoresis, the sample was prepared, packaged, and an analysis was requested according to the regulations of BGI JAPAN.

[0100] Northern blotting It was carried out to measure the amount of sRNA present in the leaves of N. benthamiana. A gel with a thickness of 2 mm was used, and the electrophoresis apparatus and gel preparation were carried out. After gel preparation, 0.5x TBE buffer was added to the electrophoresis tank. The samples were treated in a heat block at 95 °C for 3 minutes and left standing on ice. Then, they were centrifuged at room temperature, 11,000 x g for 1 minute. Then, the Urea eluted in the wells was thoroughly washed with a P-1000, and the samples were immediately applied. The same amount of 1x SDS sample Buffer as the samples was applied to both ends and unused wells for smile reduction, and 5 μL of DynaMarker, Prestain Marker for Small RNA Plus (Funakoshi) was used as the marker. Taking care that the 20 nt band of this marker did not completely run off, electrophoresis was carried out at 80 V until the bromophenol blue ran off. After electrophoresis, a stainless-steel bath was filled with 0.5x TBE, and on the 0.5x TBE, in the order of the red plate on the (+) side of the cassette → sponge → filter paper → Hybond N+ membrane (GE Healthcare) → gel → filter paper → black plate on the (-) side of the cassette, using 0.5x TBE buffer as the transfer buffer, a large transfer apparatus (Nippon Eido, NA-1518), and a power supply for transfer (Biocraft, BP-T8), transfer was carried out overnight at 4 °C and 0.04 mA. After transfer, the membrane was washed with 2xSSC for 10 minutes. After washing, the membrane was placed on a wrap, and the surface of the membrane was irradiated with UV at 120x100 Joules using a UV Cross linker (Funakoshi), the membrane was turned over, and the back surface was irradiated with UV again at 120x100 Joules to fix the RNA to the membrane. To detect U6 (about 75 nt), the membrane was cut in two at the position of the 50 nt band of the marker and detected.

[0101] Example 1: Generation of DCL-deficient mutants N. benthamiana is a model plant of the Solanaceae family, known for its high susceptibility to various plant viruses and pathogens including viroids, and its ease of inducing RNA silencing and heterologous protein expression by agroinfiltration. Therefore, in order to analyze the function of the N. benthamiana DCL gene, in this study, DCL mutants were created by genome editing.

[0102] Generation of NbDCL3-deficient mutants by genome editing To generate N. benthamiana in which the function of DCL3 is lost and 24 nt siRNA is not produced, resulting in no TGS, first two gRNAs targeting DCL3 were designed (Figure 3), and pWAT735-FLAG-hcoCas9-NbDCL3-gRNA1,2 containing each gRNA and Cas9 protein on the T-DNA was created. Agrobacterium transformed with this was inoculated onto leaf fragments of wild-type N. benthamiana, and callus induction and regeneration were performed. Seeds were collected and sown from the acclimated T0 generation, and in the T1 generation transformants, null segregation of the transgene and the mutation status were confirmed. Lines #5-2 and #5-10 were obtained with pWAT735-FLAG-hcoCas9-NbDCL3-gRNA1, and lines #2-4 and #2-8 were obtained with pWAT735-FLAG-hcoCas9-NbDCL3-gRNA2, for a total of 4 lines (Figure 11). By sequence analysis, two types of mutations introduced into the DCL3 gene were confirmed: one derived from pWAT735-FLAG-hcoCas9-NbDCL3-gRNA1 with a 1-base insertion of T at the 5th base upstream from the PAM sequence on the genome of DCL3 (Figure 9), and the other derived from pWAT735-FLAG-hcoCas9-NbDCL3-gRNA2 with a 1-base deletion of A at the 3rd or 4th base upstream (Figure 10). The former was designated as the DCL3 mutant (dcl3-1), and the latter as dcl3-2. In both mutants, the base sequence was changed due to the introduced insertion mutation, resulting in a frameshift and the generation of a premature stop codon, so it was predicted that a complete DCL3 protein would not be translated.

[0103] Confirmation of the loss of function of DCL3, DCL2 / 4A / 4B To confirm that 24 nt siRNA is not generated in the dcl3 mutant and 21 and 22 nt siRNA are not generated in the dcl2 / 4a / 4b mutant, and that the functions of each DCL gene are defective, the accumulation level of GFP protein and the detection of sRNA derived from GFP mRNA were performed in each dcl mutant induced with IR-PTGS (Figure 12). pBIC-GFP and pBIC-dsGFP were introduced into wild-type N. benthamiana and dcl3-1, dcl3-2, and dcl2 / 4a / 4b by agroinfiltration. In addition, pBIC-p35 was used as the EV, and pBIC-p35 and pBIC-GFP were introduced as controls without introducing dsGFP. Two days after inoculation, samples of protein and total RNA recovered from the plants were subjected to western blotting and northern blotting to measure the accumulation levels. At this time, a decrease in the signal indicating the accumulation level of GFP protein was observed in the wild type introduced with IR-PTGS. The GFP signal disappeared in dcl3-1 and dcl3-2, and a weak signal was detected in dcl2 / 4a / 4b. At this time, weak 21, 22, and 24 nt siRNA were detected in the wild type introduced with IR-PTGS, a large amount of 21 and 22 nt siRNA were detected in dcl3-1 and dcl3-2, and more 24 nt siRNA were detected in dcl2 / 4a / 4b than in the wild type. These results strongly suggest that each DCL gene is completely defective in dcl3-1, dcl3-2, and dcl2 / 4a / 4b. In subsequent experiments, mainly dcl3-2 was used.

[0104] Generation of NbDCL mutants by crossing Crosses were made between dcl3-2 and dcl2 / 4a / 4b with the aim of creating various DCL mutants. Since dcl2 / 4a / 4b has shorter pistils and lower fertility compared to dcl3-2, and is considered to have a low probability of self-pollination, dcl3-1 was used as the male and dcl2 / 4a / 4b as the female. Pollen of dcl3 collected from the opened flowers was rubbed onto the stigmas of pre-flowering dcl2 / 4a / 4b for artificial pollination. As a result of the cross, individuals (#1, #2, #3) in which the mutation was introduced into dcl3-2 were obtained in the F1 generation plants. From the seeds obtained by growing these, many mutants were obtained in the F2 generation plants that germinated. Also, #3-2 in which DCL2, DCL3, DCL4a were homozygous and DCL4b was heterozygous and deleted was obtained in this generation. In #3-2-14 of the F3 generation, dcl2 / 3 / 4a / 4b in which DCL2, DCL3, DCL4a, and DCL4b were homozygously deleted was obtained. Similarly, dcl2 was obtained by crossing dcl2 / 4b (#3-2-) with WT.

[0105] Establishment of the dcl3-2 mutant screening system In order to select the target mutants from the crossed plants, it is necessary to confirm the genotypes of many plants. Therefore, a system was established to screen dcl3-2 more quickly and inexpensively than by sequence analysis. Using primers with mismatches, restriction enzyme sites were created in the PCR products (Figs. 19, 20).

[0106] Example 2: Functional analysis of the DCL gene Functional analysis of each DCL gene was performed using the created plants. In this study, in addition to RNA silencing and heterologous protein expression induced during agroinfiltration, phenotypic analysis and comprehensive analysis of sRNAs were carried out.

[0107] Transient expression experiment using agroinfiltration In addition to dcl3-2 with defective TGS, dcl2 / 4a / 4b with defective PTGS, and dcl2 / 3 / 4a / 4b with defective TGS and PTGS, a Luciferase assay was performed to evaluate RNA silencing induced during agroinfiltration in dcl2 and dcl4a / 4b, each lacking the function of DCL2 and 4. pAT006-Fluc-iFAD2 was agroinfiltrated into wild-type and DCL mutants of N. benthamiana to introduce Firefly luciferase (Fluc). In addition, pAT006 was introduced as an EV. Six days after inoculation, Fluc activity was measured. The relative values were evaluated based on the wild-type values. As a result, activities comparable to those of the wild type were confirmed in dcl2 and dcl4a / 4b, approximately three times higher in dcl3-2, approximately twelve times higher in dcl2 / 4a / 4b, and approximately 430 times higher in dcl2 / 3 / 4a / 4b (Figure 21).

[0108] Phenotypic analysis of dcl mutants Due to the functional deficiency of the DCL gene, characteristics such as short pistils and slender leaf blades were observed in dcl2 / 4a / 4b and dcl2 / 3 / 4a / 4b. Therefore, phenotypic observations and statistical analysis using Dunette's test were performed. On the 41st day after sowing, the growth, leaf blade shape, and flower morphology of wild-type (WT), dcl2, dcl3, dcl4a / 4b, dcl2 / 4a / 4b, and dcl2 / 3 / 4a / 4b were observed and compared with the wild type (Figure 22). For the growth comparison, the length of the main stem (plant height) was measured 41 days after sowing for each plant. As a result, WT was 7.40 ± 1.18 cm, dcl2 was 7.45 ± 1.62 cm, dcl3-2 was 7.01 ± 1.07 cm, dcl4a / 4b was 7.4 ± 1.00 cm, dcl2 / 4a / 4b was 6.81 ± 1.31 cm, and dcl2 / 3 / 4a / 4b was 4.41 ± 1.28 cm (Figure 23). For the leaf blade shape, the length of the leaf and the width of the widest part were measured, and the value obtained by dividing the width by the length was calculated. As a result, when WT was 1.01 ± 0.01, dcl2 was 1.10 ± 0.06, dcl3-2 was 1.00 ± 0.01, dcl4a / 4b was 1.0 ± 0.05 cm, dcl2 / 4a / 4b was 0.6 ± 0.04, and dcl2 / 3 / 4a / 4b was 0.6 ± 0.02 (Figure 24). For the flower morphology, the shape of the corolla lobes and the length of the pistil were observed. Changes in the morphology of the corolla lobes were observed in dcl2 / 4a / 4b and dcl2 / 3 / 4a / 4b (Figure 25). For the length of the pistil, after dissecting the petals and measuring the length of the pistil and the stamens, the value obtained by dividing the length of the pistil by the length of the stamens was calculated. As a result, WT was 0.83 ± 0.03, dcl2 was 0.80 ± 0.027, dcl3-2 was 0.80 ± 0.03, dcl4a / 4b was 0.80 ± 0.04, dcl2 / 4a / 4b was 0.57 ± 0.10, and dcl2 / 3 / 4a / 4b was 0.58 ± 0.14 (Figure 26). Also, dcl2 / 4a / 4b and dcl2 / 3 / 4a / 4b are sterile, but they can produce seeds by self-pollination as in the case of crossing (3.1.3). From the above results, it became clear that the plant height decreases in dcl2 / 3 / 4a / b, and abnormal flower morphology is observed in plants lacking both DCL2 and 4.

[0109] Example 3: Comprehensive analysis of sRNAs in dcl mutants To examine the effects of loss-of-function mutations in each DCL and the RNA silencing pathway on plants, we performed a comprehensive analysis of sRNAs using RNA extracted from sprouts.

[0110] The number of sRNA reads present in each mutant The reads of 15 - 45 nt RNAs present in each DCL mutant were compared with those of the wild type. In dcl3-1, the 21 nt sRNA was 1.543-fold, the 22 nt sRNA was 1.548-fold, and the 24 nt sRNA was 0.150-fold. In dcl3-2, the 21 nt sRNA was 1.369-fold, the 22 nt sRNA was 1.439-fold, and the 24 nt sRNA was 0.154-fold. In dcl2 / 4a / 4b, the 21 nt sRNA was 0.844-fold, the 22 nt sRNA was 0.609-fold, and the 24 nt sRNA was 0.501-fold. In dcl2 / 3 / 4a / 4b, the 21 nt sRNA was 1.904-fold, the 22 nt sRNA was 0.818-fold, and the 24 nt sRNA was 0.141-fold (Figure 27) (Table 4).

[0111]

Table 4

[0112] TAS3-derived tasiRNA mapping Based on the database of N. benthamiana (Sol Genomics Network Genome Ver. 2.6.1), we analyzed sRNAs mapped to the TAS3 locus with sequences homologous to tasiRNA, which is considered to be the cause of the phenotype of RNA silencing-deficient A. thaliana. No difference was observed between dcl3-1, 2 and the wild type. In dcl2 / 4a / 4b, it was confirmed that the 21, 22 nt sRNAs decreased and the 24 nt sRNAs were mapped. In dcl2 / 3 / 4a / 4b, mapping of sRNAs around 21 nt was confirmed (Figures 28 - 30).

Industrial Applicability

[0113] The genome-edited individuals with the DCL gene of the present invention disrupted are extremely useful in that transient expression in their leaves enables highly efficient foreign protein expression in a shorter period than in wild-type plants. It is possible to highly express high-value-added proteins such as antibodies and interferons, and proteins that are difficult to express in other species (for example, peroxidase of Japanese horseradish). Therefore, it can be expected to be used in various applications, such as highly expressing high-value-added inspection and therapeutic proteins such as antibodies for industrial use, and highly expressing proteins for structural analysis and biochemical analysis.

Claims

1. A Solanaceae plant cell having loss-of-function mutations in the DCL2 gene, the DCL3 gene, and the DCL4 gene, and having an improved ability to express an exogenous gene.

2. The cell of claim 1 , having a loss-of-function mutation in the DCL4a and DCL4b genes.

3. The cell according to claim 1 or 2, wherein the Solanaceae plant is a plant belonging to the genus Nicotiana.

4. The cell according to any one of claims 1 to 3, which does not contain any foreign gene.

5. A plant having the cell according to any one of claims 1 to 4.

6. (1) introducing a nucleic acid encoding a target protein into the cell according to any one of claims 1 to 4 or the plant according to claim 5; and (2) expressing the protein in the cell or plant into which the nucleic acid has been introduced in (1) A method for producing a target protein, comprising:

7. The method according to claim 6, further comprising: (3) isolating the target protein from the cells or plant expressing the target protein obtained in (2).

8. The method according to claim 6 or 7, wherein the step (1) comprises a step of infecting a cell or a plant body with an Agrobacterium or a plant virus vector carrying a nucleic acid encoding a target protein.

9. The method according to any one of claims 6 to 8, wherein the nucleic acid comprises a sequence encoding a replication system of viral origin or a plant viral vector.

10. (1) a step of infecting a cell according to any one of claims 1 to 4 or a plant according to claim 5 with a virus and propagating the virus; and (2) A step of recovering the virus propagated in the step (1) A method for propagating a virus, comprising:

11. The method of claim 10, wherein the virus is an attenuated plant virus.

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