JPWO2014123157A1 - Rhizoctonia resistance gene - Google Patents

Abstract

Rice FOX Arabidopsis thaliana strains resistant to both tomato spotted bacterial pathogens and cruciferous vegetable anthrax fungi were infected with rhizobonia. A strain showing resistance to bacteria was also selected. The selected lines were found to overexpress the OsPSR1 gene. Therefore, we tried to produce rice that overexpresses OsPSR1. As a result, it was confirmed that the rice was resistant to Rhizoctonia.

Description

  The present invention relates to a method for imparting resistance to Rhizoctonia bacteria to plants. The present invention also relates to a transformed plant having resistance to Rhizoctonia and a method for producing the plant.

Increasing plant disease resistance and producing disease-resistant crops is effective in reducing pesticide cultivation. The three major diseases of rice are blast, white leaf blight and coat blight. Of these, many effective resistance genes are known for blast and white leaf blight.
The inventors of the present invention have so far described more than 20,000 Arabidopsis thaliana strains (Rice FOX Arabidopsis thaliana, Non-Patent Document 1) that have over-expressed full-length cDNA of rice, by using the Pseudomonas syringae pv. Tomato DC3000, Pst 3000) and the cruciferous vegetable anthracnose fungus ( Colletotrichum higginsianum ), screening for resistance to both fungi, and identifying more than 10 rice genes responsible for it (non- Patent Documents 2 and 3). Transformed rice that overexpresses one of these genes, BSR1, showed strong resistance to bacterial leaf blight and blast fungus (Patent Documents 1 and 2). However, few effective resistance genes are known for rice blight caused by Rhizoctonia, a basidiomycete.

Kondou, Y. et al., (2009) Plant J. 57: 883-894 Dubouzet, J. et al., (2011) Plant Biotechnology Journal 9: 466-485 Abstracts of 9th International Symposium of Rice Functional Genomics (2011) S12-4 Ito, T. et al., (2000) Plant Cell 12: 1541-1550 Fang, W. et al., (2012) Plant J. 70: 929-939 Nakamura, H. et al., (2007) Plant Mol Biol. 65: 357-371 Toki, S. et al., (2006) Plant J. 47: 969-76

WO 2010/023974 US Patent Application Publication Number 2011-0258737

  This invention is made | formed in view of such the present condition, and makes it a subject to provide the method of providing the resistance with respect to Rhizoctonia bacterium of a pathogenic basidiomycete to a plant. Specifically, it is an object of the present invention to provide a method for imparting resistance to Rhizoctonia bacteria on a plant by a gene having a function of imparting resistance to Rhizoctonia bacteria on pathogenic basidiomycetes. Another object of the present invention is to provide a plant having resistance to Rhizoctonia transformed with the gene and a method for producing the plant.

The inventors of the present invention have intensively studied to solve the above problems. That is, first, the present inventors infected Rhizoctonia fungi with rice FOX Arabidopsis thaliana strains that showed resistance to both the tomato spotted leaf bacterial fungus and the Brassicaceae vegetable anthrax fungus that are bacteria. In addition to both tomato spotted bacterial fungi and cruciferous vegetable anthrax fungi, strains that were resistant to Rhizoctonia were selected. The selected lines were found to overexpress the OsPSR1 gene. Therefore, the present inventors tried to produce rice that overexpresses OsPSR1 . As a result, it was confirmed that the rice was resistant to Rhizoctonia.
The present invention is based on such knowledge and provides the following [1] to [8].
[1] A method of imparting resistance to Rhizoctonia bacteria to a plant, comprising the following steps (a) and (b):
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA encoding a protein having a function of imparting resistance, and (iv) a DNA that hybridizes under stringent conditions with a DNA comprising the base sequence described in SEQ ID NO: 1 or 2, comprising: DNA having a function of imparting resistance to bacteria.
[2] A drug for imparting resistance against Rhizoctonia bacteria to a plant, which contains DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA as an active ingredient;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, A DNA encoding a protein having a function of imparting resistance, and (d) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2, which is applied to plants DNA having a function of imparting resistance to bacteria.
[3] A transformed plant having resistance to Rhizoctonia, into which a DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA is introduced;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, A DNA encoding a protein having a function of imparting resistance, and (d) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2, which is applied to plants DNA having a function of imparting resistance to bacteria.
[4] A plant that is resistant to Rhizoctonia, which is a descendant or clone of the transformed plant according to [3].
[5] A breeding material for the transformed plant according to [3] or [4], which is resistant to Rhizoctonia bacteria.
[6] A cell isolated from the transformed plant according to [3] or [4], which is resistant to Rhizoctonia.
[7] An organ isolated from the transformed plant according to [3] or [4], which is resistant to Rhizoctonia.
[8] A method for producing a transformed plant that is resistant to Rhizoctonia, including the following steps (a) and (b):
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA encoding a protein having a function of imparting resistance, and (iv) a DNA that hybridizes under stringent conditions with a DNA comprising the base sequence described in SEQ ID NO: 1 or 2, comprising: DNA having a function of imparting resistance to bacteria.

According to the present invention, a novel gene OsPSR1 which provides a trait resistant to Rhizoctonia is provided. Arabidopsis overexpressing this gene was resistant to Rhizoctonia, and rice overexpressing this gene was resistant to blight caused by Rhizoctonia.

Figure 1 is a graph showing the degree of resistance to R.solani in OsPSR1 overexpressing Arabidopsis thaliana. Columbia (Col-0) indicates a wild type. OsPSR1 : OX-3, -4 indicates a strain overexpressing OsPSR1 . *** indicates that there is a significant difference at the 0.1% level compared to Col-0 and vector control. FIG. 2 is a graph showing the degree of resistance to a) Pst 3000 and b) C. higginsianum in OsPSR1- overexpressing Arabidopsis thaliana. WT indicates wild type. Eil-0 is a C. higginsianum resistant Arabidopsis ecotype. OsPSR1 : OX-3, -4 indicates a strain overexpressing OsPSR1 . * Indicates that there is a significant difference at the 5% level compared to WT. *** indicates that there is a significant difference at the 0.1% level compared to WT and vector control. FIG. 3 is a graph showing a) plant height and b) flower size of OsPSR1- overexpressing Arabidopsis thaliana. Columbia (Col-0) indicates a wild type. OsPSR1 : OX-1, -2, -5, -6 indicates a strain overexpressing OsPSR1 . ** indicates a significant difference at the 1% level compared to Col-0. FIG. 4 is a graph showing the degree of resistance to blight in OsPSR1- overexpressing rice. The results of resistance tests using a) the leaf tip half and b) the leaf proximal half are shown respectively. The length of lesions on the 8th day after inoculation was shown. Nipponbare shows the wild type. OsPSR1: OX represents rice (T0 generation) that overexpresses OsPSR1 . * Indicates that there is a significant difference at the 5% level compared to Nipponbare. FIG. 5 is a graph showing the degree of resistance to tomato spotted bacterial disease in OsPSR1- overexpressing tomatoes. WT indicates wild type (tomato variety Microtom). OsPSR1: OX represents a tomato overexpressing OsPSR1 . * Indicates that there is a significant difference at the 5% level compared to WT.

The present invention relates to a method for imparting resistance against Rhizoctonia bacteria to plants using the OsPSR1 gene. The present invention also provides a transformed plant having resistance to Rhizoctonia obtained by the method. Specifically, the present invention provides a method for imparting resistance to Rhizoctonia bacteria to a plant including the following steps (a) and (b), and a transformed plant obtained by the method.
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA encoding a protein having a function of imparting resistance, and (iv) a DNA that hybridizes under stringent conditions with a DNA comprising the base sequence described in SEQ ID NO: 1 or 2, comprising: DNA having a function of imparting resistance to bacteria.

In the present specification, the DNA described in (i) to (iv) above is referred to as “the DNA of the present invention” or “ OsPSR1 gene”. The protein encoded by the DNA of the present invention is referred to as “protein of the present invention” or “OsPSR1 protein”.
In the present invention, “comprising” means both “comprising” and “consisting of”.

The nucleotide sequences of the OsPSR1 gene cDNA are shown in SEQ ID NOs: 1 and 2, and the amino acid sequence of the protein (OsPSR1 protein) encoded by the coding region of these nucleotide sequences is shown in SEQ ID NO: 3.
The nucleotide sequence of SEQ ID NO: 1 is known as DDBJ Accession No: AK072163. The coding region of the protein in the base sequence described in SEQ ID NO: 1 is from the 106th base to the 1761st base (including a stop codon).
On the other hand, the base sequence of SEQ ID NO: 2 is known as RAPDB Accession No: Os08g054730 (Os08t054730-01). The coding region of the protein in the base sequence described in SEQ ID NO: 2 is from the 104th base to the 1759th base (including a stop codon). In the base sequence described in SEQ ID NO: 2, the GT at the beginning of the base sequence of SEQ ID NO: 1 is deleted.

The form of DNA used in the present invention is not particularly limited, and may be cDNA or genomic DNA. Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For example, genomic DNA was prepared from the target plant by designing an appropriate primer pair from the known base sequence information (SEQ ID NO: 1, 2) of the OsPSR1 gene (DDBJ Accession No: AK072163, RAPDB Accession No: Os08g054730). It can be prepared by PCR using genomic DNA as a template and screening a genomic library using the resulting amplified DNA fragment as a probe. Similarly, by designing a primer pair, performing PCR using cDNA or mRNA prepared from the target plant as a template, and screening the cDNA library using the resulting amplified DNA fragment as a probe, the OsPSR1 of the present invention A cDNA encoding the protein can be prepared. Furthermore, if a commercially available DNA synthesizer is used, the target DNA can be prepared by synthesis.

  As long as the DNA of the present invention has a function of imparting resistance to Rhizoctonia bacteria in plants, not only DNA encoding the OsPSR1 protein (SEQ ID NO: 3) but also a protein structurally similar to the protein. Encoding DNA (eg, mutants, derivatives, alleles, variants and homologs) can also be used. Such DNA includes, for example, DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 3. DNA encoding a protein having a function of imparting resistance to Rhizoctonia bacteria to plants is included.

  Methods well known to those skilled in the art for preparing DNA encoding proteins with modified amino acid sequences include, for example, the site-directed mutagenesis method (Kramer, W. and Fritz, HJ Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA. Methods in Enzymology. 154, 1987, 350-367.). In addition, the amino acid sequence of the encoded protein may be mutated in nature due to the mutation of the base sequence. Thus, even if the DNA encoding a protein having an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence of natural OsPSR1 (SEQ ID NO: 3), the plant is resistant to Rhizoctonia. As long as it encodes a protein having a function of imparting sex, it is included in the DNA of the present invention.

  The number of amino acids to be modified is not particularly limited, but is generally within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (eg, within 5 amino acids, within 3 amino acids). The amino acid modification is preferably a conservative substitution. The hydropathic index (Kyte, J. and Doolittle, RF J Mol Biol. 157 (1), 1982, 105-132.) And Hydrophilicity value (US Pat. No. 4,554,101) for each amino acid before and after modification are as follows: , Preferably within ± 2, more preferably within ± 1, and most preferably within ± 0.5.

  Moreover, even if the base sequence is mutated, it may not be accompanied by amino acid mutation in the protein (degenerate mutation), and such a degenerate mutant is also included in the DNA of the present invention.

  Examples of DNA encoding a protein structurally similar to the OsPSR1 protein (SEQ ID NO: 3) include hybridization technology (Southern, EM Journal of Molecular Biology. 98, 1975, 503.) and polymerase chain reaction (PCR) technology ( Saiki, RK et al. Science. 230, 1985, 1350-1354 .; Saiki, RK et al. Science, 239, 1988, 487-491.), Prepared using in silico hybridization technology, etc. It is also possible to use it. That is, for those skilled in the art, the DNA base sequence (SEQ ID NO: 1, 2) or a part thereof encoding the OsPSR1 protein (SEQ ID NO: 3) is used as a probe, and the OsPSR1 protein (SEQ ID NO: 3) is encoded. Isolation of DNA having high homology with DNA encoding OsPSR1 protein (SEQ ID NO: 3) from rice and other plants using oligonucleotides that specifically hybridize to DNA as primers is there. In this way, DNA that can be isolated by hybridization technology or PCR technology, has the function of imparting resistance to Rhizoctonia bacteria to plants, or the same function as OsPSR1 protein (function to impart resistance to Rhizoctonia bacteria to plants) DNA encoding a protein having the above is also included in the DNA of the present invention.

  In order to isolate such DNA, the hybridization reaction is preferably carried out under stringent conditions. Those skilled in the art can appropriately select stringent hybridization conditions. An example is 25% formamide, 50% formamide under more severe conditions, 4 × SSC, 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / ml denatured in a hybridization solution containing denatured salmon sperm DNA at 42 ° C. After overnight prehybridization, a labeled probe is added and hybridization is performed by incubating overnight at 42 ° C. The cleaning solution and temperature conditions in the subsequent cleaning are, for example, “2 × SSC, 0.1% SDS, 50 ° C.”, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.” The more severe conditions are “2 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe conditions are “0.2 × SSC, 0.1% SDS, 65 ° C.” It can be carried out at about “° C.”. Thus, isolation of DNA having high homology with the probe sequence can be expected as the hybridization conditions become more severe. 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.

  A plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors. The isolated DNA is considered to have high homology with the amino acid sequence of the OsPSR1 protein (SEQ ID NO: 3) at the amino acid level. High homology means a sequence of at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) in the entire amino acid sequence. Refers to the identity of The identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST (Karlin, S. and Altschul, SF Proc Natl Acad Sci US A. 87 (6), 1990, 2264-2268 .; Karlin, S. and Altschul, SF Proc Natl Acad Sci US A. 90 (12), 1993, 5873-5877.). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul, S.F. et al. J Mol Biol. 215 (3), 1990, 403-410.). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known. The homologue of DNA encoding the OsPSR1 protein (SEQ ID NO: 3) obtained by such a method is highly likely to be functional DNA.

  The DNA of the present invention may be in a form inserted into a vector. The vector is not particularly limited as long as it can express the inserted gene in plant cells. For example, a vector having a promoter for constitutive gene expression in plant cells (for example, potato chitinase gene SK2 promoter, cauliflower mosaic virus 35S promoter, etc.) or inducibly activated by external stimulation It is also possible to use a vector having a promoter.

  Examples of plant cells into which the DNA or vector is introduced include monocotyledonous plants such as rice, wheat, barley, corn, sorghum, and sugarcane, dicotyledonous plants such as Arabidopsis, rapeseed, tomato, soybean, potato, and torenia. However, it is not limited to these.

  The form of the plant cell into which the DNA or vector is introduced is not particularly limited as long as it can regenerate the plant body, and includes, for example, suspension culture cells, protoplasts, leaf sections, and callus.

  The introduction of the DNA or vector into plant cells can be carried out by methods known to those skilled in the art such as polyethylene glycol method, electroporation method, Agrobacterium-mediated method, particle gun method and the like. it can. In the method involving Agrobacterium, for example, according to the method of Nagel et al. (Nagel, R. et al. FEMS Microbiol Lett. 67, 1990, 325-328.) The DNA can be introduced into a plant cell by infecting the plant cell with the Agrobacterium by direct infection method or leaf disk method.

The regeneration of plant bodies from plant cells can be performed by methods known to those skilled in the art depending on the type of plant. For example, for rice, the method of Fujimura et al. (Fujimura. Et al. Tissue Culture Lett. 2, 1995, 74.) can be mentioned, and for wheat, Harris et al. (Harris, R. et al. Plant Cell Reports. 7 1988, 337-340) and Ozgen et al. (Ozgen, M. et al. Plant Cell Reports. 18, 1998, 331-335). For barley, Kihara and Funatsuki (Kihara, M. and Funatsuki, H. Breeding Sci. 44, 1994, 157-160.) and Lurs and Lorz (Lurs, R. and Lorz, H. Theor. Appl. Genet. 75, 1987, 16-25.) In the case of maize, the method of Shillito et al. (Shillito, RD, et al. Bio / Technology, 7, 1989, 581-587.) And Gordon-Kamm et al. (Gordon-Kamm, WJ et al. Plant Cell. 2 (7), 1990, 603-618.). For sorghum, the method of Wen et al. (Wen, FS, et al. Euphytica. 52, 1991, 177-181.) And Hagio (Hagio, T. Breeding Sci. 44, 1994, 121-126.) Ishida Y et al. (Ishida Y, Saito H, Ohta S, Hiei Y, Kom ari T, Kumashiro T. (1996), Nat Biotechnol. 14 (6): 745-50), but is not limited thereto.
For Arabidopsis, the method of Akama et al. (Akama. Et al. Plant Cell Reports. 12, 1992, 7-11.) Can be mentioned. For rape, Wang et al. (Wang, YP et al. Plant Breeding. 124, 2005, 1-4.), And in the case of tomato, Koblitz and Koblitz (Koblitz, H and Koblitz, D. Plant Cell Reports. 1, 1982, 143-146.) And Morgan and Cocking (Morgan , A. and Cocking, ECZPflanzenpysiol. 106, 1982, 97-104.), And for soybeans, Lazzeri et al. (Lazzeri, PA et al., Plant Mol. Biol. Rep. 3, 1985, 160- 167.) and the method of Ranch et al. (Ranch, JP et al., In Vitro Cell Dev. Biol. 21, 1985, 653-658.), Visser et al. (Visser, RGF et al. Theor. Appl. Genet. 78, 1989, 594-600.) Torenia, Aida, R et al. (Aida, R. and Shibata, M. (1995), Breed. Sci. 45: 71-74), but is not limited thereto.

  In addition, when regenerating a plant from a plant cell into which the gene of the present invention has been introduced, it is preferable to remove the T0 plant regenerated from callus from the hormone-free medium and place it in a hydroponic solution. In normal transformed rice, roots emerge when the regenerated T0 plant is transplanted to a hormone-free medium and placed for 1-2 weeks. However, roots do not appear in plants overexpressing the gene of the present invention. In the present invention, the roots can be gradually rooted and elongated by enlarging the above-ground part of the plant body on a hormone-free medium and then transferring it to a hydroponic solution (Mr. Kimura's B solution or the like). After the roots are fully extended, they can be transplanted to soil and cultivated in the same way as ordinary transformed rice. That is, in this invention, a plant body can be reproduced | regenerated by growing with a hydroponic liquid prior to cultivation with soil.

  Once a transformed plant into which the DNA or vector is introduced into the genome is obtained, offspring or clones can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible.

  Whether or not a plant is resistant to Rhizoctonia can be determined by comparison with a control. In the present invention, the control means a plant body of the same species as the plant body of the present invention, in which the DNA of the present invention is not overexpressed. The control in the present invention is not limited at all as long as it is a plant of the same species as the plant of the present invention and the DNA of the present invention is not overexpressed. Therefore, the control of the present invention includes, for example, a plant into which a DNA different from the DNA encoding the OsPSR1 protein of the present invention has been introduced. Examples of such a plant include, for example, a plant of the same species as the transformed plant of the present invention, a plant transformed with a DNA other than the DNA of the present invention, and the function of the DNA of the present invention. A plant transformed with a DNA introduced with a deletion mutation, a plant transformed with a DNA converted to a function-inhibited form of the DNA of the present invention, a DNA fragment of a region insufficient for functional expression of the DNA of the present invention The plant body transformed with is mentioned, but it is not limited to these.

Usually, the rice blight resistance test (resistance test for Rhizoctonia) is performed using rice in the reproductive growth stage before heading. However, in rice transformed with OsPSR1 of the present invention, as described above, root growth does not occur even when cultured in a hormone-free medium after redifferentiating T0 plants from callus by the Agrobacterium method. . Therefore, it is difficult to match the growth stage of the transformed plant of the present invention with that of the wild type. Therefore, in the present invention, it is preferable to determine whether the plant body is resistant to Rhizoctonia using the length of lesions of cut leaves as an index. Whether or not the plant body is resistant to Rhizoctonia can be determined by measuring the lesion length of the cut leaves of the plant infected with Rhizoctonia and comparing it with the control (wild-type plant). . Infection with Rhizoctonia can be performed by cultivating Rhizoctonia and then dropping the bacterial solution onto a cut leaf or applying it to a plant body. Plants transformed with OsPSR1 are less resistant to Rhizoctonia if the lesion length in the transformed plant or the ratio of lesion length to leaf length is statistically significantly shorter than those in the control. It can be determined to have resistance.

Rhizoctonia is a highly acne pathogen and causes diseases such as rice wilt, potato black rot, sugar beet root rot, buckwheat leaf rot, and tomato seedling wilt. The life cycle is completed only with mycelium and forms mycelia when cultured for a long time. Rhizoctonia solani can be exemplified as Rhizoctonia bacterium in the present invention, but it is not limited as long as it has the above-mentioned characteristics.

Furthermore, the present invention provides a transformed plant that is resistant to Rhizoctonia. The plant body is obtained by introducing an OsPSR1 gene having a function of imparting resistance to Rhizoctonia bacteria to a plant or a vector containing the gene into a plant cell and regenerating the plant body from the plant cell. These specific methods are described above.
Once a transformed plant into which the DNA of the present invention has been introduced into the chromosome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. In addition, cells, organs, and propagation materials (eg, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) are isolated from the plant body, its progeny or clones, and the plant body is based on them. Mass production is also possible. The present invention includes a plant cell into which the DNA of the present invention has been artificially introduced, a plant containing the cell, an organ of the plant (eg, a flower, leaf, root, stem, etc.), progeny and clone of the plant And propagation material of the plant and its progeny and clones. These plant cells, plants containing the cells, organs of the plants, progeny and clones of the plants, and propagation materials of the plants and their progeny and clones are resistant to Rhizoctonia. These can be used as materials for imparting resistance to Rhizoctonia bacteria to plants.

The present invention includes an agent for conferring resistance to Rhizoctonia bacteria on a plant, which includes an OsPSR1 gene or a vector that holds the gene in an expressible manner. The form of DNA used for the drug of the present invention is not particularly limited, and may be cDNA or genomic DNA. Moreover, as long as it is possible to confer resistance to Rhizoctonia bacteria when introduced into a plant, not only DNA encoding OsPSR1 protein, but also DNA encoding a protein structurally similar to the protein ( For example, mutants, derivatives, alleles, variants, and homologs) can also be used.
The DNA contained in the drug of the present invention may be in a form inserted into a vector. The vector is not particularly limited as long as it can express the inserted gene in plant cells. For example, a vector having a promoter for constitutive gene expression in plant cells (for example, potato chitinase gene SK2 promoter, cauliflower mosaic virus 35S promoter, etc.) or inducibly activated by external stimulation It is also possible to use a vector having a promoter.
The drug of the present invention may be the above-described DNA or the vector itself into which the DNA is inserted, or may be a mixture of these with other components for introduction into plant cells. For example, the DNA in the present invention includes the DNA, a vector into which the DNA has been inserted, Agrobacterium into which the DNA has been introduced, and a biochemical reagent or solution containing these.

In addition, as long as resistance is provided to Rhizoctonia bacteria, this invention also includes what provides resistance with respect to filamentous fungi or bacteria other than Rhizoctonia bacteria. Examples of such bacteria include, but are not limited to, tomato bacterial leaf blight and fungi such as cruciferous vegetable anthrax. That is, the present invention also provides the following [1] to [8].
[1] A method for imparting resistance to a filamentous fungus and bacterium according to (1) or (2) or a filamentous fungus according to (3), comprising the following steps (a) and (b):
(1) Rhizoctonia fungus, tomato spotted bacterial fungus and cruciferous vegetable anthrax fungus,
(2) Rhizoctonia fungus and tomato spotted bacterial disease,
(3) Rhizoctonia bacteria and cruciferous vegetables anthrax,
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 3, comprising: (1) Or a DNA encoding a protein having a function of imparting resistance to the filamentous fungus and bacteria described in (2) or the filamentous fungus described in (3), and (iv) a base sequence described in SEQ ID NO: 1 or 2 A DNA that hybridizes under stringent conditions with a DNA comprising a plant, and that imparts resistance to a filamentous fungus and bacterium according to (1) or (2) or a filamentous fungus according to (3) DNA having
[2] A filamentous fungus and bacterium according to (1) or (2), which contains, as an active ingredient, a DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA: 3) A drug for imparting resistance to filamentous fungi according to
(1) Rhizoctonia fungus, tomato spotted bacterial fungus and cruciferous vegetable anthrax fungus,
(2) Rhizoctonia fungus and tomato spotted bacterial disease,
(3) Rhizoctonia bacteria and cruciferous vegetables anthrax,
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, Or a DNA encoding a protein having a function of imparting resistance to the filamentous fungus and bacteria described in (2) or the filamentous fungus described in (3), and (d) a base sequence described in SEQ ID NO: 1 or 2 A DNA that hybridizes under stringent conditions with a DNA comprising a plant, and that imparts resistance to a filamentous fungus and bacterium according to (1) or (2) or a filamentous fungus according to (3) DNA having
[3] The filamentous fungus and bacterium according to (1) or (2), wherein DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA is introduced, or (3) Transgenic plants having resistance to filamentous fungi;
(1) Rhizoctonia fungus, tomato spotted bacterial fungus and cruciferous vegetable anthrax fungus,
(2) Rhizoctonia fungus and tomato spotted bacterial disease,
(3) Rhizoctonia bacteria and cruciferous vegetables anthrax,
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, Or a DNA encoding a protein having a function of imparting resistance to the filamentous fungus and bacteria described in (2) or the filamentous fungus described in (3), and (d) a base sequence described in SEQ ID NO: 1 or 2 A DNA that hybridizes under stringent conditions with a DNA comprising a plant, and that imparts resistance to a filamentous fungus and bacterium according to (1) or (2) or a filamentous fungus according to (3) DNA having
[4] A progeny or clone of the transformed plant according to [3], which is resistant to the filamentous fungus and bacteria according to (1) or (2) or the filamentous fungus according to (3) Plant body;
(1) Rhizoctonia fungus, tomato spotted bacterial fungus and cruciferous vegetable anthrax fungus,
(2) Rhizoctonia fungus and tomato spotted bacterial disease,
(3) Rhizoctonia bacteria and cruciferous vegetables anthrax.
[5] A propagation material for a transformed plant according to [3] or [4], wherein the material is a fungus and bacterium according to (1) or (2) or a filamentous fungus according to (3) Propagation material of resistant transformed plants;
(1) Rhizoctonia fungus, tomato spotted bacterial fungus and cruciferous vegetable anthrax fungus,
(2) Rhizoctonia fungus and tomato spotted bacterial disease,
(3) Rhizoctonia bacteria and cruciferous vegetables anthrax.
[6] A cell isolated from the transformed plant according to [3] or [4], wherein the filamentous fungus and bacterium according to (1) or (2) or the filamentous fungus according to (3) Cells that are resistant to;
(1) Rhizoctonia fungus, tomato spotted bacterial fungus and cruciferous vegetable anthrax fungus,
(2) Rhizoctonia fungus and tomato spotted bacterial disease,
(3) Rhizoctonia bacteria and cruciferous vegetables anthrax.
[7] An organ isolated from the transformed plant according to [3] or [4], wherein the filamentous fungus and bacteria according to (1) or (2) or the filamentous fungus according to (3) Organs that are resistant to;
(1) Rhizoctonia fungus, tomato spotted bacterial fungus and cruciferous vegetable anthrax fungus,
(2) Rhizoctonia fungus and tomato spotted bacterial disease,
(3) Rhizoctonia bacteria and cruciferous vegetables anthrax.
[8] A transformed plant that is resistant to the filamentous fungus and bacterium according to (1) or (2) or the filamentous fungus according to (3), comprising the following steps (a) and (b): Manufacturing method of
(1) Rhizoctonia fungus, tomato spotted bacterial fungus and cruciferous vegetable anthrax fungus,
(2) Rhizoctonia fungus and tomato spotted bacterial disease,
(3) Rhizoctonia bacteria and cruciferous vegetables anthrax,
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 3, comprising: (1) Or a DNA encoding a protein having a function of imparting resistance to the filamentous fungus and bacteria described in (2) or the filamentous fungus described in (3), and (iv) a base sequence described in SEQ ID NO: 1 or 2 A DNA that hybridizes under stringent conditions with a DNA comprising a plant, and that imparts resistance to a filamentous fungus and bacterium according to (1) or (2) or a filamentous fungus according to (3) DNA having

In the present invention, tomato spotted bacterial pathogen refers to Pseudomonas syringae pv. Tomato . Tomato spotty bacterial disease occurs in tomato fruit, fruit pattern, petiole and leaf blades. In the leaves, there are many needle-sized brown spots along the veins, or round spots with halos. The spots merge to form large lesions, and in severe cases the leaves die. Fruits, fruit stalks, and petiole have chewy spots or small spots.
In the present invention, the cruciferous vegetable anthrax fungus refers to an ascomycete fungus, Colletotrichum higginsianum . Brassicaceae vegetable anthracnose fungi cause spots on the leaves of cruciferous vegetables such as turnips, komatsuna, radish, and chinensai. Under high humidity, the lesions spread out and cause leaf rot. If it is intense, it will rot so that the seedling will melt.

The present inventors have also found that rice seeds that overexpress OsPSR1 are large and that the rice has extremely low fertility. Therefore, the present invention also provides the following [1] to [8].
[1] A method for increasing the size of rice seeds, comprising the following steps (a) and (b):
(A) a step of introducing DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into rice cells; and (b) rice into which the DNA or vector has been introduced in step (a). Regenerating rice from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 3, and the size of rice seeds DNA that encodes a protein having a function of increasing the length and (iv) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2, DNA that has the function of increasing the size.
[2] A drug for increasing the size of rice seeds, comprising DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA as an active ingredient;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, wherein the size of rice seeds A DNA encoding a protein having a function of increasing the length and (d) a DNA that hybridizes under stringent conditions with a DNA comprising the base sequence set forth in SEQ ID NO: 1 or 2, comprising: DNA that has the function of increasing the size.
[3] Transformed rice with an increased seed size into which a DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA is introduced;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, wherein the size of rice seeds A DNA encoding a protein having a function of increasing the length and (d) a DNA that hybridizes under stringent conditions with a DNA comprising the base sequence set forth in SEQ ID NO: 1 or 2, comprising: DNA that has the function of increasing the size.
[4] A transformed rice having an increased seed size, which is a descendant or clone of the transformed rice according to [3].
[5] A breeding material for transformed rice according to [3] or [4], wherein the seed is increased in size.
[6] A cell isolated from the transformed rice according to [3] or [4].
[7] An organ isolated from the transformed rice according to [3] or [4].
[8] A method for producing transformed rice having increased seed size, comprising the following steps (a) and (b):
(A) a step of introducing DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into rice cells; and (b) rice into which the DNA or vector has been introduced in step (a). Regenerating rice from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 3, and the size of rice seeds DNA that encodes a protein that increases the thickness and (iv) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2, wherein the size of rice seeds is determined DNA with increasing function.

Alternatively, the present invention also provides the following [1] to [8].
[1] A method for reducing the fertility of rice, including the following steps (a) and (b):
(A) a step of introducing DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into rice cells; and (b) rice into which the DNA or vector has been introduced in step (a). Regenerating rice from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA that encodes a protein having a function of decreasing, and (iv) DNA that hybridizes under stringent conditions with DNA containing the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and reduces the fertility of rice DNA having the function of
[2] A drug for reducing fertility of rice, which contains DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA as an active ingredient;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA that encodes a protein having a function of decreasing, and (d) DNA that hybridizes under stringent conditions with DNA containing the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and reduces the fertility of rice DNA having the function of
[3] Transformed rice with reduced fertility into which DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA has been introduced;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA that encodes a protein having a function of decreasing, and (d) DNA that hybridizes under stringent conditions with DNA containing the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and reduces the fertility of rice DNA having the function of
[4] A transformed rice with reduced fertility, which is a descendant or clone of the transformed rice according to [3].
[5] A breeding material for transformed rice according to [3] or [4], wherein the breeding material for transformed rice has reduced fertility.
[6] A cell isolated from the transformed rice according to [3] or [4].
[7] An organ isolated from the transformed rice according to [3] or [4].
[8] A method for producing transformed rice with reduced fertility, comprising the following steps (a) and (b):
(A) a step of introducing DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into rice cells; and (b) rice into which the DNA or vector has been introduced in step (a). Regenerating rice from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA that encodes a protein having a function of decreasing, and (iv) DNA that hybridizes under stringent conditions with DNA containing the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and reduces the fertility of rice DNA having the function of
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited to these Examples.
1. Materials and methods
(1) Rice FOX Arabidopsis lines tomato Madaraha bacteria fungus, the screening of rice FOX Arabidopsis lines that are resistant to cruciferous vegetables anthracnose fungus and Rhizoctonia fungus using T ₂ seeds of rice FOX Arabidopsis lines.

(2) Selection for resistance to tomato spotted bacterial disease
Pseudomonas syringae pv. Tomato DC3000 ( Pst 3000) was inoculated into rice FOX Arabidopsis thaliana strains at a bacterial concentration of (0.5-2) × 10 ⁸ CFU / ml by the improved Dip method (Dubouzet et al., 2011). The FOX strain 3 weeks after sowing was immersed in the above bacterial solution, and selected based on the presence or absence of survival 6 days later.

(3) C. higginsianum was used for cruciferous vegetable anthrax resistant selection . Infection was performed by the improved dip method used for Pst 3000 selection. The conidia concentration of the infectious solution is 10 ⁵ to 10 ⁶ conidia / ml. A strain that was resistant to the cruciferous vegetable anthrax was selected using the presence or absence of survival 6 days after infection.

(4) Rhizoctonia resistant selection The rice sheath blight fungus was used for R. solani . Three days after passage into a 9 cm petri dish of PDA medium (Nissui), the bacteria spread over the whole and the whole medium were transferred to a mortar, and crushed and stirred with 50 ml of sterilized water to prepare an inoculum. Arabidopsis thaliana was cultivated in soil with a humidity of almost 100%. The inoculum was inoculated in drops of 5 μl per rosette leaf and 3 leaves per plant. After inoculation, the plants were cultivated under humidified conditions, and the lesion length was measured 7 days later. Evaluation was made by comparing the lesion length to the leaf length.

(5) Quantitative evaluation of resistance to tomato spotted bacterial fungus and cruciferous vegetable anthrax
The quantitative evaluation of Pst 3000 resistance was performed by the method described in Dubouzet et al., (2011). Quantitative evaluation of C. higginsianum resistance was performed by inoculating 10 μl drop of the same bacterium into rosette leaves at a concentration of 10 ⁶ conidia / ml, and measuring the lesion length 15 days later.

(6) Production of transgenic rice Nipponbare was used as the rice variety. PRiceFOX (Nakamura et al. 2007) was used as an expression vector in rice. Rice transformation was performed by high-speed transformation method (Toki et al. 2006) using Agrobacterium strain EH105.

(7) Rice sheath blight resistance test The second and third leaf blades were cut into leaf tip-side halves and leaf base-side halves, respectively, and used for inoculation. OsPSR1 : OX rice used T0 plants. Cut leaves were placed on a wet sheet in a petri dish, and 20 μl of the above Rhizoctonia inoculum was dropped per leaf. After inoculation, the lesion length was measured 8 days after being placed under humidified conditions at 25 ° C. for 14 hours.

(8) Production of transformed tomatoes Tomato varieties were dwarf tomato microtoms. The EHA105 strain was used for Agrobacterium. The tomato cotyledons 7 days after sowing were pre-cultured for 2 days in a preculture medium (MS medium containing 1 mg / L zeatin, 3% sucrose, 0.8% agar) and used as explants. Agrobacterium was inoculated by immersing the explants in the bacterial suspension for 3 minutes and then co-cultivating them on the preculture medium for 2 days. The explants after co-cultivation are transplanted to a selection medium (pre-culture medium containing 10 mg / L hygromycin and 200 mg / L carbenicillin), and then a new selection medium every two weeks until adventitious buds are redifferentiated through callus. The cells were cultured while being transplanted. The regenerated adventitious buds were rooted on a rooting medium (1/2 MS medium containing 1% sucrose and 0.8% agar), and then transplanted to a culture soil placed in a pot.

(9) Tomato spotty bacterial disease resistance test Approximately 5 weeks after sowing, the tomato plant was immersed in Pst 3000 bacterial solution (5 × 10 ⁸ cfu / ml) for 20 seconds, and the disease symptoms of the inoculated leaves were observed after 6 days. . For quantitative assay, soak in a Pst 3000 bacterial solution (2 × 10 ⁸ cfu / ml) for 20 seconds, grind the inoculated leaves after 3 days, dilute the ground solution and place on a plate. The number of bacteria was counted.

2. result
(1) Selection of Tomato Spotted Bacterial Bacterial Resistance Screening About 21,000 rice FOX Nazuna lines were screened, and finally 85 lines showing Pst 3000 resistance were selected.

(2) Selection of resistance to cruciferous vegetable anthrax fungi The above-mentioned 85 strains were further subjected to a C. higginsianum resistance test to determine whether there were strains exhibiting combined resistance against both strains. As a result, compound resistance was confirmed in 18 lines. Genomic DNA was extracted from these strains, and the gene was identified by amplifying the inserted rice full-length cDNA by PCR and determining the terminal nucleotide sequence.

(3) Production of reintroduced over-expressing Arabidopsis thaliana After identifying the above 18 strains of inserted genes, some cDNAs were obtained from the Rice Genome Resource Center of the National Institute of Agrobiological Resources and used for the production of rice FOX It was ligated to the SfiI site of pBIG2113SF vector (Kondou et al., 2009). The resulting plasmid was introduced into Agrobacterium GV3101 and introduced into Arabidopsis thaliana by the Floral dip method to produce a reintroduced overexpressing Arabidopsis thaliana line.

(4) Rhizoctonia resistance selection The R.solani resistance test was further performed using the above reintroduction lines to obtain lines showing resistance. The line showing resistance was a reintroduction line of OsPSR1 (AK072163, Os08g0547300) (FIG. 1). Furthermore, the quantitative evaluation method confirmed that the OsPSR1 reintroduced line showed Pst3000 resistance and C. higginsianum resistance (Fig. 2). Hlast homology analysis revealed that OsPSR1 encodes a novel cytochrome P450 protein. Among the known proteins, the homology with Arabidopsis CYP78A9 (AT3G61880) and CYP78A6 (AT2G46660) was the highest. Although there are reports that these genes are involved in morphogenesis of reproductive organs such as flowers and pods and seeds, there is no report regarding disease resistance (Ito et al., 2000; Fang et al., 2012).

(5) OsPSR1-overexpressing Arabidopsis morphology
OsPSR1 overexpressing Arabidopsis thaliana plant height was higher than WT (Fig. 3a) and flowers were larger (Fig. 3b). The flowers were large in petals, sepals, stamens and pistils. The sheath was long, the seeds were large and the fertility was low. The hypocotyl and petiole were long, and the leaves were growing flat.

(6) Production of overexpressed rice and disease resistance test Regenerated seedlings of rice overexpressing the OsPSR1 gene (AK072163) could be obtained on a hormone-free medium by the method described in 1. (6) above. The seedlings of the expression did not get root for some reason, and these could not be transplanted to the soil. Therefore, when the regenerated seedlings were transferred to a hydroponic solution (Kimura B solution) and hydroponically cultivated, root rooting and elongation were gradually observed. After the roots were fully extended, they were transplanted to soil and cultivated in the same manner as ordinary transformed rice. The rice blight resistance test was performed in the T0 generation of rice that highly expresses OsPSR1, and rice (Nipponbare) at almost the same size and growth stage was used as a control. After inoculation with the fungus, the symptoms of WT increased in Nipponbare, whereas the overexpressing body showed almost no expansion. Therefore, rice overexpressing the OsPSR1 gene was shown to be resistant to blight (FIG. 4). The OsPSR1- overexpressing rice seeds were large and extremely low in fertility.

(7) Production of overexpressed tomato and disease resistance test
A tomato (microtom) overexpressing the OsPSR1 gene (AK072163) was prepared, and a tomato spotty bacterial disease resistance test was performed in the T1 generation. After inoculation, the symptom increased in the WT tomato leaves, whereas the overexpressing body showed almost no expansion. Furthermore, when the number of viable bacteria in the plant body was measured, it was significantly less in the overexpressing body. Therefore, it was shown that OsPSR1 overexpressing tomato is resistant to tomato spotted bacterial disease (FIG. 5). The form of overexpressed tomato was not significantly different from WT.

According to the present invention, a novel gene OsPSR1 is provided that gives plants such as rice and Arabidopsis thaliana a trait resistant to the pathogenic basidiomycete R. solani . Introduction of OsPSR1 into plants can be expected to impart resistance to a plurality of pathogenic bacteria including Rhizoctonia. The present invention enhances disease resistance of plants to produce disease-resistant crops, and is useful in the agricultural field of reduced agricultural chemicals.

Claims (8)

A method of imparting resistance to Rhizoctonia bacteria to a plant, comprising the following steps (a) and (b):
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA encoding a protein having a function of imparting resistance, and (iv) a DNA that hybridizes under stringent conditions with a DNA comprising the base sequence described in SEQ ID NO: 1 or 2, comprising: DNA having a function of imparting resistance to bacteria. A drug for imparting resistance to Rhizoctonia bacteria to a plant, which contains DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA as an active ingredient;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, A DNA encoding a protein having a function of imparting resistance, and (d) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2, which is applied to plants DNA having a function of imparting resistance to bacteria. A transformed plant having resistance to Rhizoctonia bacterium into which a DNA selected from the group consisting of (a) to (d) below or a vector containing the DNA is introduced;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, A DNA encoding a protein having a function of imparting resistance, and (d) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2, which is applied to plants DNA having a function of imparting resistance to bacteria. A plant body that is resistant to Rhizoctonia, which is a descendant or clone of the transformed plant body according to claim 3. A propagation material for a transformed plant according to claim 3 or 4, wherein the propagation material is resistant to Rhizoctonia. A cell isolated from the transformed plant according to claim 3 or 4, which is resistant to Rhizoctonia. An organ isolated from the transformed plant according to claim 3 or 4, which is resistant to Rhizoctonia. A method for producing a transformed plant that is resistant to Rhizoctonia bacteria, comprising the following steps (a) and (b):
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(Ii) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1 or 2,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, DNA encoding a protein having a function of imparting resistance, and (iv) a DNA that hybridizes under stringent conditions with a DNA comprising the base sequence described in SEQ ID NO: 1 or 2, comprising: DNA having a function of imparting resistance to bacteria.

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