JP6486661B2 - Rice having resistance to blast and production method thereof - Google Patents

Description

  The present invention relates to a plant having disease resistance and a production method thereof.

  Damage caused by plant diseases is often affected by environmental conditions. For example, damage caused by diseases such as rice blast is known to increase when rice is exposed to low temperatures, such as the year of cold damage. In recent years, damage caused by blast disease is not significant in 1993 and 2003. did. It is also known that blast infection is likely to occur around dawn when the temperature drops. This is thought to be because the resistance mechanism of rice is weakened by low temperatures. Therefore, the development of plants exhibiting high disease resistance that is not affected by environmental conditions is beneficial for improving productivity in agriculture.

  It is known that the salicylic acid (SA) signaling pathway is activated by a chemical resistance inducer and plays a central role in defense against diseases such as rice blast (Non-patent Documents 1 and 2). ). The transcription factor WRKY45 plays an indispensable role in this process, and over-expressing WRKY45 can improve plant disease resistance (Non-Patent Documents 1 and 2). Patent Document 1 describes that a monocotyledonous plant having both disease resistance and agricultural traits can be produced using a combination of a WRKY45 gene and a promoter gene.

  However, the effect of improving disease resistance via the SA signaling pathway is inhibited by activation of abscisic acid (ABA) signaling under low temperature conditions, but the mechanism is unknown (Non-patent Documents 3 and 4). ).

  On the other hand, OsMPK6, which is one of rice MAP kinases, is known to be activated in response to SA and phosphorylate WRKY45 protein (Non-patent Document 5). In general, MAP kinases are required for dual-phosphorylation at the threonine (Thr) and tyrosine (Tyr) residues within the TEY signature sequence, whereas OsMPK6 is located at positions 225-227. It has an arrangement (Non-patent Document 6).

  Non-Patent Document 7 describes that in Arabidopsis thaliana, protein tyrosine dephosphorylation enzyme (PTPase) AtPTP1 dephosphorylates the phosphorylated tyrosine residue of MAP kinase (MAPK) in vitro, AtPTP1 and protein Disease resistance is enhanced by the functional deficiency of MKP1, which is a protein phosphatase having the activity of dephosphorylating both phosphorylated threonine residues or phosphorylated serine residues and phosphorylated tyrosine residues It is described.

  Previous studies on human PTP1B, a typical protein tyrosine phosphatase, show that after human PTP1B binds to phosphorylated tyrosine residues in other proteins, the phosphorylated tyrosine residue phosphate group As a result of the transfer to the 215th cysteine residue of PTP1B, it is shown that the phosphorylated tyrosine residue in the other protein is dephosphorylated (Non-patent Document 8). The presence of a thiol group in the cysteine residue is essential for the transfer of the phosphate group. It has also been shown that the 181st asparagine residue in human PTP1B promotes the transfer of a phosphate group from a phosphorylated tyrosine residue to the cysteine residue as a general Lewis base (Non-patent Document 9). Flint et al. (Non-patent Document 10) show that the enzyme activity of a mutant enzyme in which the 215th cysteine residue or 181st asparagine residue of PTP1B is substituted with another amino acid residue based on the results of mutant analysis of human PTP1B. Has almost disappeared. Based on this finding, Xu et al. (Non-Patent Document 11) conducted a mutant analysis of AtPTP1, which is a protein tyrosine phosphatase of Arabidopsis thaliana, and found the 265th AtPTP1 corresponding to the 215th cysteine residue in human PTP1B. Reported that the enzymatic activity of mutant AtPTP1 in which the 234th aspartic acid residue of AtPTP1 corresponding to the 181st aspartic acid residue in cysteine residue or human PTP1B was replaced with another amino acid residue was lost. Yes.

International Publication No. 2012/121093

M.M. Shimono et al. , Plant Cell 19, 2064 (2007) M.M. Shimono et al. Mol. Plant Pathol. 13, 83 (2012) A. Robert-Seiliantz, M .; Grant, J.M. D. Jones, Annu. Rev. Phytopathol. 49, 317 (2011). M.M. Fujita et al. Curr. Opin. Plant Biol. 9,436 (2006) Y. Ueno et al. , Plant Signal Behav. 8, e24510 (2013) N. G. Anderson, J.M. L. Maller, N.M. K. Tonks, T.M. W. Sturgill, Nature 343, 651 (1990) S. Bartels et al. , Plant Cell 21, 2884 (2009) Bradford et al. , Science 263, 1397-1404 (1994) Jia et al. , Science 268, 1754-1758 (1995). Flint et al. , Proc. Natl. Acad. Sci. USA 94, 1680-1685 (1997) Xu et al. Plant Cell 10, 849-857 (1998)

  Patent Document 1 does not focus on maintaining and improving disease resistance even under severe environmental conditions such as low temperatures. Further, in Non-Patent Documents 1 to 11, protein tyrosine phosphatase is involved in maintaining or improving disease resistance in various plants, in particular, protein tyrosine phosphatase is an environmental condition such as low temperature. It is not described that it is involved in maintaining or improving disease resistance that is not affected.

  An object of the present invention is to provide a plant having a high disease resistance, particularly a plant having a high disease resistance not affected by environmental conditions, and a high disease resistance, particularly a plant having a high disease resistance not affected by environmental conditions. It is to provide a technique for granting.

The present invention provides the following inventions.
[1] A disease-resistant plant in which expression or activity of one or more protein tyrosine phosphatase genes is suppressed.
[2] The suppression according to [1] above, wherein the suppression of expression of one or more protein tyrosine phosphatase genes is suppression by RNAi method, antisense method, co-suppression method or RNA-directed DNA methylation method plant.
[3] The plant according to [1] above, wherein the suppression of the activity of one or more protein tyrosine phosphatase genes is suppression by introduction of one or more mutations.
[4] The plant according to [3], wherein the one or more mutations include at least a mutation in the protein coding region of the protein tyrosine phosphatase gene.
[5] One or more mutations are amino acid residues contained in protein tyrosine phosphatase, which corresponds to the 258th cysteine residue of OsPTP1, and / or the 166th cysteine of OsPTP2 The plant according to [3] or [4] above, comprising at least a codon mutation of a protein tyrosine phosphatase gene encoding a cysteine residue corresponding to the residue.
[6] The plant according to any one of [1] to [5], which is a monocotyledonous plant.
[7] The plant according to any one of [1] to [6], which is rice.
[8] A method for producing a disease-resistant plant, which suppresses the expression or activity of one or more protein tyrosine phosphatase genes.
[9] The plant production according to [8], wherein the suppression of expression of one or more protein tyrosine phosphatase genes is performed by RNAi method, antisense method, co-suppression method or RNA-directed DNA methylation method. Method.
[10] The method for producing a plant according to [8] above, wherein the activity of one or more protein tyrosine phosphatase genes is suppressed by introducing one or more mutations.
[11] A plant progeny or clone produced by the method according to any one of [8] to [10] above, wherein the expression or activity of one or more protein tyrosine phosphatase genes is suppressed. A plant having disease resistance.

  According to the present invention, high disease resistance can be imparted to plants. The disease-resistant plant brought about by one embodiment of the present invention exhibits remarkable disease resistance even at the time of cold damage. Therefore, compared with the case of cultivating conventional plants, the effect of the resistance inducer at a low temperature is also achieved. Can last. Therefore, the present invention can improve plant productivity including rice, and is useful in fields such as agriculture and food industry where plants are involved.

FIG. 1 is a diagram showing a model of the salicylic acid pathway. FIG. 2 shows anti-pTEpY antibody (upper), anti-OsMPK6 (upper) using wild-type (WT) rice callus, osmpk6 gene mutant rice callus, and wild-type (WT) rice leaf after or without salicylic acid (SA) treatment. (Middle panel) and immunoblot assay results with anti-PEPC antibody (bottom panel). The arrowheads in FIG. 2 indicate OsMPK6 bands, and * and ** indicate bands of MAP kinases other than OsMPK6, respectively. FIG. 3 shows the results of phosphorylation assay using mutant K96R and mutant K96R / T225A / Y227D using MBP-labeled OsMPK6 (upper) and the results of Coomassie brilliant blue (CBB) staining as a loading control (lower). FIG. In FIG. 3, the arrowhead indicates the MBP-OsMPK6 band, and * indicates the GST-MKK10-2D band. FIG. 4 shows the results of phosphorylation assay using MBP-labeled OsMPK6 (MBP-MPK6) of wild type (WT), mutant T225A and mutant Y227D (upper panel), and Coomassie brilliant blue (CBB) staining as a loading control. It is a figure which shows the result of (lower stage). FIG. 5 shows anti-pTEpY antibody (upper), anti-OsMPK6 antibody (lower) using an extract of GVG-MKK10-2D transformed rice (lines # 3 and # 14) after dexamethasone treatment (Dex) or mock treatment. It is a figure which shows the time-dependent change in the immunoblot assay by. FIG. 6 shows the WRKY45 transcription level (relative to ubiquitin transcription level: average of three measurements) in GVG-MKK10-2D transformed rice (lines # 3 and # 14) after dexamethasone treatment (Dex) or mock treatment. And standard deviation) over time. FIG. 7 shows dexamethasone-treated or untreated M. pneumoniae. It is a figure which shows the number of lesions per leaf blade (n = 12 biological repetition: mean and standard deviation) in GVG-MKK10-2D (lines # 3 and # 14) inoculated with oryzae. In FIG. 7, * indicates that there is a significant difference at P <0.1 (Student t test). FIG. 8 shows immunoblotting assay with MKK10-2D transcription level, WRKY45 transcription level and anti-pTEpY antibody in GVG-MKK10-2D rice (line # 3) after dexamethasone treatment (Dex) and / or abscisic acid (ABA) treatment. As a result, immunoblot assay results using anti-phosphotyrosine (pY) antibody, immunoblotting assay results using anti-phosphorylated threonine (pT) antibody, and immunoblotting assay results using anti-OsMPK6 antibody are shown. Are shown in order from the top. Arrowheads indicate the position of OsMPK6. FIG. 9 shows MKK10-2D transcription level (relative to ubiquitin transcription level: 3 in GVG-MKK10-2D plants (lines # 3 and # 14) after dexamethasone treatment (Dex) and / or abscisic acid (ABA) treatment. It is a figure which shows a time-dependent change of the result of the measurement value of 1 time, and a standard deviation; an upper stage) and the result of WRKY45 transcription | transfer level (relative level with respect to a ubiquitin transcription level: the average of 3 measurements, and a standard deviation; the lower stage). FIG. 10 shows the result of immunoblotting assay with anti-pTEpY antibody in anti-pTEpY antibody in GVG-MKK10-2D plant (line # 14) after dexamethasone treatment (Dex) and / or abscisic acid (ABA) treatment. It is a figure which shows the result of the immunoblot assay by an antibody, the result of the immunoblot assay by an anti- phosphorylated threonine (pT) antibody, and the result of the immunoblot assay by an anti- OsMPK6 antibody, respectively, These are shown in order from the top. Arrowheads indicate the position of OsMPK6. FIG. 11 shows WRKY45 transcription level (relative to ubiquitin transcription level: 6 or more biology) in wild-type rice treated with Bay11-7082 or vanadic acid, salicylic acid (SA) and abscisic acid (ABA: 100 μM). It is a figure which shows the average and standard deviation of an iterative repetition. In FIG. 11, *** indicates that there is a significant difference (P <0.01: Student t test). FIG. 12 shows WRKY45 transcription levels (relative to ubiquitin transcription levels: mean and standard deviation) in wild-type rice treated with Bay11-7082 or vanadic acid, salicylic acid (SA) and abscisic acid (ABA). It is. In FIG. 12, * indicates a significant difference at P <0.05, and ** indicates a significant difference at P <0.001 (Student t test). FIG. 13 is a diagram schematically showing a structure of a construct PTP-wkd for simultaneously knocking down OsPTP1 and OsPTP2 genes by RNAi method. FIG. 14 is a diagram showing an alignment of OsPTP1, OsPTP2, and AtPTP1. In FIG. 14, the box indicates the retained amino acid sequence, and * indicates the Cys residue essential for the catalyst. FIG. 15 is a diagram showing the results of immunoblotting assays with anti-pTEpY antibody and anti-OsMPK6 antibody in WT plants and PTP-wkd plants after SA treatment under ABA treatment. FIG. 16 shows the results of the phosphorylation assay (MBP-MPK6) of wild-type MBP-PTP1 (WT) and MBP-PTP2 (WT), and mutant MPB-PTP1 (CS) and MPB-PTP2 (CS) (upper panel). FIG. 4 is a diagram showing the results of Coomassie brilliant blue (CBB) staining (middle stage) as a loading control and the results of MBP-WRKY45 phosphorylation assay (MBP-WRKY45). FIG. 17 shows WRKY45 transcription level (upper), OsPTP1 transcription level (middle) in wild-type rice (WT) and OsPTP1 / 2 knockdown rice (PTP-wkd) treated with salicylic acid (SA) and abscisic acid (ABA). ) And OsPTP2 transcription level (bottom) (respectively relative to the ubiquitin transcription level: average and standard deviation of three technical repeats). 18 shows WT, WRKY45-overexpressing plants (W45-ox) and PTP-wkd plants treated with B. and / or ABA treated or untreated. After inoculation with Oryzae, M. It is a graph which shows the quantitative value of oryzae 28S rDNA. Rice leaves which were respectively treated at normal temperature (30 ° C.) or low temperature treatment (15 ° C.) were used. In FIG. 18, * indicates P <0.1, ** indicates significant difference at P <0.05, and *** indicates significant difference at P <0.005 (Student t test). FIG. 19 is a diagram showing an alignment of amino acid sequences of protein tyrosine phosphatases deduced from cDNA sequences of plant protein tyrosine phosphatase genes. The plant species from which the cDNA is derived is shown on the left side of each sequence. The plant species are as follows, and the parenthesis shows the Genbank Accession No. of each cDNA data. Indicates. A thaliana: Arabidopsis (AT1G71860), G max: soybean (AJ006308), P sativum: pea (AJ005589), P vulgaris: green beans (AY603965), S lycopersicum: tomato (BT014264), O 106 sativa PTP2: rice (AK243006), T aestivum 1: wheat (BT009319), T aestivum 2: wheat (BT009636), Z mays 1: maize (BT041145), Z mays 2: maize (BT065365), P9901G ). Amino acid residues in the amino acid sequence are indicated by single letter symbols. A “-” in the amino acid sequence indicates a gap inserted to maximize homology between amino acid sequences. The cDNAs derived from T aestivum 1, T aestivum 2 and Z mays 2 are incomplete cDNAs that do not contain a translation initiation codon, and the figure shows the amino acid sequence of the protein portion encoded by each cDNA. Figure 20 shows the results after inoculating M. oryzae on each rice line (wild type rice (Nipponbare; NB) and PTP-kd rice (# 3 and # 11)) treated with BTH and / or 250 mM NaCl. It is a graph which shows a blast disease resistance (relative value with respect to the ubiquitin transcription level of M. oryzae 28S rDNA in an inoculated leaf) The result in a figure was calculated by statistically processing each experimental result of three or more repeated experiments. Mean value and standard deviation.

  The plant of the present invention is a plant having disease resistance in which the expression or activity of one or more protein tyrosine phosphatase genes is suppressed.

  Protein tyrosine phosphatases (protein tyrosine phosphatases, PTPases) are enzymes that dephosphorylate phosphorylated tyrosine residues. As the present inventors show in the following examples, protein tyrosine phosphatases are involved in the expression of disease resistance in plants.

  FIG. 1 is a diagram schematically showing the salicylic acid pathway of rice (Oryza sativa). In the salicylic acid pathway, WRKY45 is phosphorylated with the activation of MAP kinase (OsMPK6), and expression is further induced by self-regulation, leading to disease resistance expression. As shown in the Examples below, the inventors have induced functional expression of protein tyrosine phosphatases (OsPTP1 and OsPTP2) via abscisic acid (ABA) under low temperature conditions (for example, 15 ° C. or lower). Occurs, and OsMPK6 is dephosphorylated and inactivated. Therefore, information transmission downstream from OsMPK6 is stopped, in other words, protection against a disease dependent on WRKY45 does not occur, and disease resistance does not develop. As shown in the examples in the latter part, protein tyrosine phosphatase dephosphorylates and inactivates MAP kinase (OsMPK6) in the salicylic acid pathway. Therefore, disease resistance can be imparted to or enhanced by suppressing the expression or activity of the protein tyrosine phosphatase gene.

  As used herein, an amino acid residue may be represented by a three letter symbol (eg, Tyr represents a tyrosine residue) or a one letter symbol (eg, Y represents a tyrosine residue).

In the present invention, OsPTP1 includes mutants, derivatives, variants or homologs thereof, and means the following (A1) to (C1):
(A1) a protein having the amino acid sequence of SEQ ID NO: 1;
(B1) a protein having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having protein tyrosine phosphatase activity; as well as,
(C1) A protein having an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 1 and having protein tyrosine phosphatase activity.

In the present invention, OsPTP2 includes mutants, derivatives, variants or homologs thereof, and means the following (A2) to (C2):
(A2) a protein having the amino acid sequence of SEQ ID NO: 2;
(B2) a protein having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2 and having protein tyrosine phosphatase activity; as well as,
(C2) A protein having an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 2 and having protein tyrosine phosphatase activity.

  The amino acid sequences of SEQ ID NOs: 1 and 2 are the amino acid sequences of rice OsPTP1 and OsPTP2, respectively. Thus, (B1) and (C1) are intended to be variants, derivatives, variants or homologs of (A1), and (B2) and (C2) are variants, derivatives, variants or homologues of (A2). Intended.

  In (B1) and (B2), the number of amino acid residues that may be deleted, substituted, inserted and / or added is, for example, within 50, within 40, within 30, within 20 and within 10 It is preferably 5 or less, more preferably 4 or less, still more preferably 3 or less, still more preferably 2 or less, and particularly preferably 1 or less.

  In (B1) and (B2), it is preferable that the amino acid side chain before mutation is preserved in the amino acid residue after mutation. Amino acids are classified according to the properties of side chains as follows: hydrophobic amino acids (alanine (A), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), Tyrosine (Y), tryptophan (W), valine (V), etc.); hydrophilic amino acids (arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q) ), Glycine (G), histidine (H), lysine (K), serine (S), threonine (T), etc.); amino acids having an aliphatic side chain (glycine (G), alanine (A), isoleucine (I) ), Leucine (L), valine (V), proline (P), etc.); amino acids having a hydroxyl group-containing side chain (serine (S), threonine (T), tyrosine (Y), etc.) Amino acids having side chains containing sulfur atoms (cysteine (C), methionine (M), etc.); amino acids having side chains containing carboxylic acid and amide (aspartic acid (D), asparagine (N), glutamine (Q), glutamic acid Amino acids having base-containing side chains (arginine (R), histidine (H), lysine (K), etc.); amino acids having acid-containing side chains (aspartic acid (D), glutamic acid (E), etc.) ); Amino acids having β-branched side chains (threonine (T), valine (V), isoleucine (I), etc.); and amino acids having aromatic-containing side chains (histidine (H), phenylalanine (F), tryptophan) (W), tyrosine (Y), etc.).

  In (C1) and (C2), the amino acid sequence identity is 70% or more in the entire amino acid sequence or a region necessary for functional expression, for example, 80% or more, 90% or more, 95% or more, preferably It is 96% or more, More preferably, it is 97% or more, More preferably, it is 98% or more, More preferably, it is 99% or more. Amino acid sequence identity can be determined using algorithms such as BLAST (Proc. Natl. Acad. Sci., USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90; 5873, 1993). , BLASTN, BLASTX and the like (Altschul SF et al., J. Mol. Biol. 215: 403, 1990). Examples of parameters for determining an amino acid sequence using BLASTX include score = 50 and wordlength = 3. Examples of parameters used when determining a base sequence using BLASTN include score = 100 and wordlength = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program may be used.

  Protein tyrosine phosphatase gene cDNAs of plants other than rice include kidney beans (Phaseolus vulgaris; GenBank accession number, AY603965), peas (Pisum sativum; GenBank accession number, AJ005589), soybean (Glycine max; GenBank accession number) , AJ006308), Arabidopsis thaliana (GenBank accession number, AF055635), moth orchid (Phalaenopsis amabilis; GenBank accession number, GU119901), wheat (Triticum aestivum; BT009636), corn (Zea mays; GenBank accession number, BT041145 and BT065365), tomato (Lycopersicon esculentum; GenBank accession number, BT014264) are exemplified. Since the amino acid sequences of the proteins encoded by these cDNAs have high homology with the amino acid sequences of OsPTP1 and OsPTP2, respectively, the proteins encoded by these cDNAs are OsPTP1 or OsPTP2 homologous proteins (Orthologous proteins). It is thought that there is (FIG. 19). In the present invention, the protein tyrosine phosphatase gene is a gene encoding protein tyrosine phosphatases and their homologous proteins in the above plant species.

  The protein tyrosine phosphatase gene whose expression or activity is suppressed in the present invention is usually an endogenous protein tyrosine phosphatase gene. That is, it is a protein tyrosine phosphatase gene that the plant originally has. When the plant has only one endogenous protein tyrosine phosphatase gene, its expression or activity may be suppressed. When the plant has two or more kinds of endogenous enzyme genes, it is sufficient that the expression or activity of one or more of them is suppressed, and the expression of all of them may be suppressed. It is preferable that the expression or activity of more than one species is suppressed. Moreover, it is only necessary that the expression or activity of at least one allele among alleles (alleles) of one endogenous protein tyrosine phosphatase gene possessed by a plant cell is suppressed, and the expression or activity of both alleles. Is preferably suppressed. Furthermore, unless contrary to the object of the present invention, the expression and activity of genes other than protein tyrosine phosphatase genes may be simultaneously suppressed.

  The kind of plant which this invention makes object is not specifically limited, What is necessary is just a plant which originally has 1 or more types of protein tyrosine phosphatases. The plant may be either a dicotyledonous plant or a monocotyledonous plant, such as rice plant (such as rice), kidney bean plant (such as kidney bean), pea plant (such as pea), soybean plant (such as soybean), Arabidopsis plant (such as Arabidopsis) Arabidopsis etc.) , Allium (such as leek), Spinach (such as spinach), Japanese radish (such as radish), Brassica (such as Chinese cabbage, cabbage, broccoli, cauliflower, turnip), Broad genus (such as broad bean), Honey genus (such as bee) , Cucumber (cucumber, melon, etc.), watermelon Mosquitoes etc.), pumpkin genus (such as squash) will be exemplified. Among these, monocotyledonous plants are preferable, rice plants are more preferable, and rice (Oryza sativa) is more preferable.

  Rice (Oryza sativa) may be any of Japonica species (Oryza sativa subsp. Japonica), Indica species (Oryza sativa subsp. Indica), and Javanica species (Oryza sativa subiva sub. Rice can be either sticky rice or sticky rice. In addition, there is no particular question regarding the rice varieties, the difference between sticky rice and sticky rice.

  The expression or activity of the protein tyrosine phosphatase gene is suppressed, for example, that the protein tyrosine phosphatase gene is not expressed, the expression level of the gene is low compared to a normal plant, It is exemplified that the activity of the gene product is reduced.

  Whether the expression of the protein tyrosine phosphatase gene is suppressed depends on whether the amount of mRNA of the gene in the plant according to the present invention or the amount of translation product (protein tyrosine phosphatase) of the gene in the control plant is It can be determined by examining whether or not it is reduced compared to the amount of mRNA or the amount of translation product. The control plant is a plant in which the expression or activity of the protein tyrosine phosphatase gene is not suppressed, and is usually a plant that has not been subjected to artificial suppression treatment. For example, in a plant having disease resistance obtained by introducing a mutation into rice (variety: Nipponbare), the control plant is rice (Nipponbare) before the mutation is introduced.

  For example, when the expression of protein tyrosine phosphatase gene is suppressed by RNAi method, antisense method, co-suppression method or RNA-directed DNA methylation method, protein tyrosine is expressed by the method described in Example 6. Total RNA was extracted from the corresponding sites (for example, leaves) of the plants in which the expression of the phosphatase gene is suppressed and cDNA was synthesized, and quantified using these as templates and appropriate primer DNA. By performing the PCR method, the amount of protein tyrosine phosphatase gene mRNA in both can be compared. In order to standardize the amount of total RNA used as a sample, for example, the amount of endogenous ubiquitin gene mRNA in the sample is measured in the same manner, and the amount of protein tyrosine phosphatase gene mRNA and ubiquitin gene mRNA in both samples is measured. It is preferable to determine the ratio and use this quantitative ratio as an index for quantitative comparison of the protein tyrosine phosphatase gene mRNA in both. In order for the expression of protein tyrosine phosphatase gene to be suppressed in a plant, the amount of the gene mRNA or the translation product in the plant is compared with the amount of the gene mRNA or the translation product in the control plant. Thus, the range may be in the range of 50% to 0%, the range is preferably 30% to 0%, and the range is more preferably 10% to 0%.

  Examples of the method for examining the amount of a protein tyrosine phosphatase gene translation product in a plant include an immunoblot assay and an ELISA assay using an antibody specific for the translation product, and an immunoblot method is more preferred.

  Whether the activity of the protein tyrosine phosphatase encoded by the protein tyrosine phosphatase gene into which the mutation is introduced can be determined according to the method described in Non-Patent Document 11.

  That is, the case of examining the mutation introduced into the OsPTP1 gene is as follows. The first to 987th sequences of the base sequence of SEQ ID NO: 3 were inserted into the polylinker site of pMAL-c5x vector (New England Biolabs, Inc.), and a fusion protein (MBP-PTP1 (MBP-PTP1 ( A vector pMAL-PTP1 (WT) expressing WT)) is produced. The target mutation (base substitution, deletion, etc.) is introduced into the sequence encoding OsPTP1 in this vector by a method well known to those skilled in the art. As a result, this vector pMAL-PTP1 (mutation) that expresses a fusion protein (MBP-PTP1 (mutation)) of OsPTP1 and maltose binding protein having a mutation on the amino acid sequence due to the target mutation is obtained. A soluble extract is obtained by a method well known to those skilled in the art from Escherichia coli transformed with pMAL-PTP1 (WT) and pMAL-PTP1 (mutation), respectively. MBP-PTP1 (WT) and MBP-PTP1 (mutation) are each purified from the extract using an amylose resin column.

A ³² P-labeled casein or myelin basic protein is prepared as follows as a substrate for protein tyrosine phosphatase. Casein or myelin basic protein (50 micrograms), ³² P-labeled-γ-ATP (50 microcuries) and human c-Src (25 units, manufactured by Upstate Biotechnology Inc.) in 100 microliters (25 mM Tris) _{-HCl (pH7.2), 5mM MnCl 2} , 0.5mM EGTA, 0.05mM Na 3 VO 4, 25mM Mg (OAc) 2) 30 ℃ in reacted 1 hour. The reaction solution was loaded onto a reverse layer column (Sep-Pak-18, manufactured by Waters), washed with 0.1% trifluoroacetic acid, and ³² P-labeled casein or myelin basic protein was eluted with acetonitrile. To do. The eluted protein is lyophilized, dissolved in phosphate buffer (50 mM Tris-HCl (pH 7.0), 2 mM dithiothreitol) and stored at low temperature.

The phosphatase activity of MBP-PTP1 (WT) and MBP-PTP1 (mutant) is determined by measuring the release of ³² P from ³² P-labeled substrate protein. A substrate protein (2 × 10 ⁴ cpm) labeled with ³² P in a 100 microliter reaction solution (50 mM Tris-HCl, 2 mM dithiothreitol) containing MBP-PTP1 (WT) or MBP-PTP1 (mutation) (5 ng / ml) And incubate at 30 ° C. The reaction is stopped by adding twice the amount of 25% trifluoroacetic acid to the reaction solution at regular intervals. The reaction solution is centrifuged, and the count of ³² P in the supernatant is measured with a scintillation counter. The radioactivity of ³² P in the supernatant is plotted against the reaction time. The range in which the radioactivity of ³² P in the supernatant increases in proportion to the reaction time, for example, in the supernatant from 0 minutes after the start of the reaction, 20 minutes after the start of the reaction, or 30 minutes after the start of the reaction. The rate of dephosphorylation can be determined from the rate of increase in the radioactivity of ³² P.

  In order to suppress the activity of the protein tyrosine phosphatase gene into which the mutation has been introduced, a fusion protein containing the protein tyrosine phosphatase moiety into which the mutation has been introduced (for example, MBP-PTP1 (mutation)) The dephosphorylation rate measured by the above-described method according to the above may be in the range of 0% to 50% compared to the dephosphorylation rate due to the wild-type fusion protein (for example, MBP-PTP1 (WT)), The range is preferably 0% to 20%, more preferably the range is 0% to 10%, and even more preferably the range is 0% to 5%. Moreover, it is particularly preferable that the enzyme activity of the fusion protein containing the protein tyrosine phosphatase portion into which the mutation is introduced is substantially lost.

  The protein tyrosine phosphatase gene means a gene encoding protein tyrosine phosphatase. In the present specification, a gene is usually an endogenous gene. In the present specification, a gene may mean a polynucleotide, that is, a polymer of nucleotides. The polynucleotide may be DNA (cDNA, genomic DNA, etc.) or RNA (mRNA, etc.). DNA and RNA may be either double-stranded or single-stranded unless otherwise specified. The single-stranded DNA and RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand) unless otherwise specified. The polynucleotide may be a chemically synthesized polynucleotide. Chemical synthesis methods include commercially available polynucleotide modification kits (e.g., QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene), KOD-Plus Site-Directed Mutagenesis Kit (Toyobo), TransformerKeten Dirtite Dirtite Dimentitite Kit And a method using polymerase chain reaction (PCR).

When the protein tyrosine phosphatase is OsPTP1, examples of the tyrosine phosphatase gene include the following (a1) to (e1):
(A1) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 1;
(B1) a protein having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having protein tyrosine phosphatase activity Encoding gene;
(C1) a gene encoding a protein having an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 1 and having protein tyrosine phosphatase activity;
(D1) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or a polynucleotide having the 1st to 987th nucleotide sequence of the nucleotide sequence of SEQ ID NO: 3; and (e1) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 Alternatively, it hybridizes with a complementary polynucleotide of the polynucleotide consisting of the nucleotide sequence 1 to 987 of the nucleotide sequence of SEQ ID NO: 3 or a probe thereof under stringent conditions, and has protein tyrosine phosphatase activity A gene that encodes a protein.

When the protein tyrosine phosphatase is OsPTP2, examples of the protein tyrosine phosphatase gene include the following (a2) to (e2):
(A2) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 2;
(B2) a protein having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2 and having protein tyrosine phosphatase activity Encoding gene;
(C2) a gene encoding a protein having an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 2 and having protein tyrosine phosphatase activity;
(D2) a base sequence encoding the amino acid sequence of SEQ ID NO: 2 or a polynucleotide having the 1st to 714th base sequence of the base sequence of SEQ ID NO: 4; and (e2) a base sequence encoding the amino acid sequence of SEQ ID NO: 2 Alternatively, it hybridizes under stringent conditions with a complementary polynucleotide of the polynucleotide consisting of the 1st to 714th nucleotide sequences of the nucleotide sequence of SEQ ID NO: 4 or a probe thereof, and has protein tyrosine dephosphorylating enzyme activity A gene that encodes a protein.

  The amino acid sequences of SEQ ID NOs: 1 and 2 are the amino acid sequences of rice OsPTP1 and OsPTP2, respectively. Of the base sequence of SEQ ID NO: 3, the 1-987th base sequence and of the base sequence of SEQ ID NO: 4 are the base sequences of the regions encoding rice OsPTP1 and OsPTP2, respectively. Thus, (b1), (c1), and (e1) are intended to be variants, derivatives, variants, or homologs of (a1), and (b2), (c2), and (e2) are (a2) ) Variants, derivatives, variants or homologs.

  The number and type of amino acid residues which may be deleted, substituted, inserted and / or added in (b1) and (b2) are as described above. The identity of the amino acid sequences in (c1) and (c2) is also as described above.

  The codons constituting each gene in (d1) and (e1) and (d2) and (e2) may be codons encoding the corresponding amino acids.

  In (e1) and (e2), stringent conditions refer to conditions in which a double-stranded polynucleotide specific to the base sequence is formed and a non-specific double-stranded polynucleotide is not formed. In other words, diffusions with high homology, for example, temperatures ranging from the melting temperature (Tm value) of a perfectly matched hybrid to a temperature 15 ° C. lower, preferably 10 ° C. lower, more preferably 5 ° C. lower. It can also be said that the conditions for hybridization in Stringent conditions include the following conditions: 6M urea, 0.4% SDS, conditions that hybridize under conditions of 0.5 × SSC; and 6M urea, 0.4% SDS,. Conditions for hybridizing for 16 to 24 hours under 1 × SSC conditions. A person skilled in the art may refer to Molecular Cloning (see Sambrook, J. et. Al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive 9). Thus, such a gene can be easily obtained. The base sequence of the polynucleotide can be determined by a method such as the dideoxy method. In addition, the identity of the base sequence can be analyzed using the algorithm such as BLAST described above and the program such as BLASTN and BLASTX.

  Disease resistance means resistance to disease (resistance, tolerance), resistance to disease is less likely to be lower than control plants, no symptoms of disease, less symptoms of disease, Inhibiting the growth of pathogens. In the present invention, disease resistance preferably means disease resistance when a plant is grown at a low temperature (for example, 15 ° C. or lower), and the plant is grown at a low temperature and in the presence of a plant activator. More preferably, it means inhibiting the growth of pathogens inoculated in

  Inhibition of protein tyrosine phosphatase gene expression or activity is usually carried out by an artificial method. Examples of methods for suppressing gene expression include suppression of transcription of protein tyrosine phosphatase gene and suppression of translation of transcribed mRNA.

  Examples of methods for suppressing transcription of protein tyrosine phosphatase genes include the following: RNA-directed DNA methylation, insertion, deletion or alteration of sequences that regulate transcription such as promoters and enhancers; and transcription Protein or compound binding to genes Of these methods, the RNA-directed DNA methylation method is preferred.

  Examples of the method for suppressing translation of mRNA transcribed from a protein tyrosine phosphatase gene include RNAi method, antisense method, co-suppression method, and ribozyme method. In the present invention, among these methods, the RNAi method is preferable.

  In the RNAi (RNA interference) method, double-stranded RNA is introduced into plant cells to cause RNA interference, and the protein tyrosine phosphatase gene mRNA is degraded.

  The double-stranded RNA may be any double-stranded RNA capable of degrading mRNA complementary to the protein tyrosine phosphatase gene, and is a gene encoding tyrosine phosphatase activity in vivo or in cells. Double-stranded RNA that can generate short interfering RNA (siRNA) that can bind to complementary mRNA is preferred. When the protein tyrosine phosphatase gene is the OsPTP1 gene, the double-stranded RNA is, for example, a part of one selected from the above (a1) to (e1) (for example, continuous 10 bases or more, 20 bases) Including a base sequence comprising 30 bases or more, 40 bases or more, 50 bases or more, 60 bases or more, 70 bases or more, 80 bases or more, 90 bases or more, 100 bases or more, 200 bases or more) and its complementary sequence Double stranded RNA is preferred. When the protein tyrosine phosphatase gene is the OsPTP2 gene, the double-stranded DNA includes, for example, a partial base sequence selected from the above (a2) to (e2) and a complementary sequence thereof 2 Single-stranded RNA is preferred.

  The upper limit of the length of double-stranded RNA is usually 15 bases or more, for example, 20 bases or more, 30 bases or more, 40 bases or more, 50 bases or more, 60 bases or more, 70 bases or more, 80 bases or more, 90 bases or more, 100 The number of bases is preferably 200 bases or more. The lower limit of the length of the double-stranded DNA is usually 500 bases or less, such as 450 bases or less, 400 bases or less, preferably 350 bases or less, more preferably 300 bases or less.

  The double-stranded RNA may be a gene construct (cassette) partially having a single-stranded structure. For example, a trimming sequence (GUS linker or the like) may be arranged between the sequences constituting the double strand, and the trimming sequence may connect each strand. In addition, a promoter (such as a ubiquitin promoter) and a terminator (such as NOS) may be linked to each of the two strands.

  Examples of a method for producing a double-stranded RNA corresponding to a protein tyrosine phosphatase gene include a method by simultaneous transcription of a template DNA using T7 RNA polymerase and a method using a commercially available kit.

  In the RNAi method, an RNAi vector is usually used. Any RNAi vector can be used as long as it can introduce double-stranded DNA into a cell and can generate siRNA in the cell, and reverse repeat of the 3 ′ untranslated sequence (utr) of the protein tyrosine dephosphorylation gene cDNA. A vector capable of expressing an RNA containing a sequence in a cell (for example, the vector of FIG. 13) is preferable. Commercially available RNAi vectors may be used.

  Examples of methods for introducing RNAi vectors into plant cells include the Agrobacterium method, the polyethylene glycol method, and the electroporation method.

  In the antisense method, antisense nucleotides are introduced into plant cells to degrade mRNA of a gene encoding a protein tyrosine phosphatase.

  The antisense nucleotide may contain an RNA sequence complementary to at least a part of a protein tyrosine phosphatase gene (sense sequence). The antisense nucleotide is at least a part of a base sequence complementary to the coding region of the protein tyrosine phosphatase gene (for example, continuous 10 bases or more, 20 bases or more, 30 bases or more, 40 bases or more, 50 bases or more, 60 The base region is preferably 70 bases or more, 80 bases or more, 90 bases or more, 100 bases or more, 200 bases or more), and more preferably the entire coding region. When the protein tyrosine phosphatase is OsPTP1, it preferably includes at least a part of a base sequence complementary to the coding regions (a1) to (e1) described above, and may include the entire coding region. More preferred. When the protein tyrosine phosphatase is OsPTP2, at least a part of the base sequence complementary to the coding region (a2) to (e2) described above (for example, 10 or more consecutive bases, 20 or more bases, 30 bases) 40 bases or more, 50 bases or more, 60 bases or more, 70 bases or more, 80 bases or more, 90 bases or more, 100 bases or more, 200 bases or more), and preferably includes the entire coding region. More preferred. In addition, the antisense nucleotide includes a base sequence having an identity to the above exemplified base sequence of, for example, 70% or more, 80% or more, 90% or more, or 95% or more.

  The length of the antisense nucleotide is preferably 5 bases or more, and more preferably 15 bases or more. The upper limit is preferably 5000 bases or less, more preferably 2500 bases or less, and even more preferably 500 bases or less. The method for preparing the antisense nucleotide is not particularly limited, and may be designed and prepared using a commercially available kit.

  In the antisense method, an expression cassette for introducing an antisense nucleotide may be used. The expression cassette includes, for example, DNA encoding an antisense nucleotide (RNA) and, if necessary, additional sequences (promoter sequence and terminator sequence). Examples of the method for introducing an expression cassette into a plant cell include a microinjection method, an electroporation method, a particle gun method, a method of introducing via a vector and Agrobacterium.

  In the ribozyme method, a ribozyme that cleaves a transcription product of a protein tyrosine phosphatase gene is introduced. Ribozymes can be designed to cleave the mRNA of the target gene. When introducing a ribozyme into a plant cell, a promoter (cauliflower mosaic virus (CaMV) 35S promoter or the like) and another trimming ribozyme acting in cis for trimming in the cell may be arranged as necessary. Thereby, only a ribozyme part can be correctly cut out from RNA containing the transcribed ribozyme.

  In the co-suppression method, transcription or the like is suppressed by co-suppression by introducing a polynucleotide having a base sequence identical or similar to the base sequence of the protein tyrosine phosphatase gene. The polynucleotide to be introduced includes a protein tyrosine phosphatase gene, a mutant, derivative, variant or homologue thereof. When the protein tyrosine phosphatase is OsPTP1, (a1) to (e1) described above are preferable. When the protein tyrosine phosphatase is OsPTP2, (a2) to (e2) described above are preferable.

  The RNA-directed DNA methylation method (RdDM method) refers to a double-stranded RNA or hairpin RNA having the same sequence as a promoter sequence or an enhancer sequence having a function of activating gene expression or a partial sequence thereof. Introduction into a cell induces DNA methylation in the promoter sequence or enhancer sequence, and as a result, to the sequence of transcriptional activator proteins that can specifically bind to the promoter sequence or enhancer sequence. (Mette, MF, et al., Transgenic silencing and promoter methylation trigg) red by double-strandedRNA.EMBO Journal 19,5194-5201 (2000);. Matzke, M & Birchle J.RNAi-mediated pathways in the nucleus.Nature Reviews Genetics6,24-35 (2005)). As a method for introducing DNA methylation into a promoter sequence or an enhancer sequence, an inverted repeat sequence (usually several hundreds (about 200 to 300) base sequence) of those sequences or a part of the sequence (usually a number between them) Examples thereof include a method of producing a transformant using a vector that expresses RNA containing a 100 base spacer sequence) or transiently expressing the inverted repeat RNA.

  Usually, a sequence having promoter activity is contained in the region from the 5 'end of the protein coding region of the endogenous gene to about 1000 bases upstream thereof. Therefore, as an example, as a method for inducing DNA methylation in the promoter region of the OsPTP1 gene or OsPTP2 gene by the RdDM method and suppressing the expression of these genes, the upstream of the 5 'end of the protein coding region of these genes Examples include a method of introducing a repetitive RNA containing a sequence of up to about 1000 bases (for example, the 1st to 1000th sequence of SEQ ID NO: 5 and the 1st to 983th sequence of SEQ ID NO: 6) or a partial sequence thereof into a cell. It is done. A vector for expressing the inverted repeat RNA in a cell can be prepared by the same method as that for the RNAi vector. The base sequence of protein tyrosine phosphatase gene cDNA of each plant species is disclosed. Therefore, a person skilled in the art can screen a genomic DNA library using a method well known to those skilled in the art as a probe, or a partial sequence thereof, or can be prepared based on the base sequence of cDNA. By performing inverse PCR using genomic DNA as a template using the obtained PCR primers, protein tyrosine phosphatase genes of each plant species are obtained, and the promoter sequences of the respective genes are obtained by determining the base sequences thereof. be able to. Expression of the protein tyrosine phosphatase gene in each plant can be suppressed by the RdDM method using such a sequence.

  DNA methylation induced by the RdDM method is maintained even if a double-stranded RNA that induces DNA methylation or a double-stranded RNA expression vector that expresses it is not present in the plant body, and is transmitted to the progeny plant body ( . Mette, M.F et al. Transcriptional silencing and promoter methylation triggered by double-stranded RNA.EMBO Journal 19,5194-5201 (2000);. Matzke, M & Birchler J.RNAi-mediated pathways in the nucleus.Nature Reviews Genetics 6 , 24-35 (2005)). Therefore, a plant body in which the expression of the protein tyrosine phosphatase gene is suppressed by the RdDM method is crossed with a wild type plant body, and the plant-derived protein in which the expression of the protein tyrosine phosphatase gene is suppressed by the RdDM method Protein tyrosine dephosphorylation is obtained by obtaining a plant having an allele of the tyrosine dephosphorylation gene and having no vector in the genome for expressing the inverted repeat RNA used in the RdDM method in the cell. Although the expression of the oxidase gene is suppressed, a plant that does not contain exogenous genes can be obtained.

  The method for suppressing protein tyrosine phosphatase gene expression is preferably RNAi method, antisense method, co-suppression method or RdDM method, more preferably RNAi method or RdDM method.

  Examples of a method for suppressing the activity of a protein tyrosine phosphatase gene include the following methods: a method of introducing one or a plurality of mutations into a protein tyrosine phosphatase gene; Examples thereof include a method of allowing a compound (protein, amino acid, etc.) that binds to a phosphorylase to act, and a method of transforming a gene having a dominant negative trait of a protein tyrosine phosphatase gene into a plant.

  Examples of mutation introduction into a protein tyrosine phosphatase gene include one or more changes selected from the group consisting of substitution, deletion, insertion and addition of bases constituting the gene. Examples of methods for introducing substitution, deletion, insertion and addition of bases include irradiation with high-energy electromagnetic waves (radiation, ultraviolet rays, etc.), mutagenic chemical substances (nitroso compounds (for example, nitrosoguanidine), base analogs (for example, BrdU). Compounds), alkylating agents (eg, N-ethyl-N-nitrosourea (ENU), methyl methanesulfonate (EMS), polycyclic aromatic hydrocarbons (eg, benzopyrene, chrysene), DNA intercalators (eg, odor Ethidium chloride), a method of introducing random mutations into genomic DNA by treatment with a DNA cross-linking agent (for example, cisplatin, mitomycin C), active oxygen), transcription activation-like effector nuclease (TALEN) (Japanese translations of PCT publication No. 2012-514976) No. 2013-513389), zinc finger nuclease (Japanese Patent No. 4350907, Japanese Patent No. 4555292), CRISPR / Cas9 (Jinek et al., A programmable dual-endi- bative DNA in adductive bacteria. 337, 816-821 (2012); Mali et al., RNA-guided human gene engineering via Cas9. Science 339, 823-826 (2013)). The introduction method and the introduction method of transposon are exemplified. Method using the beam mutagenesis or site-specific DNA degrading enzymes are preferred.

  In the method using site-directed mutagenesis, the mutation (for example, substitution) introduction site may be a site where the activity of the protein tyrosine dephosphorylation enzyme encoded by the gene after the mutation introduction is suppressed, and the gene encodes after the mutation introduction. It is more preferably a site where the activity of protein tyrosine phosphatase disappears. Such mutation sites include at least a site in the protein coding region, a cysteine residue corresponding to the 265th cysteine residue of AtPTP1, and / or an asparagine residue corresponding to the 234th aspartic acid residue of AtPTP1. The part to include is illustrated. The number of mutations to be introduced may be one or more. The upper limit is usually 50, 40, 30, 30, 20, 10 or less, preferably 5 or less, more preferably 4 or less, and even more preferably 3 or less. And even more preferably no more than 2 and particularly preferably 1. Examples of the introduced mutation include a frameshift mutation, a nonsense mutation, and a missense mutation. Among these, a frameshift mutation or a nonsense mutation is preferable.

The following method can be illustrated as a preferable method for producing a plant in which the activity of the protein tyrosine phosphatase gene is suppressed by introducing a mutation into the protein tyrosine phosphatase gene. However, the method for producing a plant in which the activity of the endogenous protein tyrosine phosphatase gene is suppressed by introducing a mutation into the protein tyrosine phosphatase gene is not limited thereto. Hereinafter, in the present specification, a cysteine residue corresponding to the 265th cysteine residue of AtPTP1 and an aspartic acid residue corresponding to the 234th aspartic acid residue of AtPTP1 in the plant protein tyrosine phosphatase are referred to below. They are sometimes referred to as “cysteine residues involved in enzyme activity” and “aspartic acid residues involved in enzyme activity”, respectively:
(1) By deleting a DNA sequence containing at least a sequence (codon) encoding a cysteine residue involved in enzyme activity or an aspartic acid residue involved in enzyme activity in a protein tyrosine phosphatase gene of a plant A plant in which the activity of the endogenous protein tyrosine phosphatase gene is significantly suppressed can be produced;
(2) A base substitution in a sequence (codon) encoding a cysteine residue involved in enzyme activity or an aspartic acid residue involved in enzyme activity in a plant protein tyrosine phosphatase gene. Thus, by introducing a mutation that results in an amino acid substitution in the protein as a result of the base substitution, a plant body in which the activity of the gene is greatly suppressed can be produced;
(3) A cysteine residue involved in the enzyme activity from the amino terminus of the protein tyrosine phosphatase encoded by the gene relative to the plant protein tyrosine phosphatase gene By introducing a nonsense mutation or a frameshift mutation in the region encoding the amino acid sequence up to the base, a plant body in which the activity of the protein tyrosine phosphatase gene is greatly suppressed can be produced;
(4) The amino acid sequences of protein tyrosine phosphatases of many plant species have high homology, and amino acid residues other than cysteine residues involved in enzyme activity or aspartate residues involved in enzyme activity Is also presumed to be important for the enzyme activity of the enzyme. Therefore, the activity of protein tyrosine phosphatase can also be suppressed by mutations that cause substitution or deletion of these amino acid residues.
By the methods (1) to (3) above, the amino acid residue contained in the protein tyrosine phosphatase, the cysteine residue corresponding to the 258th cysteine residue of OsPTP1, and / or the 166th position of OsPTP2 A codon mutation of a protein tyrosine phosphatase gene encoding a cysteine residue corresponding to the cysteine residue of can be introduced.

  The mutation at the target site of the protein tyrosine phosphatase gene is, for example, TALEN (Japanese Patent Publication No. 2012-514976, Japanese Patent Publication No. 2013-513389), Clusters of legally spaced short repeats / Cas9 (CRISPR / Cas9). ) (Jinek et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science 337, 816-821 (2012); Mali et al., RNA-guided human 9 , Site-specific DNA-degrading enzymes exemplified by zinc finger nucleases (Patent Nos. 4350907 and 4555292) are introduced into plant cells, and protein tyrosine dephosphorylating enzymes are produced by these site-specific DNA-degrading enzymes. It can be introduced by causing double-strand breaks of DNA at a desired position in the gene. This is because, as a result of repair of the double-strand break, base substitution, base deletion, or base insertion occurs at or near the cleavage site with a relatively high probability.

  TALEN is a region referred to as transcription activation-like effect repeats (TALE repeats) that specifically binds to a DNA having a nuclear translocation signal sequence from the N-terminal side, usually 14 to 20 bases in length, and a restriction enzyme It is a protein consisting of a DNA degrading enzyme region derived from Fok1. Usually, the first recognition sequence and the second recognition sequence are set in the genome sequence on both sides of the target site to be introduced with the mutation. The length of the first recognition sequence and the second recognition sequence is usually 14 to 20 bases, respectively. The distance between the first recognition sequence and the second recognition sequence is usually 14 to 18 bases. By introducing the first and second TALENs having the activity of specifically binding to the first recognition sequence and the second recognition sequence, respectively, into the plant cell, the first TALEN and the second TALEN are respectively Specifically binds to the first recognition sequence and the second recognition sequence, resulting in double-strand breaks in DNA between the first recognition sequence and the second recognition sequence. Thereby, base substitution, base deletion or insertion can be introduced into the site (Japanese Patent Publication No. 2012-514976; Japanese Patent Publication No. 2013-513389; Wei et al., Journal of Genetics and Genomics 40, 281-289). (2013); Zhang et al., Plant Physiology 161, 20-27 (2013)). Since the first and second recognition sequences can be set basically without any sequence restriction, TALEN can cause double-strand breaks in DNA at any position on the genomic DNA. Usually, DNA cleavage by TALEN occurs only at one location in the genomic DNA due to the high sequence specificity of TALEN. Further, a TALE repeat having an activity of specifically binding to the set first or second recognition sequence is designed based on a known method (Japanese Patent Publication No. 2012-514976; Japanese Patent Publication No. 2013-513389). A person skilled in the art can produce a vector expressing TALEN having the designed TALE repeat using techniques well known to those skilled in the art. Currently, in Japan, Wako Pure Chemical Industries, Ltd., Life Technologies Japan Co., Ltd. and others provide contract manufacturing services for TALEN, which specifically bind to the first and second recognition sequences, respectively. Production of the first and second TALENs can also be commissioned.

  A zinc finger nuclease is a protein comprising a region called a zinc finger region having an activity of specifically binding to DNA having a specific sequence, as in TALEN, and a DNA degrading enzyme region derived from the restriction enzyme Fok1 (Patent No. 4350907). No., Japanese Patent No. 4555292). As in the case of TALEN, both the first zinc finger nuclease that specifically binds specifically to the first recognition sequence and the second zinc finger nuclease that specifically binds to the second recognition sequence are planted together. By introducing into a cell, a mutation can be introduced into a target site on the genomic DNA.

  As a method for introducing TALEN or zinc finger into a plant cell, a method of transforming a plant cell with an expression vector that expresses these proteins in the plant cell is usually used, and a messenger RNA encoding these proteins. Alternatively, TALEN or zinc finger can be introduced into plant cells by injecting these proteins themselves into cells.

  In the method using CRISPR / Cas9, a sequence having a length of 20 bases is usually set on the 5 ′ side of the position to introduce a mutation on genomic DNA, and a guide RNA having a sequence complementary to this sequence is used. An expression vector that expresses a protein in which a nuclear translocation signal is added to Cas9, which is a DNA-degrading enzyme derived from bacteria, and an expression vector that expresses a protein are added into plant cells, whereby the complex of guide RNA and Cas9 is introduced into the recognition sequence. This is a method of causing DNA double strand breaks near the 3 ′ end of the above recognition sequence by specifically binding to (Jinek et al., A programmable dual-RNA-guided DNA endocrinese in adaptive bacterial affinity. Science 33. 7, 816-821 (2012); Mali et al., RNA-guided human gene engineering via Cas 9. Science 339, 823-826 (2013)). By this method, a mutation is introduced in the vicinity of the cleavage site of genomic DNA as in the case of using TALEN.

  Thus, for example, a protein tyrosine encoded by a gene that is a mutation in the protein coding region of the gene relative to a plant protein tyrosine phosphatase gene using a site-specific DNA degrading enzyme such as TALEN. To introduce a nonsense mutation or a frameshift mutation in the region encoding the amino acid sequence from the amino terminus of phosphatase to the cysteine residue involved in the enzyme activity, a protein tyrosine encoded by the gene on genomic DNA TALEN designed to cleave DNA in a site-specific manner within the region encoding the amino acid sequence from the amino terminus of phosphatase to the cysteine residue involved in enzyme activity was introduced into plant cells or plants Later, the cleavage site that occurred during DNA break repair The base substitutions at the vicinity thereof may be selection of plants cells or plants having a nonsense mutation or frameshift mutation is the result of the deletion or insertion of bases.

  So far, modification of endogenous genes using TALEN has been reported for Arabidopsis, tobacco, rice, and Brachypodium distantachon (Chen K. et al., TALENs: Customizable molecular DNA ssisoesme. of Genetics and Genetics 40, 271-279 (2013)). So far, modification of endogenous genes using zinc finger nuclease has been reported for Arabidopsis, soybean, tobacco, and corn. (For Arabidopsis and soybeans, Sander J. et al., Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nature Methods 8, 67-69 (11); Rapid “open-source” engineering of customized zinc-finger nuclease for high efficient gene modification. Molecular Cell 31, 294-301 (2008); ification in the crop species Zea mays using zinc-finger nuclease.Nature 459,437-441 (2009)). Therefore, when TALEN is used in the present invention, the conditions described in these documents may be applied.

  In addition, as a method of obtaining a plant having a mutation at a specific site on the genome, for example, a specific site within a protein tyrosine phosphatase gene, genomic DNA of the plant is obtained by irradiation with a high-energy electromagnetic wave or treatment with a mutagenic substance. And a method of selecting a plant individual having a mutation at the specific site after introducing a random mutation. In order to select a plant individual having a mutation at the specific site, first, genomic DNA is extracted from a plurality of plant individuals subjected to the mutagenesis treatment according to a method well known to those skilled in the art, DNA containing the sequence of the specific site as a template is amplified by PCR. The presence or absence of mutations in DNA amplified by PCR can be examined, for example, by determining the base sequence of the amplified DNA. Alternatively, the mutation can be rapidly detected by the TILLING method (Patent No. 4213214) using CelI nuclease derived from celery.

  So far, Arabidopsis, Oven (Avena sativa), Orchid (Brassica oleracea), Rapeseed (Brassica rapa), Melon, Soybean, Barley, Lotus japonicus, Rice, Tomato, Sorghum, Wheat, etc. And the selection of mutants using the TILLING method has been carried out, and as a result, many mutant plants having mutations in the endogenous gene have been obtained (Kurowaka, M. et al.). TILLING-a shortcut in functional genomics.Journal of Applied Genetics 52, 371-390 (2011)).

  A plant having a mutation in the protein tyrosine phosphatase gene obtained in this way is crossed with a wild type plant, and a plant that is a progeny plant and has a mutation in the protein tyrosine phosphatase gene is selected. By repeating this crossing with a wild type plant, a plant having a mutation in the protein tyrosine phosphatase gene but having the DNA sequence of the other genomic region derived from the wild type plant can be produced. .

  When the protein tyrosine phosphatase is OsPTP1, one or more residues (for example, 228th, 234th, 258th) from the amino terminus of the amino acid sequence of SEQ ID NO. It is preferable that a mutation is introduced into the residue. Further, it is preferable that a mutation is introduced into at least the 228th aspartic acid residue and / or the 258th cysteine residue, more preferably a mutation is introduced into at least the 258th cysteine residue, and at least the 228th position. It is more preferable that the aspartic acid residue and / or the 258th cysteine residue are substituted with other residues (eg, aspartic acid residue and serine residue, respectively), and at least the 258th cysteine residue is other. Even more preferably, it is substituted with a residue (for example, a serine residue). When the protein tyrosine phosphatase is OsPTP2, it is mutated to one or more residues contained in the amino acid sequence of SEQ ID NO: 2 at positions 1 to 166, particularly 136 to 166 (for example, the 136th and 166th positions). Residues) are preferably introduced. Further, it is preferable that a mutation is introduced into at least the 136th aspartic acid residue and / or the 166th cysteine residue, more preferably a mutation is introduced into the 166th cysteine residue, and at least the 136th cysteine residue. More preferably, the aspartic acid residue and / or the 166th cysteine residue is substituted with another residue (for example, an alanine residue or a serine residue, respectively), and the 166th cysteine residue is another residue. Even more preferably, it is substituted with (for example, a serine residue). In the above description, the reason why the C-terminal of the preferable mutagenesis site is the 166th in SEQ ID NO: 2 is that the activity is lost when the 265th cysteine of AtPTP1 (the 258th in OsPTP1) is mutated. . Similarly, the fact that the C-terminal of the mutagenesis site is the 136th position of SEQ ID NO: 2 is based on the fact that it corresponds to the 234th aspartic acid of AtPTP1 (the 228th position in OsPTP1).

  Even if the protein tyrosine phosphatase is an enzyme other than OsPTP1 and OsPTP2, it is an amino acid residue contained in the enzyme, and corresponds to the 258th cysteine residue of OsPTP1, and / or OsPTP2 It is also preferable that a mutation including at least a codon mutation of a protein tyrosine phosphatase gene (for example, a mutation to a serine residue), which encodes a cysteine residue corresponding to the 166th cysteine residue, is introduced. . In the region encoding the protein tyrosine phosphatase gene from the amino terminus of the protein coding region to the cysteine residue corresponding to the 258th cysteine residue of OsPTP1 and / or the 166th cysteine residue of OsPTP2. It is preferred that a mutation is introduced.

  Examples of the compound that binds to protein tyrosine phosphatase include compounds that specifically bind to protein tyrosine phosphatase, an antibody against protein tyrosine phosphatase (eg, monoclonal antibody), a part of the antibody (Eg, variable regions) are preferred.

  A plant activator means a substance that enhances the immunity of a plant and improves disease resistance. In the present invention, the plant activator is preferably related to the activation of MAP kinase, more preferably related to the activation of MAP kinase 6, and indirectly by activating MAP kinase kinase together with salicylic acid. More preferred are those involved in activation and indirectly involved in activation of MAP kinase 6 through activation of MAP kinase kinase by activation of salicylic acid by activating the salicylic acid pathway. Examples of such plant activators include benzothiadiazole (BTH), probenazole (PBZ), validamycin A (VMA), validoxylamine A (VAA), thiazinyl, and isothianyl.

  Although the causative factor of a disease is not specifically limited, As a causative factor, a filamentous fungus, bacteria, and a virus are illustrated. Examples of the filamentous fungi include algae (flagellate, zygomycetes, etc.), incomplete fungi, ascomycetes, basidiomycetes, and the like. The pathological condition in the disease of filamentous fungi includes formation of black grains (mycorrhiza (mycelium lump)), formation of lesions, decay of stems and leaves, withering, and formation of bumps. The diseases of filamentous fungi in rice include rice blast, brown rice disease, brown sclerotia, yellow dwarf disease, brown leaf blight, cinnamon mold, staphylococcal sclerosis, black smut, red sclerotia. Illustrated. Examples of bacterial diseases include the formation of yellowish discolored spots, the decay of leaves and stems, withering, and the formation of tumorous tumors. Examples of diseases caused by bacteria in rice include downy mildew, brown streak disease, white leaf blight, inner brown browning disease, blight blight and seedling blight. Examples of diseases caused by viruses include mosaic symptoms (light spots on flowers or leaves), malformation (atrophy, deformation, etc.), reversal of small brown gangrene, growth inhibition (yellowing and downsizing of the whole strain). The Examples of diseases caused by viruses in rice include black stripe atrophy, transition yellowing disease, and dwarf disease.

  The disease targeted in the present invention is preferably a disease caused by a filamentous fungus and a disease caused by a bacterium, more preferably blast, mildew, powdery mildew, and rust, and more preferably blast. In the present invention, the target disease may be one type or two or more types.

  The disease resistant plant of the present invention may be a plant itself (first generation) that has been subjected to a treatment for suppressing the expression or activity of one or more protein tyrosine phosphatase genes. As long as the expression or activity of the phosphatase gene is suppressed, it may be a progeny after the second generation.

  The plant of the present invention may be the whole plant, or a part or a processed product. Some plant parts include organs (eg, roots, stems, leaves, flowers, seeds, fruits), tissues (eg, epidermis, sieves, soft tissues, xylem, vascular bundles, hairy roots), cells, cells Examples (eg, protoplasts), cultured cells and tissues (eg, callus, shoot primordia, shoots, polyblasts) are exemplified. The processed plant products are usually processed food products and processed food products, and are preferably processed seed products. Examples of processed rice products include cooked rice, rice cake, fried rice, rice flour, white flour, confectionery (rice crackers, arabe, cookies, etc.), sake, vinegar, oil, and rice bran.

  According to the present invention, there is provided a disease resistance-imparting composition comprising, as an active ingredient, one or more protein tyrosine phosphatase gene expression or activity inhibitors. Thereby, disease resistance can be efficiently provided to a plant. The component of the protein tyrosine phosphatase gene expression or activity inhibitor is not particularly limited as long as it can suppress the expression or function of the protein tyrosine phosphatase gene. However, a nucleic acid that causes RNA interference (protein tyrosine phosphatase) Examples include short interfering RNA (siRNA) that binds complementarily to mRNA of a gene or double-stranded RNA containing the same, mutation introduction kits, nucleotides such as ribozymes and antisense nucleotides, and proteins such as antibodies, and siRNA is preferred. . In the disease resistance-imparting composition, components other than the active ingredient are not particularly limited, and examples thereof include a carrier (base) for the expression or activity inhibitor to exert its function in plants, specifically, , Preservatives, stabilizers, excipients, buffers, solvents, colorants, binders.

  According to the present invention, disease resistance can be imparted to a plant by suppressing the expression or activity of one or more protein tyrosine phosphatase genes contained in the plant. The method for suppressing the expression or activity of the enzyme gene is usually an artificial method, and examples and preferred examples thereof are as described above.

  In the present invention, a plant having disease resistance can be produced by suppressing the expression or function of one or more protein tyrosine phosphatase genes contained in the plant. In the production method of the present invention, a plant cell (for example, callus) or plant tissue subjected to a treatment for suppressing the expression or activity of one or more protein tyrosine phosphatase genes is obtained, and the plant body from the plant cell The method of reproducing | regenerating is illustrated. The treatment for suppressing the expression or activity of the enzyme gene is usually an artificial treatment, and examples and preferred examples thereof are as described above for the method for suppressing the expression or activity of the gene.

  In the present invention, the conditions for growing a disease-resistant plant are not particularly limited, and may be the same as those for normal plant growth, but it is preferable to perform a plant activator treatment. Thereby, the transcription | transfer of WRKY45 in a salicylic acid pathway is accelerated | stimulated, and disease resistance can be improved. Moreover, it is preferable that temperature conditions are low temperature. Since the disease-resistant plant of the present invention suppresses MAP kinase activation inhibition caused by environmental conditions such as low temperature in the salicylic acid pathway as occurs in normal plants, the object of the present invention is sufficiently exerted. Because. According to the present invention, not only the plant itself but also its progeny or clone can be obtained by the above production method or breeding method, and the progeny and clone also express or express one or more protein tyrosine phosphatase genes. It can be used as a plant having disease resistance in which is suppressed.

  In the present invention, a disease resistant plant can be selected by determining whether the expression or activity of one or more protein tyrosine phosphatase genes contained in the plant is suppressed. That is, in the sample plant, if the expression or activity of one or more protein tyrosine phosphatase genes is suppressed compared to the control plant, the plant is selected as a plant having disease resistance. can do. Thereby, the plant which has disease resistance can be selected easily, and agricultural production can be made efficient.

  The present invention will be specifically described with reference to examples. The description of the materials and methods used in the examples are summarized in the latter part.

Example 1 [Modulation of Salicylic Acid Pathway by MAP Kinase]
In rice, it was confirmed by the following test that phosphorylation of rice MAP kinase 6 (OsMPK6) mediates salicylic acid (SA) signaling.

(1A) The following test was conducted to elucidate SA-responsive phosphorylation of endogenous OsMPK6 in rice. Rice callus and leaves were treated with 1 mM SA, and a protein having a double phosphorylated TEY motif and a protein having OsMPK6 were detected by an anti-pTEpY antibody and an anti-OsMPK6 antibody, respectively. The anti-pTEpY antibody is phosphor-specific and recognizes a dual phosphorylated TEY signature sequence.

  One of the bands (arrowhead in FIG. 2) represented OsMPK6. This is because the osmpk6 gene mutant does not show the band, and the anti-OsMPK6 antibody detects a band having the same mobility (in electrophoresis). The result of (1A) shows that SA induces phosphorylation of OsMPK6.

  The bands that migrated earlier in FIG. 2 (* and ** in FIG. 2) were presumed to be another double phosphorylated MAPK, and this band was present in the osmpk6 gene mutant callus. This suggests a mechanism that compensates for the loss of OsMPK6.

(1B) Rice MAP kinase kinase 10-2 (OsMKK10-2), which is one of rice MAPK kinases, phosphorylates and activates OsMPK6 in vitro. Therefore, it was investigated which site of OsMKK10-2 phosphorylates OsMPK6.

WT with maltose binding protein (MBP) labeled OsMPK6 (MBP-MPK6), mutant K96R with kinase activity of MBP-MPK6, other mutants Y227D and T225A, and three mutations Mutant K96R / Y227D / T225A was prepared, and each was treated with a constitutively active form of glutathione-S-transferase-labeled OsMKK10-2 (GST-MKK10-2D). The presence or absence was confirmed. K96R is a mutant form in which Lys ^{96 of} MBP-MPK6 is substituted with Arg. Y227D and T225A are mutant types in which Thr ²²⁵ and Tyr ²²⁷ of MBP-MPK6 are substituted with Asp, respectively. K96R / Y227D / T225A is a mutant form of MBP-MPK6 in which Lys ⁹⁶ is replaced with Arg, Thr ²²⁵ is replaced with Asp, and Tyr ²²⁷ is replaced with Asp.

As a result, the constitutively activated form of GST-MKK10-2D phosphorylated MBP-MPK6 (WT), K96R, Y227D and T225A (FIGS. 3 and 4), but both Thr ²²⁵ and Tyr ²²⁷ were replaced. The mutant K96R / Y227D / T225A was not phosphorylated (FIG. 3). The result of (1B) indicates that OsMKK10-2 double phosphorylates the TEY signature in OsMPK6 but does not phosphorylate other sites.

(1C) The following test was performed to confirm whether OsMKK10-2D phosphorylates the TEY motif. A transgenic plant (GVG-MKK10-2D) in which OsMKK10-2D can be induced by dexamethasone (Dex) was created. Two independent strains (# 3 and # 14) of GVG-MKK10-2D were treated with Dex, respectively, and each protein was identified by immunoblotting with anti-pTEpY antibody and anti-OsMPK6 antibody. In FIG. 5, “mock” indicates a result of an extract from a plant that was subjected to the same treatment except that the Dex treatment was not performed. In leaves, the amount of phosphorylated OsMPK6 protein increased during Dex treatment (FIG. 5).

(1D) Subsequently, GVG-MKK10-2D plants (lines # 3 and # 14) were treated with Dex or mock, and the relative level of the WRKY45 transcription level to the ubiquitin transcription level was measured by RT-qPCR. The transcription level of WRKY45 increased in parallel with the amount of phosphorylated OsMPK6 protein (FIG. 6). Note that the same results were obtained in two independent experiments.

  The results of (1C) and (1D) indicate that OsMKK10-2D phosphorylates the TEY motif to activate OsMPK6 and activate WRKY45.

(1E) GVG-MKK10-2D plants that have been pre-treated with Dex or not pre-treated with Dex, oryzae was inoculated. As a result, the Dex-treated GVG-MKK10-2D plant showed stronger blast resistance than the control plant (FIG. 7). The result of (1E) shows that OsMKK10-2D expression induces blast resistance.

  Since OsMPK6 phosphorylates WRKY45, the results of (1A) to (1E) suggest that phosphorylation / activation of OsMPK6 by OsMKK10-2D mimics activation of the SA pathway and has an equivalent effect And lead to activation of the WRKY45-dependent defense response.

Example 2 [Regulation of Salicylic Acid Pathway by Protein Tyrosine Dephosphorylating Enzyme]
In the response to abscisic acid (ABA), it was confirmed in the following test that protein tyrosine phosphatase 1 (OsPTP1) and protein tyrosine phosphatase 2 (OsPTP2) dephosphorylate OsMPK6.

(2A) In order to verify the influence of ABA on phosphorylation of OsMPK6, a GVG-MKK10-2D rice plant (line # 3) that had been pretreated with 10 μM ABA or untreated was treated with 10 μM Dex. The transcription levels of MKK10-2D and WRKY45 (relative to the transcription level of ubiquitin) were analyzed by RT-qPCR. In order to further investigate the effect of ABA on the phosphorylation state of OsMPK6, phosphorylation of Thr and Tyr was carried out using specific antibodies against the respective phosphorylated amino acids (anti-phosphorylated Thr antibody and anti-phosphorylated Tyr antibody), respectively. analyzed.

  Double phosphorylation almost disappeared in the presence of ABA, and at the same time the transcription level of WRKY45 decreased (FIGS. 8 to 10). This suggests that ABA antagonizes SA-responsive OsMPK6 phosphorylation. It was also found that phosphorylated tyrosine residues decreased but phosphorylated threonine residues did not decrease (FIGS. 8 and 10).

  From the results of (2A), it is clear that OsMKK10-2D-induced WRKY45 transcription and TEY phosphorylation are reduced by ABA treatment.

(2B) WT rice plants were treated with 1 mM SA and 30 or 100 μM ABA in the presence or absence of 50 μM Bay11-7082, or in the presence or absence of 2 mM vanadic acid.

  PTPase inhibitors vanadic acid and Bay11-7082 (see N. Krishnan, G. Bencze, P. Cohen, N.K. Tonks, FEBS J, (2013).) Counteracted ABA-responsive suppression of WRKY45 expression. (FIGS. 11 and 12). The result of (2B) suggests that PTPase is involved in OsMKK10-2D-induced WRKY45 transcription suppression by ABA treatment.

  When the results of (2A) to (2B) are combined, it is suggested that tyrosine dephosphorylation of OsMPK6 by PTPase is involved in suppression of SA signaling by ABA.

Example 3 [Inhibition of protein tyrosine phosphatase gene expression by RNA interference]
It was examined whether the ABA-induced suppression of the WRKY45 defense response was mitigated by RNA knockdown of OsPTP1 / 2.

(2C) The rice genome is OsPTP1 (Rice Annotation Project Database (RAP Database: http://rapdb.dna.affrc.go.jp/), MSU RicenProGet jp :). LOC_Os12g07590) and OsPTP2 (LOC_Os11g07850.1) in //rice.plantbiology.msu.edu/analyses_search_loc.shtml). In order to investigate their function in the dephosphorylation of OsMPK6 caused by ABA, in order to produce a transgenic rice in which both OsPTP1 and OsPTP2 genes are knocked down by RNA interference (OsPTP1 / 2 double knockdown), the following conditions are used: The gene construct PTP-wkd was prepared in (Fig. 13). The DNA sequence of the 3 ′ untranslated region of the OsPTP1 gene is expressed as the u7 primer (5′-CACCCGGGTATCCCTAAGGCAGGA-3 ′: SEQ ID NO: 7) and the u8 primer (5′-AAATGAATTCAGTTTAACCACTACTACTACTCTTTAATTCCGT-3 ′: SEQ ID NO: 8), OsPTP2 gene The DNA sequence of the translation region was PCR amplified using the u9 primer (5′-TTAGTAGTTTAAACTGAATCATTTCTATGAGAACAATCAGT-3 ′: SEQ ID NO: 9) and the u10 primer (5′-AGGCCTGGGTGGGCAGGAGAAGCG-3 ′: SEQ ID NO: 10). Next, these two reaction products are mixed and further PCR amplified using u7 and u10 primers to obtain a DNA fragment in which the 3 ′ untranslated region of the OsPTP1 gene and the OsPTP2 gene is fused. Inserted into pANDA vector via D-TOPO (Miki, D. & Shimamoto, K. Simple RNAi vectors for stable and transient preparation in gene function in 4th, 4th, 4th, 4th, 4th, 4th, 4th, 4th). The prepared construct was transformed into rice through Agrobacterium (Toki, S., Hara, N., Ono, K., Onodera, H., Tagiri, A., Oka, S. & Tanaka, H Early infection of scutellum tissue with Agrobacterium allo high-speed transformation of rice. Plant J. 47, 969-976 (2006)).

(2D) In order to confirm whether PTP knockdown antagonizes SA-induced inhibition of OsMPK6TEY phosphorylation by ABA, the following test was performed. WT rice and OsPTP1 / PTP2 double knockdown rice (PTP-wkd rice) were treated with 1 mM SA in the presence or absence of 100 μM ABA, and phosphorylation of OsMPK6 was immunodetected over time. In the absence of ABA, the TEY phosphorylation level of OsMPK6 increased after SA treatment in WT rice. On the other hand, the TEY phosphorylation level of OsMPK6 in PTP-wkd rice was higher than that in WT rice before SA treatment, and was maintained after SA treatment. In the presence of ABA, TEY phosphorylation level of OsMPK6 did not increase in WT rice even after SA treatment, whereas in PTP-wkd rice, it significantly increased after SA treatment (FIG. 15). .

Example 4 [Inhibition of protein tyrosine phosphatase gene activity by mutagenesis]
It was examined whether or not the tyrosine dephosphorylation activity of OsMPK6 was lost by introducing a mutation into OsPTP1 / 2.

A DNA consisting of the 1st to 987th base sequences of the base sequence of SEQ ID NO: 3 and a DNA consisting of the 1st to 714th base sequences of the base sequence of SEQ ID NO: 4 were respectively prepared as multis of pMAL-c5x (New England Biolabs, Inc.). It inserted in the cloning site and produced pMAL-PTP1 (WT) and pMAL-PTP2 (WT). pMAL-PTP1 (WT) expresses a fusion protein (MBP-PTP1 (WT)) in which the full length of OsPTP1 is fused to the C-terminal side of maltose-binding protein, and pMAL-PTP2 (WT) is the C-terminal of maltose-binding protein. A fusion protein (MBP-PTP2 (WT)) in which the full length of OsPTP2 is fused to the side is expressed. from pMAL-PTP1 (WT) and pMAL-PTP2 (WT) to a fusion protein of OsPTP1 and maltose binding protein (MBP-PTP1CS) containing a Cys to Ser substitution and OsPTP2 and maltose containing a Cys to Ser substitution Expression vectors pMAL-PTP1CS and pMAL-PTP2CS, each expressing a fusion protein (MBP-PTP2CS) with a binding protein, were prepared according to the manual of QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene) according to the conditions at the latter stage. Of MBP-PTP1 in vitro (WT), the MBP-PTP2 (WT) or variants thereof (MBP-PTP1CS and MBP-PTP2CS) in accordance MBP-MPK6 and phosphorylation activity of MBP-WRKY45, the respective protein ³² Observed by P-bonding. In MBP-PTP1CS, a mutation is introduced into the 258th Cys of OsPTP1. In MBP-PTP2CS, a mutation is introduced into the 166th Cys of OsPTP2.

  The amount of MBP-MPK6 phosphorylated by GST-MKK10-2D was significantly reduced in the presence of MBP-PTP1 (WT) or MBP-PTP2 (WT) compared to the presence of maltose binding protein. (FIG. 16).

  In Arabidopsis PTP1 (AtPTP1), certain Cys residues are essential for activity. In MBP-PTP1 (WT) and MBP-PTP2 (WT), a mutant (MBP-PTP1CS) in which the Cys residues corresponding to Cys of AtPTP1 (258th of OsPTP1 and 166th of OsPTP2; FIG. 14) are replaced with Ser. Alternatively, when phosphorylation assay of MBP-MPK6 by GST-MKK10-2D is performed in the presence of MBP-PTP2CS), phosphorylated MBP-MPK6 does not decrease (MBP-PTP1CS and MBP-PTP2CS in FIG. 16). Of Cys residues of MBP-PTP1 and MBP-PTP2 were shown to be essential for the phosphatase activity of MBP-PTP1 and MBP-PTP2. For example, the dephosphorylation enzyme activity of MBP-PTP1 (WT) represents the activity of the OsPTP1 portion of the fusion protein, and the maltose binding protein portion of the fusion protein is dephosphorylation of MBP-PTP1 (WT). Since it is considered that the enzyme activity is not affected, OsPTP1 and OsPTP2 have the activity to dephosphorylate OsMPK6, and the 258th (OsPTP1) and 166th (OsPTP2) Cys residues are present for the activity. Each was shown to be essential.

  Furthermore, as a result of the bimolecular fluorescence complementation assay, it was found that OsPTP1 and OsPTP2 interact with OsMPK6 in rice cells in rice cells.

  The results of this example show that OsPTP1 and OsPTP2 have the activity to dephosphorylate OsMPK6, and suppress the expression of OsPTP1 and OsPTP2 by RNA interference, thereby inhibiting the dephosphorylation of OsMPK6 by ABA and It shows that it is possible to prevent a decrease in the WRKY45-dependent defense response.

  The cysteine residue corresponding to the 265th cysteine residue of AtPTP1 and the aspartic acid residue corresponding to the 234th aspartic acid residue are not only Arabidopsis thaliana and rice, but also soybean, pea, green beans, tomato, wheat, corn Is completely conserved in the amino acid sequence of the protein tyrosine phosphatase, suggesting that the cysteine residue and the aspartate residue are conserved in the plant protein tyrosine phosphatase ( FIG. 19). When the results of Example 4 and these findings are combined, a cysteine residue corresponding to the 265th cysteine residue of AtPTP1 and the 234th aspartic acid residue of AtPTP1 in protein tyrosine phosphatases of many plant species Aspartate residues corresponding to the phosphatase activity of the enzyme are essential for the enzyme dephosphorylating enzyme activity. Therefore, in these enzymes, the cysteine residue corresponding to the 265th cysteine residue of AtPTP1 or the 234th aspartate residue of AtPTP1 The dephosphorylating enzyme activity of the mutant lacking the aspartic acid residue corresponding to the group or the mutant in which the cysteine residue or the aspartic acid residue is substituted with another amino acid residue is the activity of the wild-type enzyme It strongly suggests that it is greatly reduced or almost disappeared.

Example 5 [Specificity of regulation by PTP knockdown]
It was examined by the following test how knockdown of OsPTP1 / 2 affects the inhibition of WRKY45 expression induction by SA by ABA.

  After treating WT and PTP-wkd with 1 mM SA and 0 or 30 μM ABA, each transcription level of WRKY45, OsPTP1 and OsPTP2 (relative to ubiquitin) was investigated by RT-qPCR using RNA extracted from leaves. did.

  In WT plants, when treated with SA and ABA, the WRKY45 transcription level was significantly reduced compared to treatment with SA alone, whereas in PTP-wkd plants, when treated with SA and ABA, The degree of decrease in the WRKY45 transcription level relative to the case of treatment with SA alone was small (FIG. 17). This result shows that suppression of WRKY45 expression induction by SA by ABA is reduced in PTP-wkd plants.

Example 6 [resistance to blast disease]
Benzothiadiazole (BTH) is a kind of plant activator that activates defense responses in various plants including rice (M. Shimono et al., Plant Cell 19, 2064 (2007)., Y. Ueno et al.). al., Plant Signal Behav. 8, e24510 (2013) and J. Gorlach et al., Plant Cell 8, 629 (1996).). Therefore, the effect of protein tyrosine phosphatase gene knockdown on BTH-induced blast resistance in rice under various conditions was examined.

  WT rice (variety: Nipponbare) (Oryza sativa subsp. Japonica cv. Nipponbare), PTP-wkd rice, and WRKY45-overexpressing rice (W45-ox) were used as experimental materials. W45-ox is an M.I. Shimono et al. , Plant Cell 19, 2064 (2007). Each was sown in the soil in the greenhouse (Bonsol No. 2; Sumitomo Chemical Co., Ltd.) and grown for one and a half months. The growth temperature was 30 ° C./26° C. (day / night), and the relative humidity (RH) was about 60%.

  The leaf blades were cut out from each plant body, put into a 50 ml Falcon tube containing 10 ml of a drug solution described later, and sealed with surgical tape (3M; cat # 3530-1).

  The drug solution was a 0.1 × MS salt solution containing 10 μM BTH, a 0.1 × MS salt solution containing 10 μM BTH, 10 μM ABA and 100 μM fluridone, or a 0.1 × MS salt solution (Mock). Thereafter, the rice leaf blades in the falcon tube were treated as follows.

  Normal temperature (30 ° C) conditions: A 50 ml tube containing either rice leaf blades or each drug solution is grown in a growth chamber (light period: 14 hours at 30 ° C, dark period: 10 hours at 26 ° C; 60% relative humidity ) For 1 day. The drug solution was removed, and rice shoots were inoculated with blast fungus. Blast Fungus M. The culture of oryzae [Magnaporte oryzae] (race003) and the inoculation of rice plants were carried out in accordance with the description in Reference 6. Specifically, conidia of blast fungus were suspended in 0.02% Tween 20 to a concentration of 150,000 spores / ml and sprayed on rice leaves. 10 ml of the drug solution was added to the 50 ml tube, sealed with surgical tape, and placed in a 24 ° C. inoculation box (dark place) for 1 day. The drug solution was removed and 10 ml of 0.1 × MS salt solution was added, and then further 5 days in a growth chamber (light period: 30 ° C. for 14 hours, dark period: 26 ° C. for 10 hours; 60% relative humidity). After placement, DNA was extracted from the rice leaf blades.

  Low temperature (15 ° C.) conditions: A 50 ml tube containing rice leaf blades and each drug solution is placed in a growth chamber (light period: 14 hours at 15 ° C., dark period: 10 hours at 9 ° C .; 60% relative humidity ) For 2 days. The drug solution was removed and rice leaf blades were inoculated with blast fungus under the above conditions. 10 ml of the drug solution was added to the 50 ml tube, sealed with surgical tape, and placed in a 24 ° C. inoculation box (dark place) for 1 day. The 50 ml tube was placed in a growth chamber (light period: 14 hours at 15 ° C., dark period: 10 hours at 9 ° C .; 60% relative humidity) for an additional 3 days. The drug solution was removed and a 0.1 × MS salt solution was added and placed in a growth chamber (light period: 30 ° C. for 14 hours, dark period: 26 ° C. for 10 hours; 60% relative humidity) for 5 days. Thereafter, DNA was extracted from the rice leaf blades.

  Using DNA extracted under the respective conditions, Quantification of oryzae 28S rDNA was performed. The progression of blast was evaluated by quantifying by qPCR in general accordance with Reference 13. Each disease resistance assay was repeated three or more times.

  At 30 ° C., BTH induced strong blast resistance in WT rice. On the other hand, PTP knockdown did not affect blast resistance with or without BTH pretreatment (FIG. 18). In WT rice, ABA co-treatment strongly inhibited BTH-induced blast resistance (FIG. 18). However, in PTP-wkd rice, the effect of this ABA was dramatically weakened, and showed strong blast resistance comparable to that of rice plants overexpressing WRKY45 (FIG. 18). These results are consistent with the role of PTPases in mediating ABA antagonism of SA signaling. In addition, M.M. When a low-temperature treatment at 15 ° C. was performed before and after oryzae inoculation, WT rice plants showed high susceptibility to blast fungus even in the presence of BTH (FIG. 18). On the other hand, in PTP-wkd rice similarly treated at low temperature, BTH induced strong blast resistance at the same level as WRKY45 overexpressing rice. Based on all of these results, PTPase undergoes ABA signaling induced by cold to dephosphorylate and inactivate OsMPK6, thereby mediating antagonism of ABA signals to WRKY45-dependent SA pathway defense responses I think so.

[Plants and growth conditions]
Rice (Oryza sativa subsp. Japonica cv. Nipponbare) was grown on soil in the greenhouse (Bonsol No. 2; Sumitomo Chemical Co., Ltd.). The temperature during growth was 30 ° C./60° C. (day / night), and the relative humidity (RH) was about 60%.

(Construction of plasmid)
Site-directed mutagenesis was performed using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) according to its instructions. To obtain GVG-MKK10-2D, OsMKK10-2D cDNA (see Reference 1) was subcloned into the pINDEX vector (see Reference 2). To obtain PTP-wkd, the 3 ′ untranslated regions of the OsPTP1 gene and the OsPTP2 gene were each PCR amplified. The primers used for the amplification of the 3 ′ untranslated region of the OsPTP1 gene were u7 primer (5′-CACCCGGGTATCCCTAAGGCAGGA-3 ′: SEQ ID NO: 7) and u8 primer (5′-AAATGATTCAGTTTAAACCACTACTACTCTCTTTTAATTCCGT-3 ′: SEQ ID NO: 8). . The primers used for the amplification of the 3 ′ untranslated region of the OsPTP2 gene were u9 primer (5′-TTAGTAGGTTTAAACTGAATCATTTCTATGAGAACAACATGT-3 ′: SEQ ID NO: 9) and u10 primer (5′-AGGCCTGGGTGGGCAGGAGAAGAGCG-3 ′: SEQ ID NO: 10). . Next, second-stage overlap PCR was performed using the u7 primer, u10 primer and the reaction product of the first-stage reaction. The fusion gene amplified by this reaction was cloned into pENTR / D-TOPO (Invitrogen) and subsequently introduced into a pANDA vector (see Reference 3).

[Rice transformation]
Rice callus was subjected to Agrobacterium transformation as described in References 4 and 5. Plants were regenerated from transformed calli by selection for hygromycin resistance.

[Chemical processing of leaves for protein or RNA analysis]
The chemical treatment of the leaves was carried out in accordance with the description in Reference 6. Leaf blades collected from the four-leaf rice were cut into pieces about 0.5 cm in length and submerged in a solution containing chemicals prepared in 0.002% Silwet L-77. The leaf fragments were incubated in the light at 30 ° C. for a certain time. Abscisic acid (ABA) was administered one hour prior to salicylic acid (SA) or dexamethasone (Dex) treatment. PTPase inhibitor was administered an additional hour prior to ABA administration. The treatment amount of SA was 1 mM.

[Immunoblot assay]
The double phosphorylated TEY motif and OsMPK6 contained in the protein were identified by immunoblot assay.

The immunoblot assay was generally performed according to the description in Reference 7. Extracted Complete Protein Inhibitor Cocktail (Roche Diagnostics), Protease Inhibitor Cocktails 1 and 2 (Sigma), and 50 mM Hepes-KOH containing 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.5. After centrifugation, the supernatant (containing 6-20 μg protein) was subjected to SDS-PAGE and subsequent blotting. The immunoassay was performed using SNAPid (Milipore) according to the instructions. The following antibodies were used:
Anti-pTEpY antibody: Promega, cat # V8031, 1 / 1,500 dilution;
Anti-OsMPK6 antibody: Kishi-Kaboshi, M .; et al. (See Reference 8), 1 / 1,500 dilution;
Anti-PEPC antibody: Ueno, Y. et al. et al. (See reference 9), 1 / 30,000 dilution;
Anti-pY antibody: Millipore, clone 4G10 platinum, 1 / 1,500 dilution;
Anti-pT antibody: Promega, Anti-pT183 MAPK pAb (Rabbit), 1/4000 dilution; and anti-WRKY45 antibody: Matsushita, A .; et al. (See Reference 7), 1/300 dilution.

[RT-qPCR]
Total RNA was isolated from rice leaves treated with drugs as described above using Trizol reagent (Invitrogen). CDNA was synthesized using RiverTraAce (Toyobo Co., Ltd.). According to the description in Reference Document 10, quantitative PCR was performed with a Thermal CyclerDice TP800 system (Takara Bio Inc.) using SYBR premix ExTaq mixture (Takara Bio Inc.). Table 1 shows the primer sequences used in RT-qPCR.

[In vitro phosphorylation and dephosphorylation assay]
Phosphorylation and dephosphorylation assays were performed as described in References 8 and 11 with some modifications. GST-MKK10-2D and MBP-MPK6 (wild type or mutant type) are reaction buffers (10 mM Hepes-KOH, pH 7.5, 5 mM EGTA, 20 mM MgCl) containing 0.5 mM ATP and 37 kBq [γ- ³² P] ATP. ₂ and 1 mM DTT) for 20 minutes at 25 ° C. To perform the dephosphorylation assay, MBP-PTP1 and MBP-PTP2 were added to the reaction solution, and the reaction mixture was further incubated for 20 minutes. To perform the WRKY45 phosphorylation activity assay, a reaction mixture similar to the above except that [γ- ³² P] ATP is not contained is preincubated for 20 minutes, and then MBP-WRKY45 and 37 kBq [γ- ³² P] ATP are added. The reaction was started. The mixture was further incubated for 20 minutes, followed by boiling after adding Laemmli's sample buffer, after the reaction was terminated, the reaction was analyzed by SDS-PAGE. As a loading control, Coomassie brilliant blue (CBB) staining was performed.

[Bimolecular Fluorescence Complementation Assay]
A bimolecular fluorescence complementation assay was performed as described in reference 12.

Example 7 [Blast resistance after high salt treatment]
WT rice “NB” (variety: Nipponbare) and OsPTP1 / 2 double knockdown rice (PTP-wkd rice) are grown on the soil in the greenhouse (Bonsol No. 2; Sumitomo Chemical Co., Ltd.) until 30 days after sowing. did. The growth temperature was 30 ° C./26° C. (day / night), and the relative humidity (RH) was about 60%. These rice plants were treated with 250 mM aqueous sodium chloride solution for 4 days. The leaf blades of the developed leaves were removed from each plant body, placed in a 50 ml falcon tube containing 10 ml of any of the drug solutions described below, and sealed with surgical tape. The drug solution was a 0.1 × MS salt solution or 0.1 × MS salt solution (Mock) containing 10 μM BTH.

  The leaf blades placed in the 50 ml Falcon tube were inoculated with the blast fungus according to the method described in Example 6, and then DNA was extracted from each rice leaf blade. The extraction of DNA was performed according to a known method (new version plant PCR experiment protocol, Shujunsha, 1997). Using DNA extracted from each treated rice leaf, Quantification of oryzae 28S rDNA was performed. In general, the resistance to blast was evaluated by quantification using qPCR in accordance with Reference 13. Each disease resistance assay was repeated three or more times, and the average value and standard deviation were shown in a graph in FIG.

  As a result, in NB, although strong disease resistance was observed in the presence of BTH without high salt treatment, disease resistance was weakened in the presence of BTH when high salt treatment was performed. In contrast, PTP-wkd rice maintained strong disease resistance in the presence of BTH even under high salt treatment (FIG. 20). This result shows that the present invention can impart high disease resistance to plants even under harsh environmental conditions.

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4). T.A. Fuse, T .; Sasaki, M .; Yano, Plant Biotechnology 18, 219 (2001).
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SEQ ID NO: 1: amino acid sequence of OsPTP1 SEQ ID NO: 2: amino acid sequence of OsPTP2 SEQ ID NO: 3: nucleotide sequence of cDNA of OsPTP1 1-987: coding sequence (including stop codon)
988-1243: 3′-UTR (sequences that can be used for RNAi vectors)
SEQ ID NO: 4: Base sequence of cDNA of OsPTP2 1-714: Coding sequence (including stop codon)
715-989: 3′-UTR (Sequences that can be used for RNAi vectors)
SEQ ID NO: 5: Base sequence of genomic DNA of OsPTP1 1-1000: RdDM target region 1001-1368: first exon (translation start point 1172)
1369-2589: first intron 2690-2778: second exon 2779-2893: second intron 2894-2978: third exon 2979-3073: third intron 3074-3208: fourth exon 3209-4556: fourth intron 4557 -4683: 5th exon 4684-5171: 5th intron 5172-5314: 6th exon 5315-5391: 6th intron 5392-5533: 7th exon 5534-5816: 7th intron 5817-5902: 8th exon (end) Codon 5886)
5903-6015: 8th intron 6016-: 9th exon SEQ ID NO: 6: nucleotide sequence of genomic DNA of OsPTP2 1-983: RdDM target region 984-1109: 1st exon 1110-3408: 1st intron 3409-3497: 1st 2 exons 3498-4081: second intron 4082-4172: third exon (translation start point 4166)
4173-4262: 3rd intron 4263-4345: 4th exon 4346-4495: 4th intron 4496-4636: 5th exon 4737-5902: 5th intron 5903-6028: 6th exon 6029-7528: 6th intron 7529 -7895: 7th exon 7896-8323: 7th intron 8324-: 8th exon (stop codon 8395)
SEQ ID NO: 7: u7 primer SEQ ID NO: 8: u8 primer SEQ ID NO: 9: u9 primer SEQ ID NO: 10: u10 primer SEQ ID NO: 11: Blast1 primer SEQ ID NO: 12: Blast2 primer SEQ ID NO: 13: W62kdRT1F primer SEQ ID NO: 14: W62kdRT1R primer SEQ ID NO: 15: W45RTF4 primer SEQ ID NO: 16: W45RTR4 primer SEQ ID NO: 17: Rubiq1 RT F1 primer SEQ ID NO: 18: Rubiq1 RT R1 primer SEQ ID NO: 19: u193-PTP1real primer SEQ ID NO: 20: u194-PTP1real primer SEQ ID NO: 21: p195-TP2 SEQ ID NO: 22: u196-PTP2real primer SEQ ID NO: 23 alt-RTF primer SEQ ID NO 24: Salt-RTR primer SEQ ID NO 25: NPR1-UTRT-F1 primer SEQ ID NO 26: NPR1-UTRT-R1 primer SEQ ID NO 27: T3A 2+ primer SEQ ID NO 28: T3A 2-Primer

Claims (9)

Rice having resistance to blast in which the expression or activity of protein tyrosine phosphatase genes, which are OsPTP1 and OsPTP2, is suppressed. The rice according to claim 1, wherein the suppression of expression of one or more protein tyrosine phosphatase genes is suppression by RNAi method, antisense method, co-suppression method or RNA-directed DNA methylation method. The rice of Claim 1 whose suppression of the activity of 1 or more types of protein tyrosine phosphatase genes is suppression by introduction | transduction of a 1 or several mutation. The rice according to claim 3, wherein the one or more mutations include at least a mutation in the protein coding region of the protein tyrosine phosphatase gene. One or more mutations encode cysteine residues corresponding to 258 th cysteine residues OsPTP1, and / or a cysteine residue corresponding to 166 th cysteine residues OsPTP2, a protein tyrosine phosphatase The rice according to claim 3 or 4, comprising at least a codon mutation of a gene. A method for producing rice having resistance to blast , which suppresses the expression or activity of protein tyrosine phosphatase genes that are OsPTP1 and OsPTP2 . The method for producing rice according to claim 6 , wherein the protein tyrosine phosphatase gene expression is suppressed by RNAi method, antisense method, co-suppression method or RNA-directed DNA methylation method. The method for producing rice according to claim 6 , wherein the activity of the protein tyrosine phosphatase gene is suppressed by introducing one or more mutations. A rice progeny or clone produced by the method according to any one of claims 6 to 8 , wherein the expression or activity of a protein tyrosine phosphatase gene that is OsPTP1 and OsPTP2 is suppressed. Rice with disease resistance.