JP2016052296A - Scab resistant plant, and method of making and using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique relating to a scab resistant plant, and methods of making and using it.SOLUTION: The present invention provides a scab resistant plant making method comprising the step of increasing the expression of NMNAT gene in a plant, making it possible to make a scab resistant plant.SELECTED DRAWING: None

Description

  The present invention relates to a head blight-resistant plant, a method for producing the plant, and use thereof.

  Barley fungus caused by the fungus Fusarium (Fusarium graminearum, Fusarium asiaticum, etc.) is a disease that infects many major gramineous grains such as wheat, barley, oats, and corn, and is the most important Known as one of the diseases. Fusarium head blight fungus is a common saprophytic fungus that is distributed in wheat regions around the world and is particularly damaging in areas where there is heavy rain from the flowering period to the ripening period. By infecting the ear of this mold, the growth of the grain and inhibition of the whole ear are caused, and not only the commercial value of the grain is impaired, but also the trichothecene fungal toxin produced by the mold is It is a problem from the viewpoint of food safety.

  Trichothecene fungus poison is difficult to detoxify because it is stable against changes in pH and heat, and when ingested by livestock, it causes poisoning symptoms such as nausea, vomiting, and abdominal pain, and it is extremely dangerous to death depending on the situation Is a high toxin. Trichothecene fungal toxins have a wide variety of molecular species, and can be roughly divided into four groups based on their structures. There are many strains in Fusarium head blight fungus, and the molecular species of trichothecene fungal venom produced by each strain is determined. Among these, there are two types of type A and type B that have many reports of contamination.

  Type A includes very toxic T2 toxin and the like, but has a limited contamination range. On the other hand, type B trichothecene mold poisons include deoxynivalenol (DON) and nivalenol (NIV). Type B toxicity is not as strong as type A, but the damage is more severe due to the wide range of contamination. For example, in Japan, in May 2002, the Ministry of Health, Labor and Welfare established a provisional standard value that sets the concentration of DON contained in wheat grains to 1.1 ppm or less.

  As described above, the grain afflicted with Fusarium head blight containing trichothecene mold toxin exceeding a certain standard cannot be used in any form such as brewing, processing, and feed, and is discarded. On the other hand, since the awareness of food safety is increasing, cultivation without using as much as possible the pesticide that suppresses the occurrence of head blight is required.

  In such an environment, the development of varieties resistant to head blight has become an urgent issue to be solved on a global scale. To date, the present inventors have clarified that head blight fungi, particularly DON or NIV, etc. A plurality of genes involved in susceptibility or resistance to red mold pathogens that produce type B trichothecene fungal toxins and including the step of suppressing or increasing the expression of the above-mentioned genes to produce a plant resistant to red mold The method to do is developed (for example, patent document 1).

JP 2011-172562 A

  Although the technology described above is excellent, it cannot be said that it is sufficient alone, and there has been a strong demand for the development of plants and the like that exhibit further resistance to head blight.

  This invention is made | formed in view of said problem, The objective is to provide the technique which concerns on a head blight-resistant plant, its preparation method, and its utilization.

  As a result of intensive studies to achieve the above object, the present inventors have found that nicotinamide mononucleotide (hereinafter sometimes referred to as “NMN”) induces resistance to head blight, and NMN The inventors have found novel findings such as that NMN adenyl transferase, an important enzyme in the biosynthetic pathway, induces resistance to head blight, and thus completed the present invention. That is, the present invention includes the following inventions.

(1) A method for producing a head blight-resistant plant comprising the step of increasing the expression of any gene selected from the group consisting of the following (a) to (e) in a plant:
(A) a gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 5;
(B) In the amino acid sequence described in any one of SEQ ID NOs: 1 to 5, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and has NMNAT activity A gene encoding a protein;
(C) a gene comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 1 to 5 and encoding a protein having NMAT activity;
(D) a gene comprising the base sequence described in any one of SEQ ID NOs: 6 to 10;
(E) A gene that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (a) to (d) above and encodes a protein having NMNAT activity.

  (2) A head blight-resistant plant obtained by the production method described in (1) above.

(3) A method for selecting a head blight-resistant plant comprising the step of detecting the presence or absence of the gene of any of the above (a) to (e) or the expression level of the gene in the test plant:
(4) A head blight resistant plant comprising a step of measuring an amount of at least one compound selected from nicotinamide mononucleotide, nicotinamide adenyl dinucleotide, and nicotinamide adenyl dinucleotide phosphate in a test plant. Selection method.

(5) The method for selecting a head blight-resistant plant according to (3) or (4) above, comprising at least one substance selected from the following (i) to (v): Kit to do:
(I) the gene of any one of (a) to (e) above,
(Ii) a partial fragment of (i),
(Iii) a detection instrument in which (i) or (ii) is immobilized,
(Iv) a primer set for amplifying the gene of (i),
(V) Reagents for measuring the amount of at least one compound selected from nicotinamide mononucleotide, nicotinamide adenyl dinucleotide, and nicotinamide adenyl dinucleotide phosphate.

  (6) A plant disease control agent comprising, as an active ingredient, at least one compound selected from nicotinamide mononucleotide, nicotinamide adenyl dinucleotide, nicotinamide adenyl dinucleotide phosphate, and salts acceptable in agriculture and horticulture .

  (7) An effective amount of at least one compound selected from nicotinamide mononucleotide, nicotinamide adenyl dinucleotide, nicotinamide adenyl dinucleotide phosphate, and horticulturally acceptable salts thereof, Plant disease control method including the process processed to the soil to grow.

  According to the present invention, a plant resistant to head blight can be obtained. Moreover, according to this invention, the control agent with respect to a head blight can be provided.

It is a figure which shows the result of having examined the accumulation | storage of NMN in the red mold disease susceptibility strain | stump | stock of a barley, and a low mold toxin accumulation | storage line | wire. It is a figure which shows the result of having examined the antibacterial activity of NMN with respect to a Fusarium head blight fungus. It is a figure which shows the result of having examined suppression of mold toxin production of the Fusarium head blight fungus by NMN. It is a figure which shows the result of having examined the control effect with respect to the mold of a mold | fungi at the time of spraying NMN and a related metabolite with respect to Arabidopsis thaliana. It is a figure which shows the result of having confirmed the mRNA amount of the NMNAT gene in each of a wild strain and a transformed plant. It is a figure which shows the result of having compared the disease symptom and the amount of microbial cells about the wild plant and the transformed plant which overexpresses NMNAT. It is a figure which shows the result of having confirmed the head blight prevention effect to the barley of NMN spraying.

  An embodiment of the present invention will be described in detail below. In addition, all the academic literatures and patent literatures described in this specification are incorporated by reference in this specification. Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more (including A and greater than A) and B or less (including B and less than B)”.

  As used herein, the term “gene” is used interchangeably with “polynucleotide”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. Here, the gene may be present in the form of DNA (eg, cDNA or genomic DNA) or RNA (eg, mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). Further, the gene may be chemically synthesized, and the codon usage may be changed so that expression of the encoded protein is improved. Substitutions can be made between codons encoding the same amino acid. The term “protein” is used interchangeably with “peptide” or “polypeptide”. As used herein, the base and amino acid notation uses the one-letter code or three-letter code defined by IUPAC and IUB as appropriate.

<1. Production method of plant resistant to head mold>
The method for producing a head blight-resistant plant according to the present invention only needs to include a step of increasing the expression of any of the following genes (a) to (e) in the plant, and other steps: The conditions, materials, etc. are not particularly limited.
(A) a gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 5;
(B) In the amino acid sequence described in any one of SEQ ID NOs: 1 to 5, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and has NMNAT activity A gene encoding a protein;
(C) a gene comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 1 to 5 and encoding a protein having NMAT activity;
(D) a gene comprising the base sequence described in any one of SEQ ID NOs: 6 to 10;
(E) A gene that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (a) to (d) above and encodes a protein having NMNAT activity.

  Since the genes (a) to (e) above encode proteins having a function of inducing resistance to head blight, expression of these genes in plants leads to plant head blight. Resistance can be imparted or enhanced.

  The gene (a) will be specifically described. SEQ ID NO: 1 is an amino acid sequence of a full-length 264 protein called nicotinamide mononucleotide adenyltransferase (NMNAT) derived from Arabidopsis thaliana. This protein is an enzyme having an activity of adding an adenyl group to NMN and converting it to nicotinamide adenyl dinucleotide (NAD) (hereinafter sometimes referred to as “NMNAT activity”). In the present invention, the enzyme having NMNAT activity includes those that reversibly catalyze such a reaction. NMN intends β-nicotinamide mononucleotide.

  SEQ ID NO: 2 shows the amino acid sequence of NMNAT in barley (Hordeum vulgare subsp.). This protein is a protein consisting of 251 amino acids and has NMAT activity as well. SEQ ID NO: 3 shows the amino acid sequence of NMNAT in rice (Oryza sativa Japonica Group). This protein is a protein consisting of 315 amino acids and has NMAT activity as well. SEQ ID NO: 4 shows the amino acid sequence of NMNAT in maize (Zea mays). This protein is a protein consisting of 249 amino acids and similarly has NMAT activity. SEQ ID NO: 5 shows the amino acid sequence of NMNAT in wheat (Triticum aestivum). This protein is a protein consisting of 174 amino acids and similarly has NMAT activity.

  The gene of (b) above is a functionally equivalent mutant, derivative, variant, allele, homolog, ortholog, partial peptide, or other protein of the protein having the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 As long as it is a fusion protein with a peptide, etc., and encodes a protein having NMNAT activity, its specific sequence is not limited. Here, the number of amino acids that may be deleted, substituted or added is not limited as long as the above functions are not lost. However, the number of amino acids may be deleted, substituted or substituted by a known introduction method such as site-directed mutagenesis. This number refers to the number that can be added, usually within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, more preferably within 7 amino acids, still more preferably within 5 amino acids (for example, 5, 4, 3, 2 or 1 amino acid). Further, in the specification, “mutation” mainly means a displacement artificially introduced by site-directed mutagenesis or the like, but may be a naturally occurring similar mutation.

  The amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G). , H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), a sulfur atom-containing side Amino acids having chains (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) are included (the parentheses indicate single letter amino acids). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution with other amino acids of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. Yes. Furthermore, the target amino acid residue is more preferably mutated to an amino acid residue having as many common properties as possible.

  As used herein, “functionally equivalent” intends that the protein of interest has a biological function or biochemical function equivalent (same and / or similar) to NMNAT. In this specification, the biological function and biochemical function of NMNAT include, for example, a function of adding an adenyl group to NMN to convert it to NAD and / or a function of catalyzing this reversible reaction. Can do. Biological properties may include the specificity of the site to be expressed, the expression level, and the like.

  Whether a protein into which a mutation has been introduced imparts a desired trait to a plant can be determined by introducing a gene encoding the protein into the plant and examining whether the plant exhibits resistance to head blight.

  The gene (c) is also a functionally equivalent variant, derivative, variant, allele, homolog, ortholog, partial peptide, or other protein of the protein having the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 -As a fusion protein with a peptide, etc., the specific sequence is not limited as long as it encodes a protein involved in resistance to head blight.

  Amino acid sequence homology refers to at least 80% or more, more preferably 90% or more, and even more preferably 95% or more (for example, 95%, 96%, 97) of the entire amino acid sequence (or a region necessary for functional expression). %, 98%, 99% or more). Amino acid sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. In addition, when the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known. In order to align the base sequence or amino acid sequence to be compared with each other in an optimal state, addition or deletion (for example, a gap) may be allowed.

  Regarding the gene (d), SEQ ID NO: 6 shows the base sequence (Open Reading Frame: ORF) of the gene encoding the protein consisting of the amino acid sequence shown by SEQ ID NO: 1. Similarly, SEQ ID NO: 7 shows the base sequence (ORF) of the gene encoding the protein consisting of the amino acid sequence shown by SEQ ID NO: 2. Similarly, the polynucleotides of SEQ ID NOs: 8 to 10 are genes encoding proteins having the amino acid sequences shown in SEQ ID NOs: 3 to 5, respectively.

The gene (e) is intended to be a gene that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any of the genes (a) to (d). Here, stringent conditions refer to conditions in which a so-called base sequence-specific double-stranded polynucleotide is formed and a non-specific double-stranded polynucleotide is not formed. In other words, conditions for hybridizing at a temperature ranging from the melting temperature (Tm value) of highly homologous nucleic acids, for example, perfectly matched hybrids, to 15 ° C, preferably 10 ° C, more preferably 5 ° C lower. It can be said. For example, the temperature is 60 to 68 ° C., preferably 65 ° C., more preferably in a buffer solution consisting of 0.25 M Na ₂ HPO ₄ , pH 7.2, 7% SDS, 1 mM EDTA, 1 × Denhardt's solution. Hybridize under conditions of 68 ° C. for 16 to 24 hours, and further in a buffer solution comprising 20 mM Na ₂ HPO ₄ , pH 7.2, 1% SDS, 1 mM EDTA, the temperature is 60 to 68 ° C., preferably 65 ° C. A preferable example is a condition in which washing is performed twice for 15 minutes under the condition of 68 ° C. Other examples include 25% formamide, 50% formamide under more severe conditions, 4 × SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / mL denatured salmon sperm DNA After prehybridization is performed overnight in a hybridization solution at 42 ° C., a labeled probe is added, and hybridization is performed by incubating at 42 ° C. overnight. The cleaning solution and temperature conditions for the subsequent cleaning are about “1 × SSC, 0.1% SDS, 37 ° C.”, and the more severe conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”. It can be performed at about “0.2 × SSC, 0.1% SDS, 65 ° C.”. As the hybridization washing conditions become more severe, hybridization with higher specificity is achieved. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like. This is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

  As a method for obtaining the above-mentioned gene / protein, a commonly used polynucleotide modification method may be used. That is, a polynucleotide having genetic information of a desired recombinant protein can be prepared by substituting, deleting, inserting and / or adding a specific base of a polynucleotide having protein genetic information. Specific methods for converting polynucleotide bases include, for example, commercially available kits (KOD-Plus Site-Directed Mutagenesis Kit; Toyobo, Transformer Site-Directed Mutagenesis Kit; Clontech, QuickChange Site Directed Mutagenesis Kit; Stratagene, etc. ) Or the use of the polymerase chain reaction (PCR). These methods are known to those skilled in the art.

  The gene may be composed only of a polynucleotide encoding the protein, but may be added with other base sequences. The base sequence to be added is not particularly limited, but includes a label (for example, histidine tag, Myc tag or FLAG tag), a fusion protein (for example, streptavidin, cytochrome, GST, GFP or MBP), a promoter sequence, and Examples thereof include a base sequence encoding a signal sequence (for example, an endoplasmic reticulum transition signal sequence, a secretory sequence, etc.). The site to which these base sequences are added is not particularly limited and may be, for example, the N-terminus or C-terminus of the translated protein.

  The plant to be subjected to the method of the present invention is not particularly limited as long as it is a plant that is infected with Fusarium head blight fungi, but is preferably a gramineous plant, and more preferably a wheat plant. More specifically, for example, rice, wheat, barley, rye, corn, oat, millet, sorghum, Arabidopsis thaliana, etc. Among these, rice, wheat, barley, oat and corn are more preferable.

  In the present specification, “redhead fungus” is intended to be a plant disease caused by filamentous fungi such as Fusarium spp., And is not particularly limited, but is a wheat fungus caused by Fusarium graminearum or Fusarium asiaticum. Preferably there is. The “red mold fungus” is not particularly limited as long as it is a filamentous fungus such as Fusarium spp., But Fusarium graminearum or Fusarium asiaticum is preferable.

  In the present specification, “increasing gene expression” means that the production amount of the protein encoded by the gene in the target plant may be increased. In addition to the case of transducing the gene from the outside, Including the case of increasing the expression level of an endogenous gene. The degree of the increase is not particularly limited, and as a result, the plant having increased expression of the gene compared to a control plant (for example, a non-introduced gene or a wild-type plant) has red mold disease. It suffices to show resistance to.

  When increasing the expression level of an endogenous gene, for example, by using a known mutation source, obtaining a mutant having an increased expression level of the endogenous gene, thereby producing a plant resistant to head blight. can do. For example, chemistry represented by EMS (ethyl methanesulfonic acid), 5-bromouracil, 2-aminopurine, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, and other carcinogenic compounds. It may be a method using an artificial mutagen, or a mutation is introduced into the gene of the target plant by radiation treatment or ultraviolet treatment such as X-ray, alpha ray, beta ray, gamma ray, ion beam, and the result. As such, in the plant, a strain having increased expression of the gene may be selected. These methods are known to those skilled in the art.

  The step of increasing the expression of the gene preferably includes a step of introducing a gene selected from the group consisting of the above (a) to (e) into a plant to produce a transformed plant.

  In the present invention, as plant materials to be transformed, for example, roots, stems, leaves, seeds, embryos, ovules, ovary, shoot tips, buds, pollen, etc., plant tissues and sections thereof, cells, callus, Plant cells such as protoplasts obtained by enzymatic treatment of the cells to remove the cell wall are exemplified, but not limited thereto.

  When a gene is transduced from the outside, a known method can be used, and a specific method is not limited. For example, a plant resistant to head blight can be obtained by introducing a recombinant vector into a plant and expressing the gene. Can be created. When a gene is transduced from the outside, it is preferable to use a gene derived from a plant similar to the host plant or a plant of a related species. For example, in the case of wheat, it is preferable to use a gene derived from wheat or barley.

  The “recombinant vector” is not limited as long as it contains any of the above genes (a) to (e), but also contains an appropriate promoter and the like and can express this gene in plants. preferable. In this case, the cauliflower mosaic virus 35S promoter can be exemplified as the promoter to be used, but in addition to that, the promoter of nopaline synthase gene (Pnos), the ubiquitin promoter derived from corn, the actin promoter derived from rice, the tobacco-derived PR Protein promoters can also be used.

  In the method for producing a Fusarium head blight-resistant plant of the present invention, for example, a plant having Fusarium head blight resistance can be produced by introducing the recombinant vector into a plant and expressing the gene. In the method for producing a head blight-resistant plant of the present invention, a conventionally known method can be used as a method for introducing a recombinant vector into a plant cell, and is not particularly limited. For example, polyethylene glycol method, Agrobacterium method, liposome method, cationic liposome method, calcium phosphate precipitation method, electroporation method (Electroporation) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John) Wiley & Sons. Section 9.1-9.9), lipofection method (manufactured by GIBCO-BRL), microinjection method, particle gun method, and other methods known to those skilled in the art.

  A method of regenerating a transformed plant cell by redifferentiating the transformed plant cell may be performed by a method according to the type of the plant cell, and a known method can be suitably used. For example, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be cited for rice, and the method of Shillito et al. (Bio / Technology 7: 581 (1989)) or Gorden-Kamm for maize. (Plant Cell 2: 603 (1990)). The presence of the introduced foreign gene in the transformed plant that has been regenerated and cultivated by the above method is confirmed by a known PCR method or Southern hybridization method, or by analyzing the DNA base sequence in the plant body. can do. In this case, the extraction of DNA from the transformed plant can be performed according to a known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).

  When the gene of the present invention present in the regenerated plant body is analyzed using the PCR method, an amplification reaction is performed using the DNA extracted from the regenerated plant body as a template as described above. In addition, an amplification reaction can also be performed in a reaction solution in which a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequences of the genes (a) to (e) is used as a primer and these are mixed. In the amplification reaction, DNA denaturation, annealing, and extension reactions are repeated several tens of times to obtain an amplification product of a DNA fragment containing the base sequence of the gene of the present invention. When the reaction solution containing the amplified product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and that the DNA fragments correspond to the gene of the present invention.

  The present invention also includes a method for producing a head blight resistant plant by a breeding method. Examples of the breeding method include a general breeding method (cross breeding method or the like) for crossing with a cultivar having an increased expression level of any of the genes (a) to (e). By this method, plants resistant to head blight can be produced.

When producing a head blight-resistant plant by a breeding method, it can implement suitably with reference to various well-known literature. (Cellular engineering separate volume, Plant Cell Engineering Series 15 “Experimental Protocols for Model Plants” Shujunsha, 2001, Koji Kato “2.1 Mating of Rice and Wheat” p.6-9; Retsuro Takatsuki, Yoshio Sano “2 Mating method "p.46-48)
As a preferable aspect of the breeding method, a method including the following steps (A) and (B) can be exemplified.
(A) including a step of crossing with a plant having an increased expression level of any of the genes (a) to (e).
(B) A step of selecting a plant variant in which the expression level of any of the genes (a) to (e) is increased.

More specifically, a method including the following steps can be exemplified.
(A1) A step of crossing plant A with another plant B in which the expression level of any of the genes (a) to (e) is increased to produce F1 (A2) Crossing step (B1) Crossing the plant selected in the step (B2) step (B1) and the plant A selected from the plant in which the expression level of any one of the genes (a) to (e) is increased In the method described above, in the method described above, the plant B in which the expression level of any one of the genes (a) to (e) is increased, and a plant to which resistance to head blight is imparted (these plants are referred to as “plant A”). And a trait in which the expression level of any of the genes (a) to (e) possessed by the plant B is inherited and an individual close to the plant A is selected, Perform a “backcross” that repeats crosses by A Thus, a trait in which the expression level of any of the genes (a) to (e) of the plant B is increased can be intentionally introduced.

  In the above method, if necessary, it is repeated until the entire genome other than the trait in which the expression level of any of the genes (a) to (e) is increased is homo-fixed with the target genetic trait. Can do. That is, the individual crossed by the step (B2) has a trait in which the expression level of any of the genes (a) to (e) is increased using a general DNA marker, and Plant individuals whose genome structure is close to that of plant A can be selected.

<2. Red mold resistant plant>
The head blight-resistant plant according to the present invention is not particularly limited as long as it is a plant obtained by the above-described method. In other words, it can be said to be a plant in which the expression of any of the genes (a) to (e) is increased. In particular, it is preferable that the plant is resistant to Fusarium head blight, in which any gene selected from the group consisting of the above (a) to (e) is transformed.

  The plant resistant to head blight of the present invention includes whole plants, plant organs (eg, roots, stems, leaves, petals, seeds, fruits, etc.), plant tissues (eg, epidermis, sieve, soft tissue, xylem, fibers). Tube bundles, etc.), plant cells, calli and the like, and protoplasts, shoot primordia, polyblasts, and hairy roots may also be included.

  Moreover, once a transformed plant having any of the genes (a) to (e) introduced into the genome of the plant is obtained, progeny are obtained from the plant by sexual reproduction or asexual reproduction. It is possible. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant or its progeny or clones, and mass-produce the plant based on them. Further, the head blight-resistant plant of the present invention is a progeny plant such as “T0 generation” which is a regenerated generation that has undergone transformation treatment, or “T1 generation” which is a self-propagating seed of a T0 generation plant, This includes hybrid plants and their progeny plants that have been crossed as a single parent.

  In the present invention, only Arabidopsis thaliana has been experimentally confirmed to be able to confer resistance to head blight, but the present invention is not limited to plants infecting head blight, particularly grasses, and even wheat. It is thought that it can be widely applied to arboreal plants. This is due to the following reason.

  First, Fusarium head blight fungus widely infects gramineous plants, especially wheat plants, but also in Arabidopsis. It is known that the infection form and disease symptoms are almost common between wheat and Arabidopsis (Urban et al., Plant J., 32: 961, 2002). In particular, type B trichothecene fungal toxins produced by Fusarium head blight fungus are known to inhibit protein synthesis and root elongation. These fungal toxins are similarly caused in Arabidopsis and wheat (Masuda). et al., J. Exp. Bot. 58: 1617, 2007). The AtNFXL1 gene of Arabidopsis thaliana is markedly induced by trichothecene (Asano et al., Plant J., 53: 450, 2008), but the ortholog gene of barley and wheat is induced by inoculation with Fusarium head blight fungus. (Boddu et al., Mol. Plant-Microbe Interact., 20: 1364, 2007; Jia et al., Mol. Plant-Microbe Interact., 22: 1366, 2009). Furthermore, trichothecene has elicitor activity and cell death-inducing activity in Arabidopsis and wheat (Nishiuchi et al., Mol. Plant-Microbe Interact. 19: 512, 2006; Desmond et al., Mol Plant Pathol., 9: 435, 2008).

  Next, genes similar to the genes derived from Arabidopsis thaliana whose functions have been confirmed in the examples of the present invention are widely present in grasses besides Arabidopsis thaliana. Of particular note is that Arabidopsis thaliana also exists in rice and barley, which are taxonomically distantly related plants. Therefore, it is considered that genes similar to the above-mentioned genes derived from Arabidopsis thaliana are widely present in plants infected with head blight.

  After reading this specification in consideration of the above points, those skilled in the art will recognize that similar genes in plants other than Arabidopsis thaliana are resistant to head blight as well as genes resistant to head blight in Arabidopsis thaliana. It can be understood that it has a function involved. Therefore, it can be said that by introducing the various genes described above into various plants or increasing the expression in the plants, the plant can be given resistance to head blight. In addition, the presence of the similar genes derived from Arabidopsis thaliana demonstrated in the examples has been confirmed in barley, rice, corn and wheat, but in the future, as the genome analysis of each plant proceeds, There is a high probability that similar genes will be found.

<3. Selection method and kit for plant resistant to head blight>
The method for selecting a head blight-resistant plant according to the present invention includes a step of detecting the presence or absence of the gene of any one of (a) to (e) above or the expression level of the gene in a test plant. The specific process, conditions, materials, etc. are not particularly limited. Test plants include test propagation media, plant cells, plant organs and parts in addition to plants.

This selection method can be performed, for example, by a method including the steps (a) to (c).
(A) Step of preparing a polynucleotide sample from a test plant (a) A polynucleotide comprising the above-mentioned polynucleotide and a nucleotide sequence complementary to the gene of any one of (a) to (e) above and stringent conditions The step of hybridizing with a polynucleotide that hybridizes under (c) The step of detecting the hybridization The step (a) is performed by extracting a polynucleotide such as genomic DNA or mRNA from the test plant. Can do. The part of the plant used for the preparation (extraction) of the polynucleotide is not particularly limited. When mRNA is extracted, cDNA may be prepared from mRNA by reverse transcription.

  In the step (a), the polynucleotide sample prepared above is subjected to stringent conditions using a polynucleotide comprising a base sequence complementary to any of the genes (a) to (e) as a probe or primer. This can be done by hybridizing under. Stringent conditions are as already described.

  In the step (a), when a polynucleotide comprising a base sequence complementary to any of the genes (a) to (e) is used as a probe, the step (c) is performed by ordinary Southern blotting. , Northern blotting, microarray, and hybridization detection methods such as Fluorescent In Situ Hybridization (FISH).

  On the other hand, when a polynucleotide comprising a base sequence complementary to any of the genes (a) to (e) above is used as a primer in the step (a), the step (c) includes PCR amplification. Hybridization can be detected by performing a reaction and analyzing the obtained amplification product by electrophoresis or sequencing.

When performing PCR amplification reaction, the amplified polynucleotide product can be labeled by using a primer labeled with an isotope such as ³² P, a fluorescent dye, or biotin. Alternatively, the amplification product can be labeled by adding a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin to the PCR reaction solution and performing PCR. Furthermore, labeling can also be performed by adding a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin using a Klenow enzyme or the like after the PCR reaction.

  Moreover, as another aspect of the method for selecting a head blight-resistant plant according to the present invention, at least one compound selected from NMN, NAD, and nicotinamide adenyl dinucleotide phosphate (NADP) in a test plant The method of including the process of measuring the quantity of can be mentioned.

  The amount of NMN, NAD or NADP in the test plant can be determined using a known metabolome analysis method, and the specific method is not limited. For example, the method as described in the Example mentioned later can be mentioned.

  As shown in Examples described later, all of NMN, NAD, and NADP have a function of inducing resistance to head blight. In particular, it has been suggested that NMN has a plant activator-like function that enhances the immune function of the plant itself. For this reason, selection of a plant resistant to head blight can be performed using the amount of these compounds contained in the plant as an index.

  At the time of selection, for example, the amount of NMN or the like of the test plant may be compared with the amount of NMN or the like in a known head blight-resistant plant, or the amount of NMN or the like in a known head blight-resistant plant. Can be easily selected by comparing with this threshold value. When the amount of NMN or the like in the test plant is large, it can be determined that the plant is resistant to head blight.

  The selection method according to the present invention includes not only selection of head blight-resistant plants in cultivars that have been cultivated so far, but also selection of head blight resistance in new varieties by crossing or genetic recombination techniques. .

The present invention also includes a kit for carrying out the aforementioned method for selecting a head blight-resistant plant. The configuration of the kit is not particularly limited as long as the above selection method can be performed. For example, a kit including at least one substance selected from the substances (i) to (v) shown below can be mentioned. .
(I) a partial fragment (probe) of the gene (ii) (i) of any of the above (a) to (e),
(Iii) a detection instrument in which (i) or (ii) is immobilized,
(Iv) a primer set for amplifying the gene of (i),
(V) Reagents for measuring the amount of at least one compound selected from NMN, NAD, and NADP.

  The partial fragment (ii) hybridizes with the gene (i) and is used as a probe. The design of the probe can be performed by a known method. The primer set (iv) is preferably used for PCR, and can be suitably designed according to a known technique. For example, a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequences of the genes (a) to (e) can be used as a primer. The reagents of the above (v) are not particularly limited as long as they are used for measuring NMN, NAD, or NADP. For example, various reagents used in the methods described in Examples described later There can be mentioned.

  By using a kit having these configurations, the above selection method can be easily performed.

<4. Plant Disease Control Agent, Plant Disease Control Method, Mold Toxic Reducer, and Method to Reduce Mold Poison>
The plant control agent according to the present invention only needs to contain at least one compound selected from NMN, NAD, NADP, and salts acceptable in agriculture and horticulture as an active ingredient, and other configurations are particularly limited. Not. In addition, the present invention includes a step of treating an effective amount of at least one compound selected from NMN, NAD, NADP and their agriculturally and horticulturally-salts to a plant or soil for cultivating the plant. Disease control methods are also included.

  As shown in Examples described later, NMN, NAD and NADP are compounds that induce head blight resistance, and in particular, NMN has a plant activator-like function that enhances the immune function of the plant itself. Has been suggested. Therefore, these compounds can be used as active ingredients for controlling plant diseases.

  “Plant disease control agent” means a drug having a control effect against plant diseases, and not only a drug that kills plant pathogenic microorganisms but also a drug that suppresses the growth of plant pathogenic microorganisms, plants of plant pathogenic microorganisms It also includes drugs that protect against infection. The same applies to the method.

  Although it does not specifically limit about the plant disease used as object, It is preferable to make target the red mold disease.

  Moreover, as shown in the Example mentioned later, it has been clarified by the present inventors that NMN, NAD and NADP also have an action of reducing mold poison produced by Fusarium head blight. For this reason, according to the present invention, there is provided a fungus toxin reducing agent produced by Fusarium head blight fungus, which contains at least one compound selected from NMN, NAD, NADP, and their agriculturally and horticulturally acceptable salts as an active ingredient. Is done. Furthermore, a Fusarium head blight fungus comprising a step of treating an effective amount of at least one compound selected from NMN, NAD, NADP, and an agriculturally and horticulturally acceptable salt thereof on a plant or soil on which the plant is cultivated is produced. A method for reducing mold toxins is also encompassed by the present invention.

  The “mold fungus” is a toxic substance that causes poisoning to humans or livestock among secondary metabolites produced by filamentous fungi. In this specification, a toxic substance produced by Fusarium head blight fungus is particularly referred to. Intended. Specific examples of the mold toxin include type B trichothecene mold toxin produced by Fusarium head blight fungus. Among the above-mentioned type B trichothecene fungal venoms, deoxynivalenol (DON) or nivalenol (Nivalenol; NIV) is preferable as the mold venom targeted by the present invention. When the mold poison targeted by the present invention is DON or NIV, the mold toxin reduction function of the present invention shows a higher effect.

  In the present specification, “reducing” the mold toxin is intended to reduce the concentration of the mold toxin in the plant. In order to reduce mold toxins, in addition to reducing mold toxins once generated, the mold toxin itself is inhibited by suppressing the expression of genes involved in the biosynthesis of mold toxins. Is also included.

  Hereinafter, the plant disease control agent and the mold poison reducing agent are collectively referred to as “this agent”, and the plant disease control method and the method for reducing mold toxin are referred to as “the present method”. Further, NMN, NAD, NADP and their agriculturally and horticulturally acceptable salts are referred to as “NMN etc.”.

  In this agent and the present method, when NMN or the like is used as an active ingredient, NMN or the like may be used as it is, but according to conventional methods of various dosage forms used in agriculture and horticulture, a suitable solid carrier, liquid carrier, gaseous state Mixing with carriers, surfactants, dispersants, and other formulation adjuvants, optional emulsions, solutions, suspensions, wettable powders, powders, granules, tablets, oils, aerosols and flowables Can be in dosage form.

  Examples of the solid carrier include animal and vegetable powders such as starch, sugar, cellulose powder, cyclodextrin, activated carbon, soybean powder, wheat flour, rice bran powder, wood powder, fish powder, and milk powder; talc, kaolin, bentonite, organic bentonite, carbonic acid Examples thereof include mineral powders such as calcium, calcium sulfate, sodium bicarbonate, zeolite, diatomaceous earth, white carbon, clay, alumina, silica, sulfur powder, and slaked lime.

  Examples of the liquid carrier include water; vegetable oil such as soybean oil and cottonseed oil; animal oil such as beef tallow and whale oil; alcohols such as ethyl alcohol and ethylene glycol; acetone, methyl ethyl ketone, methyl isobutyl ketone, and isophorone. Ketones; ethers such as dioxane and tetrahydrofuran; aliphatic hydrocarbons such as kerosene, kerosene and liquid paraffin; aromatic hydrocarbons such as toluene, xylene, trimethylbenzene, tetramethylbenzene, cyclohexane and solvent naphtha; chloroform, Halogenated hydrocarbons such as chlorobenzene; acid amides such as N-methyl-2-pyrrolidone, N, N-dimethylformamide, N, N-dimethylacetamide; ethyl acetate, glycerin ester of fatty acid, etc. Nitriles such as acetonitrile; esters such as sulfur-containing compounds such as dimethyl sulfoxide.

  Examples of the gas carrier include LPG, air, nitrogen, carbon dioxide gas, dimethyl ether and the like.

  Examples of the surfactant or dispersant include alkyl sulfates, alkyl (aryl) sulfonates, polyoxyalkylene alkyl (aryl) ethers, polyhydric alcohol esters, lignin sulfonate, polyoxyethylene lauryl ether. , Polyoxyethylene alkyl aryl ether, polyoxyethylene sorbitan fatty acid ester and the like.

  Examples of the adjuvant for formulation include carboxymethylcellulose, gum arabic, polyethylene glycol, calcium stearate and the like.

  The above carriers, surfactants, dispersants, and adjuvants can be used alone or in combination as required.

  Although content of the active ingredient in a formulation is not specifically limited, Although it can set suitably according to a dosage form, it is preferable to mix | blend, for example in 0.0001 to 50 weight%. This agent may be used as it is, but if necessary, it can be used after diluting to a predetermined concentration with a diluent such as water.

  In this agent and this method, other agricultural chemicals such as fungicides, insecticides, acaricides, nematicides, soil insecticides, antiviral agents, attractants, herbicides, plant growth preparations, fertilizers, etc. Furthermore, it can also be mixed, for example, it may mix and disperse | distribute or may disperse | distribute over time or simultaneously. Specific examples include those described in the Pesticide Manual (The Pesticide Manual, 13th edition published by The British Crop Protection Council) and Shibuya Index (SHIBUYA INDEX 12th edition, 2007, published by SHIBUYA INDEX RESEARCH GROUP). .

  The method of using the active ingredient in this method is not particularly limited as long as it is a method that is generally applied in agriculture and horticulture, and examples include foliage spraying, water surface application, soil treatment, nursery box application, seed disinfection, and the like. It is done.

  The amount of this drug used depends on the type of target disease and degree of disease, the type and target site of the target crop, the application method generally applied in agriculture and horticulture, as well as the application mode such as aviation spraying, ultra-small amount spraying, etc. It is not particularly limited. Although the target crop is not limited, it is preferably a gramineous plant as described above.

  In addition, it is added that the contents described in the above items <1> to <4> can be used as appropriate in other items. The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments can be appropriately combined. Embodiments are also included in the technical scope of the present invention. EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited only to this Example.

(1. Examination of NMN accumulation in strains susceptible to head blight of barley and low fungal toxin accumulation lines)
The degree of accumulation of NMN in barley of the susceptible strain of Fusarium head blight and the barley of the strain that is infected with Fusarium head blight fungus but accumulates only a low concentration of mold toxin (low mold toxin accumulation strain) was examined. Specifically, T615 (Turkey 45) was used as the susceptible strain, and U389 (Maja) and U121 (Sirius O.525) were used as the low mold toxin accumulation strain. T615 accumulates 1.2 ppm of mold venom (NIV) and U389 accumulates 0.6 ppm of mold venom (NIV).

Each barley line grown in the field was inoculated with cuttings (Hori et al., Breeding Science, 2006. 56: 25-30), and the fungus B. fungus (F. asiaticum OUZ78 strain containing 0.1% Tween 20) was used. ) Spore solution (1 × 10 ⁵ conidia / mL), and the amount of β-NMN accumulated after 2 days was examined. The amount of NMN accumulated was analyzed according to the method of Sawada et al. (Sawada et al., Plant Cell Physiol. 2009. 50: 37-47).

The results are shown in FIG. As shown in the figure, it was found that more NMN was accumulated in the barley of the low mold poison line than in the barley of the susceptible line.
Spore fluid (1 × 10 ⁵ conidia / mL)
(2. Examination of antimicrobial activity of NMN against Fusarium head blight)
The antibacterial activity of NMN against Fusarium head blight was investigated. Specifically, each test compound was administered to F. graminearum, and the growth of the fungus was monitored by MTT assay to measure the antibacterial activity of each compound against B. fungus. As a negative control, water was used, and as a positive control, metconazole (manufactured by Kureha Co., Ltd.), which is a commercially available drug against red mold. In addition to NMN, hexylene glycol, serotonin hydrochloride, trisodium isocitrate, cyanine chloride, and chromanin chloride were used as test compounds. Sterile water was used for the negative control. A 96-well plate is used to culture a spore suspension (1 × 10 ⁵ conidia / mL) of F. graminearum H3 strain (standard strain in Japan) in 1 mL of SYEP liquid medium, and the test compound is added at a concentration of 0.1 mg / mL. And shaking culture was performed in the dark at 25 ° C. and 100 rpm. The culture was stopped after 36 hours, and the growth of the fungus was quantified by MTT assay (nacalai tesque). MTT is a kind of tetrazolium salt, which is converted into blue water-insoluble formazan by dehydrogenase in the mitochondria of living cells, so it is used to measure living cells.

  The results are shown in the upper panel (graph) of FIG. As shown in the figure, neither NMN nor other test compounds showed antibacterial activity against Fusarium head blight.

  In addition, filter paper soaked with water, metconazole (indicated as “Metcona” in the figure), or NMN was placed on a petri dish seeded with red mold fungus, and the growth of the mold was observed. Specifically, a spore of F. graminearum was inoculated in the center of a synthetic low-nutrient agar (SNA) medium, and a sterilized paper disc soaked with 0.1 mg / mL NMN was placed. Sterile water was used as a negative control, and metconazole was used as a positive control, and cultured at 28 ° C. in the dark for 4 days.

  Results are shown in the lower panel of FIG. As shown in the figure, it can be seen that NMN cannot inhibit the growth of Fusarium head blight.

(3. Examination of inhibition of mold toxin production of Fusarium head blight fungus by NMN)
It was examined whether NMN suppresses the fungal toxin production of Fusarium head blight. In the H3 strain of Fusarium head blight fungus (F. graminearum), a TRI5 promoter-GFP-introduced strain in which GFP was linked in place of the TRI5 coding region downstream of the promoter region of the gene encoding the enzyme TRI5 was prepared. Enzyme TRI5 is a key enzyme in the biosynthesis of trichothecene fungal toxins, and since there is a high correlation between the expression of this gene and fungal toxin production, it is one of the most suitable genes for monitoring fungal toxin production. One. Moreover, it is known that the disrupted strain of the enzyme TRI5 hardly produces mold toxin.

  In addition to NMN, hexylene glycol, serotonin hydrochloride, trisodium isocitrate, cyanine chloride, and chromanin chloride are allowed to act on this TRI5 promoter-GFP-introduced strain in addition to NMN as a test compound. The GFP signal was measured. Specifically, the test compound was added at a concentration of 0.1 mg / mL, and shaking culture was performed in the dark at 25 ° C. and 100 rpm. After 36 hours, the culture was stopped and the GFP signal was quantified using Typhoon 9400 (GE Healthcare).

  The results are shown in FIG. As shown in the figure, NMN suppressed mold toxin production in Fusarium head blight.

(4. Examination of control effect against Fusarium head blight fungus by spraying NMN)
We examined the control effect against fungus on fungi when NMN and related metabolites (NAD, NADP, nicotinamide (NIC)) were sprayed on Arabidopsis thaliana. Specifically, after spraying water or NMN aqueous solution (100 ppm) with Silwet L77 added at 0.001% to a potted Arabidopsis thaliana, spray fungus (F. graminearum) 6 hours later (1 × 10 ⁵ conidia / mL). Thereafter, after 72 hours, Arabidopsis thaliana leaves were sampled, and the expression analysis of the plant defense gene PR-1 was quantified.

Specifically, the main leaves of Arabidopsis thaliana grown for 4 weeks after seeding under normal growth (under fluorescent light) were sprayed with NMN using an atomizer, cultured for 6 hours, and then spore solution of F. graminearum ( 1 × 10 ⁵ conidia / mL). Thereafter, the inoculated leaves of Arabidopsis thaliana cultured for 72 hours were sampled. In order to analyze the expression of the plant defense gene PR-1, total RNA was extracted from the sampling leaves, and cDNA was synthesized by reverse transcriptase. QRT-PCR was performed using the Actin2 gene as a primer for PR-1 expression analysis and a control gene.

  The results are shown in FIG. As shown in the figure, it can be seen that expression of the defense gene PR-1 is significantly induced when the scab is inoculated after spraying with the NMN aqueous solution.

Next, water, NMN aqueous solution, NAD aqueous solution, NADP aqueous solution, and NIC aqueous solution (each 100 ppm) were sprayed on a potted Arabidopsis thaliana with a 300 μL spray, and 6 hours later, the spore solution of F. graminearum (1 × 10 ⁵ conidia / mL) was injected and inoculated. Thereafter, after 72 hours, Arabidopsis leaves were sampled, and the ratio of the bacterial genomic DNA to the plant genomic DNA was quantified. Specifically, a plant sample inoculated with Fusarium head blight fungus is prepared in the same manner as described above, and the fungus and plant contained in the inoculated sample are amplified by amplifying EF1α gene of Fusarium head blight fungus and ACT2 gene of plant by real-time PCR. The amount of genome was measured. The higher the amount of gDNA of the fungus, the higher the growth ability in Arabidopsis, that is, the higher the pathogenicity.

  The results are shown in FIG. As shown in the figure, the amount of bacterial cells decreased when sprayed with NMN aqueous solution, NAD aqueous solution, NADP aqueous solution and then inoculated with Fusarium head blight. On the other hand, when inoculated with Fusarium head blight after spraying the NIC aqueous solution, the amount of bacterial cells hardly changed. From this result, NMN, NAD, and NADP show a control effect against Fusarium head blight.

(5. Examination of resistance of MNAT overexpression strain to Fusarium head blight)
It was investigated whether a transformed plant overexpressing nicotinamide mononucleotide adenylyltransferase (NMNAT) is resistant to Fusarium head blight. An MNAT overexpression construct (pK2GW7.0 + NMNAT) in which a gene encoding NMNAT was linked downstream of the 35S promoter of cauliflower mosaic virus was prepared and introduced into Arabidopsis thaliana to obtain a transformed plant.

  Specifically, the full length CDS sequence of the NMNAT gene of Arabidopsis thaliana was amplified and cloned by PCR. After confirming the base sequence, an overexpression vector was prepared by incorporating the CDS sequence of the NMNAT gene downstream of the CaMV35S promoter of the binary vector for transformation. The transformed Agrobacterium C58C1 bacterial solution was introduced into Arabidopsis thaliana by the Floral Dip method to obtain a transformed plant.

  FIG. 5 shows the results of confirming the mRNA amount of the NMNAT gene in each of the wild strain and the transformed plant by RT-PCR and qPCR. In the figure, the transformed plant is shown as 35S :: NMNAT. As shown in the figure, it was confirmed that NMNAt mRNA was overexpressed in the transformed plants.

  Next, the symptom and the amount of fungus were compared for the wild strain inoculated with Fusarium head blight and the transformed plant overexpressing NMNAT.

Specifically, F. graminearum spore solution (1 × 10 ⁵ conidia / mL) was injected and inoculated into the true leaves of Arabidopsis thaliana about 4 weeks after sowing. Thereafter, the inoculated leaves of Arabidopsis were sampled for 72 hours, and the severity of disease symptoms of the inoculated leaves was scored. The scoring was evaluated by “normal”, “change to black leaf”, “partial aerial mycelium”, and “expanded aerial mycelium”. In addition, in genomic DNA extracted from plant tissues inoculated with Fusarium head blight, the amount of Arabidopsis genomic DNA was calculated by real-time PCR using the Actin2 gene, and the amount of genomic DNA of Fusarium head blight was used using the EF1α gene. It was calculated by the real-time PCR method. The amount of genomic DNA of Fusarium head blight fungus relative to the genomic DNA of Arabidopsis thaliana was calculated as a percentage.

  The results are shown in FIGS. 6 (a) to 6 (c). FIG. 6 (a) shows a diagram comparing the disease symptoms of the wild strain and the transformed plant inoculated with Fusarium head blight. As shown in the figure, the symptom of head blight was visually confirmed on the leaves of the wild strain, but the symptom of head blight was not visually confirmed in the transformed plant.

  FIG. 6 (b) shows the results of scoring the disease symptoms of the wild strain and transformed plant inoculated with Fusarium head blight. The symptom score due to Fusarium head blight was evaluated based on the degree of aerial mycelium (total or partial) and the state of death caused by the growth of Fusarium head blight fungus. As shown in the figure, it can be seen that the disease symptoms due to head blight are suppressed in the transformed plant as compared to the wild type.

  FIG. 6 (c) shows the results of measuring the genome amounts of the fungus and the plant in the wild strain and the transformed plant inoculated with Fusarium head blight. As shown in the figure, in the transformed plant, the amount of Fusarium head blight fungus decreased significantly compared to the wild type.

(6. Confirmation of redhead disease control effect on barley by NMN spray)
Using a six-rowed barley (HES4) that is very susceptible to head blight, the effect of controlling head blight was examined when the NMN solution was sprayed. Specifically, 300 μL of water or an NMN aqueous solution (100 ppm) added with Silwet L77 at 0.001% is sprayed with a spray (atomizer) on the wheat ears of six-rowed barley (HES4) grown in the field. After 4 hours, the spore solution of F. graminearum containing 0.001% Silwet L77 was spray-inoculated (1 × 10 ⁵ conidia / mL) using the above-described cutting inoculation. After 7 days, barley ears were sampled.

  The result of visual observation is shown in FIG. In the figure, “Mock / Fg” is barley inoculated with Aspergillus oryzae after spraying with water, and “NMN / Fg” is barley inoculated with Aspergillus oryzae after spraying with NMN solution. As shown in the figure, in “Mock / Fg”, the symptom of red mold fungus (appearance of aerial hyphae) was confirmed visually, but in “NMN / Fg”, a remarkable symptom of red mold was observed. There wasn't.

  Next, for the sampled barley spikes, in the genomic DNA extracted from the plant tissue inoculated with Fusarium head blight fungus, the amount of barley genomic DNA was calculated by the real-time PCR method using the Tubulin gene. The amount was calculated by a real-time PCR method using the EF1α gene. The amount of genomic DNA of Fusarium head blight fungus relative to the genomic DNA of barley (plant) was calculated as a percentage.

  The results are shown in FIG. As shown in the figure, “NMN / Fg” significantly reduced the amount of cells compared to “Mock / Fg”.

  Next, the same sampled barley spike was quantified for mold toxin (DON) using plant tissues inoculated with Fusarium head blight fungus. Specifically, mold poison purification was carried out from barley ears based on the recommended protocol of VICAM using DON-NIV WB immunoaffynity column of VICAM, and detection by HPLC was performed.

  The results are shown in FIG. As shown in the figure, “NMN / Fg” significantly reduced the amount of mold poison compared to “Mock / Fg”.

  From the above, it was confirmed that NMN has the effect of controlling head blight on barley.

  According to the present invention, a plant resistant to head blight can be provided, so that it can be used in agriculture, horticulture, food industry and the like.

Claims (7)

A method for producing a head blight-resistant plant comprising the step of increasing the expression of any gene selected from the group consisting of the following (a) to (e) in a plant:
(A) a gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 5;
(B) In the amino acid sequence described in any one of SEQ ID NOs: 1 to 5, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and has NMNAT activity A gene encoding a protein;
(C) a gene comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 1 to 5 and encoding a protein having NMAT activity;
(D) a gene comprising the base sequence described in any one of SEQ ID NOs: 6 to 10;
(E) A gene that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (a) to (d) above and encodes a protein having NMNAT activity.   2. A head blight-resistant plant obtained by the production method according to claim 1. A method for selecting a plant resistant to head blight, which comprises the step of detecting the presence or absence of any of the following genes (a) to (e) in the test plant or the expression level of the gene:
(A) a gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 5;
(B) In the amino acid sequence described in any one of SEQ ID NOs: 1 to 5, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and has NMNAT activity A gene encoding a protein;
(C) a gene comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 1 to 5 and encoding a protein having NMAT activity;
(D) a gene comprising the base sequence described in any one of SEQ ID NOs: 6 to 10;
(E) A gene that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (a) to (d) above and encodes a protein having NMNAT activity.   Bacterial head blight resistance characterized by comprising measuring the amount of at least one compound selected from nicotinamide mononucleotide, nicotinamide adenyl dinucleotide, and nicotinamide adenyl dinucleotide phosphate in a test plant How to select sex plants. 5. The method for selecting a head blight-resistant plant according to claim 3, comprising at least one substance selected from the following (i) to (v): Kit for:
(I) the gene of any of the following (a) to (e) (a) a gene encoding a protein comprising the amino acid sequence described in any one of SEQ ID NOs: 1 to 5;
(B) In the amino acid sequence described in any one of SEQ ID NOs: 1 to 5, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and has NMNAT activity A gene encoding a protein;
(C) a gene comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in any of SEQ ID NOs: 1 to 5 and encoding a protein having NMAT activity;
(D) a gene comprising the base sequence described in any one of SEQ ID NOs: 6 to 10;
(E) a gene that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (a) to (d) above and encodes a protein having NMNAT activity,
(Ii) a partial fragment of (i),
(Iii) a detection instrument in which (i) or (ii) is immobilized,
(Iv) a primer set for amplifying the gene of (i),
(V) Reagents for measuring the amount of at least one compound selected from nicotinamide mononucleotide, nicotinamide adenyl dinucleotide, and nicotinamide adenyl dinucleotide phosphate.   A plant disease characterized by containing as an active ingredient at least one compound selected from nicotinamide mononucleotide, nicotinamide adenyl dinucleotide, nicotinamide adenyl dinucleotide phosphate, and salts thereof acceptable in agriculture and horticulture Control agent.   Plant or plant growing plant with an effective amount of at least one compound selected from nicotinamide mononucleotide, nicotinamide adenyl dinucleotide, nicotinamide adenyl dinucleotide phosphate, and salts acceptable in agriculture and horticulture The plant disease control method characterized by including the process to process to.