JP2004024160A - Process for producing disease-resistant plant with flagellin protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To impart disease resistance to rice plants. <P>SOLUTION: A flagellin-encoding gene 1141-fla1 derived from a plant pathogenic bacterium, Acidoborax aveane strain N1141, is fused to a cauliflower mosaic virus 35S promoter and introduced into rice. Individuals showing high flagellin accumulation are selected from among the obtained T<SB>0</SB>transgenic plants and selfed to yield selfed progenies T<SB>1</SB>. The T<SB>1</SB>plants are inoculated with pyricularia oryzae and tested for their resistance to rice blast to demonstrate increased blast resistance in transgenic rice plants. <P>COPYRIGHT: (C)2004,JPO

Description

【００３５】
およそ２／３の形質転換体で明らかなフラジェリンタンパク質の蓄積が検出された。これらフラジェリン発現個体の葉には発育に伴い白い斑点が現れた。また多くの個体に多少の草丈の低下が見られたが、種子稔性は正常であったので、フラジェリン発現個体のＴ_１種子を得ることが出来た。一部の個体に関しては１１４１−ｆｌａ１遺伝子（ＸｈｏＩ，ＨｉｎｄＩＩＩ　１．５ｋｂ断片）をプローブとしたゲノミックサザン分析により、導入したフラジェリン遺伝子のコピー数を推定した。
【００３６】
ｂ）ノーザン分析による防御関連遺伝子の発現分析
フラジェリンを発現した形質転換体において、防御関連遺伝子の発現が誘導されているか否かを調べるため、フラジェリンの発現した形質転換体４系統（Ｄ／１１−２０−２、Ｄ／１１−２４−３、Ｄ／１１−２７−１、Ｄ／１１−２７−２）、およびｐＳＢ２４（３５Ｓ　ｐｒｏ−ＩＧＵＳ−ＮＯＳ　ｔｅｒ、Ｋｏｍａｒｉ　ら（１９９６）Ｐｌａｎｔ　Ｊ．　１０：１６５−１７４）を導入した対照組換え体２系統（Ｑ１、Ｑ２）の葉から、フェノール−ＳＤＳ法によってＲＮＡを抽出し、ＰＡＬ遺伝子をプローブとしたノーザン分析を行なった。ＰＡＬ遺伝子は以下のようにクローニングした。ＯＳＰＡＬ（Ａｃｃｅｓｓｉｏｎ　Ｎｏ．Ｘ１６０９９）の配列を基にｃｏｄｉｎｇ　ｒｅｇｉｏｎの全長を伸ばすプライマーを設計し、Ｏｃ　ｃｕｌｔｕｒｅｄ　ｃｅｌｌ　（Ｏｒｙｚａ　ｓａｔｉｖａ　Ｌ．　Ｃ５９２４（Ｂａｂａ，Ａ．ｅｔ　ａｌ．（１９８６）Ｐｌａｎｔ　Ｃｅｌｌ　Ｐｈｙｓｉｏｌ．２７，４６３−　４７２））から、ＲＴ−ＰＣＲによりＰＡＬ遺伝子をクローニングした。塩基配列の解析を行いその遺伝子がＰＡＬであることを確認した。ＰＡＬ遺伝子は傷害や、病原菌の細胞壁由来のエリシターなどによって、転写が誘導されることが報告されている（Ｚｈｕ　ｅｔａｌ．１９９５　Ｐｌａｎｔ　Ｍｏｌ　Ｂｉｏｌ　２９：５３５−５５０）。
【００３７】
形質転換体および対照系統から抽出したｔｏｔａｌ　ＲＮＡ（１５（ｇ）を１ｘ　ＭＯＰＳ　ｂｕｆｆｅｒ（２０ｍＭ　ＭＯＰＳ−ＫＯＨ、ｐＨ７．０、５ｍＭ　ｓｏｄｉｕｍ　ａｃｅｔａｔｅ，ａｎｄ　１ｍＭ　ＥＤＴＡ）中で、ホルムアルデヒドを含む１．５％アガロースゲルで電気泳動したのち、ナイロンメンブレンＨｙｂｏｎｄ　Ｎ^＋（Ａｍｅｒｓｈａｍ）に転写した。ＰＡＬ遺伝子プローブは上述のＰＣＲ産物をｍｉｃｒｏｃｏｎ　ＹＭ−３０　（Ｍｉｌｌｉｐｏｒｅ）で精製した後、［^３２Ｐ］−ｄＣＴＰでラベルしたものを使用した。
【００３８】
その結果、調査した４系統全てにおいて、対照と比較してＰＡＬ遺伝子の転写物の蓄積量が多かった（図２）。このことから、フラジェリン導入によって形質転換イネには防御機構の少なくとも一部が作動していることが示唆された。
【００３９】
ｃ）Ｔ１世代におけるフラジェリン発現イネのいもち病抵抗性試験
１１４１−ｆｌａ１遺伝子が導入されたＴ_０世代の形質転換体のうちフラジェリンが蓄積した４系統（Ｄ／１１−２０−１、Ｄ／１１−２０−２、Ｄ／１１−２０−３、Ｄ／１１−２４−３）、およびウェスタン分析ではフラジェリン蓄積が検出限界以下であった１系統（Ｄ／１１−３１−１）を選び、それらのＴ_１世代におけるイネいもち病に対する罹病度を調査した。選抜された５つの高発現個体の種子稔性は正常で、多くの自殖種子を得ることが出来た。なお対照として、いもち病に対する圃場抵抗性が極弱の水稲品種コシヒカリ（形質転換実験の原系統）、いもち病圃場抵抗性が弱〜中の日本晴、いもち病圃場抵抗性が極強の銀河を用いた。培土を入れたシードリングケースに種子を８粒×２列で撒き、温室内で栽培し、４．８〜５．２葉期に病害検定に供試した。イネいもち病菌（Ｍａｇｎａｐｏｒｔｈｅ　ｇｒｉｓｅａ）はレース００７を用いた。接種にはいもち菌をオートミール・スクロース寒天培地上で培養（２８℃暗条件）し、菌叢成長後、２５℃で３日間近紫外光照射して形成させた分生胞子を用いた。いもち菌の接種は、０．０２％　Ｔｗｅｅｎ２０で２×１０^５個の分生胞子／ｍｌに調整した懸濁液をシードリングケース３つ当たり３０ｍｌ噴霧接種することにより行なった。噴霧接種を行なったイネは接種後２４時間加湿恒温器（ＳＬＰＨ−５５０−ＲＤＳ、日本医化器械製作所製）内で２５℃、１００％湿度条件下に保持した後、温室内へ移した。温室の設定条件は、明条件２５℃−１６時間、暗条件２２℃−８時間とした。病害抵抗性の評価は、接種６日後における接種時の最上位展開葉（第５葉）の進展性病斑数を目視で数え、病徴指数（図３）により評価した。さらに接種１６日後における接種時の最上位展開葉（第５葉）の進展性病斑面積率を、病徴レベル（図５）として評価した。結果についてはコシヒカリとの有意差検定（全群比較、Ｓｔｅｅｌ−Ｄｗａｓｓ検定）を行った。
接種６日後の結果を図４に示した。フラジェリンの発現していない系統Ｄ／１１−３１−１の病徴指数の分布は、原系統であるコシヒカリ（いもち抵抗性弱）と非常によく似ていた。病徴指数の中央値はともに６（一葉当り病斑数は多く、計測不能）であり、レンジはともに４（７−３）であった。いもち抵抗性弱〜中の日本晴においては病徴指数の中央値は５（一葉当り病斑数が２５−５０個）であり、フラジェリン蓄積系統Ｄ／１１−２０−３、Ｄ／１１−２４−３の中央値も５であったが、日本晴の病徴指数のレンジが３であったのに対し、これらフラジェリン蓄積系統のそれは４であり、病徴程度の低い、抵抗性の増強が示唆される個体の割合が多かった。いもち病圃場抵抗性が強の銀河においては、病徴指数の中央値は４（一葉当り病斑数が１０−３０個）であり、指数が３（一葉当り病斑数が３−１５個）と４のものが多く、今回供試した系統の中では最も強い抵抗性を示した。フラジェリン蓄積系統Ｄ／１１−２０−１、Ｄ／１１−２０−２の病徴指数の中央値も４、３．５であり、病徴程度の低い、抵抗性の増強が示唆される個体の割合が多かった。特にＤ／１１−２０−２の分布は銀河のそれと良く似ていた。コシヒカリに対する病徴指数の分布のノンパラメトリックな有意差検定の結果、銀河と、フラジェリンが蓄積した４系統のうち３系統（Ｄ／１１−２０−２、Ｄ／１１−２０−３、Ｄ／１１−２４−３）において、危険率１％で、コシヒカリの病徴指数分布に対する有意差が検出された。
一方接種１６日後の結果（図５）では、原系統のコシヒカリでは供試した３１個体の全個体が枯死した。日本晴とＤ／１１−３１−１、Ｄ／１１−２０−１もコシヒカリと同様な分布を示し、殆どの個体の病徴レベルが９（病斑面積率１００％）であった。これに対し、銀河では半数以上の個体が病徴レベル５以下（病斑面積率２０％以下）であった。接種６日後でコシヒカリとの有意差が見られた３系統Ｄ／１１−２０−２、Ｄ／１１−２０−３、Ｄ／１１−２４−３においては、病徴レベルの低い個体が見られ、これらの系統の病徴レベルの分布図は、統計的にはコシヒカリのそれと１％の危険率で有意な差異が検出された。以上の結果からフラジェリン導入により、イネの病害抵抗性を増強させることが出来ることが示された。
接種１６日後の各個体からタンパク質を抽出、フラジェリンタンパク質の蓄積量をウェスタン分析により解析した。さらにフラジェリン発現量といもち病抵抗性の強さとの関係を調べるため、各個体のフラジェリンの発現量と、その個体の接種６日後のいもち病病徴指数（図３）との関連を図６に示した。ウイルコクソンの順位和検定の結果、フラジェリンを発現する（＋）、しない（−）間で、いもち病病徴指数の分布に５％の危険率で有意な差が得られた。この結果は、いもち病抵抗性の増強が、フラジェリンの発現によってもたらされたことを示すものである。
【００４０】
【表２】

【００４１】
【発明の効果】
本発明により、フラジェリンをコードする遺伝子を構成的プロモーターに接続して、植物に導入することにより、その植物に病害抵抗性を付与できることが初めて明らかとなった。このフラジェリン導入植物は、タンパク質性エリシターであるフラジェリンの作用機作を解明する上で、また局部、全身獲得抵抗性の機構を解明する上で役立つと考えられる。また、誘導性のプロモーターを用いなければ困難であろうと従来考えられていたタンパク質性エリシター導入による抵抗性植物の作出を、構成的プロモーターを用いても十分適用可能であることを示し、本アプローチの適用範囲の拡大を示すことができた。フラジェリンをコードするＤＮＡ配列を、植物細胞の中で機能しうる適当な構成的、器官・時期特異性、あるいはストレスや病害虫、または化学物質で誘導されるプロモーター配列と植物細胞で機能しうるターミネーター配列の発現カセットに組み込み、植物細胞に導入、再生個体を得ることにより、病害抵抗性植物を作出するという方法は、遺伝子工学的に可能かつ有効なアプローチであることを本発明は示した。
【００４２】
【配列表】

【図面の簡単な説明】
【図１】図１は、Ｔ_０世代の形質転換イネにおけるウエスタン分析によるフラジェリンタンパク質の蓄積の検出例を示す図である。ＰＣは対照とした大腸菌発現フラジェリン（３０ｎｇ）を、Ｔは形質転換体（総可溶性タンパク質３０μｇ）を、Ｃは原系統のコシヒカリ（総可溶性タンパク質３０μｇ）をそれぞれ示す。発現量の評価は表１に記載の通りである。
【図２】図２は、Ｔ_０世代の形質転換イネにおけるノーザンブロットによるＰＡＬ遺伝子の発現を示した図である。Ｄ／１１は形質転換体を示す。対照はＧＵＳ遺伝子導入個体を用いた。
【図３】図３は、接種６〜７日後のいもち病病徴指数の模式図である。
【図４】図４は、Ｔ_１世代の形質転換イネにおける接種６日後のいもち病検定結果を示したグラフである。病徴指数の評価は図３に記載の通りである。
【図５】図５は、Ｔ_１世代の形質転換イネにおける接種１６日後のいもち病検定結果を示したグラフである。各病徴レベルにおける病斑面積率は、レベル０が０％、レベル１が１％、レベル２が２％、レベル３が５％、レベル４が１０％、レベル５が２０％、レベル６が４０％、レベル７が６０％、レベル８が８０％、レベル９が１００％である。
【図６】図６は、Ｔ_１世代の形質転換イネにおける接種６日後のいもち病抵抗性の程度とフラジェリン発現レベルの関連を示した図である。病徴指数の評価は図３に、発現量の評価は表１に記載の通りである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing a plant resistant to a pathogen.
[0002]
[Prior art]
Plants do not possess the immune system found in animals, but protect themselves from pathogens by plant-specific mechanisms. The hypersensitive response (HR) of higher plants is a dynamic resistance response on the plant side to pathogen invasion, in which plant cells at the site of infection rapidly commit suicide and locally contain the pathogen. This reaction is known to occur as a result of incompatible host-pathogen and non-host-pathogen interactions. In addition, the suicide of the cell observed here can be regarded as local programmed cell death (Dangl et al. (1996) Plant {Cell} 8: 1793-1807). In addition to the mechanisms that cause HR, other defense responses are also induced, such as reactive oxygen species generation, cell wall enhancement, phytoalexin production, and biosynthesis of defense-related proteins such as PR proteins (Hammond-Kosack and Jones ( 1996) Plant {Cell 8: 1773-1791). In addition to such local defense responses, defense responses, such as PR protein accumulation, often extend to uninfected parts of the plant, resulting in the entire plant becoming resistant. This is called systemic acquired resistance (SAR) and lasts for a few weeks or more, and the whole plant becomes resistant to secondary infection (Sticher et al. (1997) Annu Rev Rev Phytopathol 35: 235). -270).
[0003]
The first reaction on the plant side to switch on such a highly organized defense response is the recognition of a molecule called 'elicitor', which is generated directly or indirectly by invading pathogens. A complex signal cascade such as rapid generation of reactive oxygen species or reversible protein phosphorylation is considered to be important as an initial reaction of defense response (Yang et al. (1997) Genes Dev 11: 1621-). 1639). There are many types of elicitors, and they do not depend on the type of pathogen, such as chitin / chitosan, which is a cell wall component of many fungi, oligosaccharides which are degradation products of glucan, or oligogalacturonic acid derived from the cell wall of plants. A so-called non-specific elicitor and a specific elicitor specific to a non-pathogenic gene product (Avr gene product) on the pathogenic bacterium side such as AVR9 and depending on the type of pathogen, or an intermediate specificity such as elicitin and harpin (Boller (1995) Annu Rev Plant Plant Physiol Plant MoI Biol 46: 189-214).
[0004]
Flagellin is a component of the bacterial flagella flagella. Felix et al. (Plant J 18: 265-276, 1999) purified a 32 kDa protein from Pseudomonas syringae pv tobaci using a cultured tomato cell line, using alkalization of the medium as an indicator of the resistance response. This protein was identified as flagellin from the amino acid sequence. Furthermore, they found that the most conserved 15- to 22-terminal N-terminal peptide in the primary amino acid structure of flagellin in flagellated bacteria induces a similar response in several plant species, including tomato, tobacco, and Arabidopsis. I figured it out. These peptides also induced oxidative burst and ethylene production, and were the first non-specific elicitors to be proteinaceous elicitors. Recently, Asai et al. (Nature @ 415: 977-983, 2002) suggested that the 22-residue peptide (flg22) activates the flagellin receptor FLS2 (Gomez et al., Mol {Cell} 5: 1003-1011,2000) in a dependent manner. Elucidated the MAP kinase cascade. Furthermore, they reported that transient expression of WRKY29, a transcription factor of these MAP kinases and many defense-related genes, in Arabidopsis enhances resistance to bacteria and filamentous fungi.
[0005]
Flg22 does not cause extracellular alkalinization in cultured rice cells (Felix et al., Plant 18: 265-276, 1999). Che et al. (J Biol Chem 275: 32347-32356, 2000) induce cell death in cultured rice cells from the phytopathogenic bacterium Acidoborax aveane strain N1141 (monad of brown flies) in monocotyledonous hosts. A 50 kDa protein was identified. The amino acid sequence identified this protein as flagellin. However, there has been no example in which flagellin itself was introduced into a plant and a transformant was analyzed.
[0006]
[Problems to be solved by the invention]
The problem to be solved by the present invention is to create a plant having disease resistance.
[0007]
[Means for Solving the Problems]
The present inventors introduced a gene encoding a flagellin (N1141 flagellin) derived from Acidoborax aveane N1141 strain, which induces hypersensitive cell death in rice, into rice, analyzed the transformants, and found that the strain in which flagellin had accumulated It was found that the resistance to blast, the most important disease of blast, was enhanced. The present invention relates to a method for creating a plant having disease resistance using the same.
[0008]
Here, “disease resistance” means that it is difficult to get a certain disease. Specific examples include a decrease in the number of lesions, a decrease in lesion size, and a reduction in symptom. In the present invention, when an individual into which the flagellin gene has been introduced is compared with an individual into which the flagellin gene has not been introduced, it means that the individual into which the gene has been introduced is less susceptible to the diseases exemplified above.
[0009]
In a broad aspect of the present invention, the present invention provides a method for introducing disease into a plant by introducing a gene encoding a flagellin having elicitor activity in a plant or a peptide constituting a part thereof (hereinafter, referred to as a flagellin gene). A method for imparting resistance and producing a disease-resistant plant is provided. In another broad aspect, the flagellin gene introduced into the plant by the method of the invention is connected to a promoter that works in plant cells.
[0010]
In one embodiment of the present invention, the flagellin gene to be introduced into a plant is a gene encoding a flagellin derived from a plant pathogenic bacterium (Acidoborax aveane N1141 strain), and the flagellin protein has the amino acid sequence of SEQ ID NO: 1. Therefore, in one embodiment, the flagellin gene to be introduced into a plant is a gene encoding the amino acid sequence of SEQ ID NO: 1. In a desirable embodiment, the gene to be introduced into a plant is a gene encoding an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO: 1. In a more desirable embodiment, the gene to be introduced into the plant is a gene encoding an amino acid sequence having at least 70% homology with the amino acid sequence of SEQ ID NO: 1. In another embodiment, the gene to be introduced into a plant is a gene encoding an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1.
[0011]
Here, the “part” of the amino acid is defined as “A. avenae refers to the amino acid sequence of flagellin or a part of a molecule having homology to the DNA sequence of its gene. Felix et al., Pseudomonas's paper on flagellin refers to a peptide of at least 15 amino acids in the above amino acid sequence, since a peptide having a minimum of 15 residues has elicitor activity when given from the outside. More specifically, there may be mentioned the 15th amino acids at positions 37 to 51 of SEQ ID NO: 1.
[0012]
In addition, the DNA sequence of SEQ ID NO: 2 shows a gene encoding flagellin (the amino acid sequence is shown in SEQ ID NO: 1) derived from a plant pathogenic bacterium (Acidoborax aveane N1141 strain). In the method of the present invention, a gene having the DNA sequence of SEQ ID NO: 2 may be introduced into a plant, preferably, at least 90% homology with the DNA sequence of SEQ ID NO: 2, more preferably, at least 70% homology. May be introduced. In another embodiment, the gene to be introduced into a plant is a gene comprising a DNA sequence in which one or more bases have been substituted, deleted, inserted, and / or added in the DNA sequence of SEQ ID NO: 2.
[0013]
Here, the “part” of the DNA is defined as “A. avenae refers to the amino acid sequence of flagellin or a part of the DNA sequence of the gene itself. Felix et al.'S Pseudomonas flagellin article refers to 45 or more bases of the above DNA, since a peptide having a minimum of 15 residues has elicitor activity when given from the outside. More specifically, in SEQ ID NO: 2, 45 bases at positions 109 to 153 can be mentioned.
[0014]
Further, the number of "substitutions, deletions, insertions, and additions" obtained in one experiment is 10 or less. These can be converted by site-directed mutagenesis (eg, Nucl. Acids Res. 10: 6487-6500, 1982). Further, the term “deletion” includes those in which a base is deleted from the end of the base sequence and those in which a base in the middle of the base sequence is deleted.
[0015]
In order to hybridize with the DNA described in SEQ ID NO: 2 under stringent (High stringency) conditions, general hybridization such as 5 × denhardt solution, 5 × SSC, 0.5% SDS or 0.1% SDS is used. The reaction may be carried out using a solution at 32 to 68 ° C, preferably in two steps of 50 ° C and 60 ° C, or in four steps of 50 ° C, 55 ° C, 60 ° C, and 65 ° C. This condition can be carried out at a slightly lower temperature when formamide is present than when no formamide is present. For hybridization under mild (low stringency) conditions, 30% deionizedformamide, 0.6 M NaCl, 0.04 M sodium phosphate (pH 7.4), 2.5 mM EDTA, and 1% SDS are used. Should be performed. Further, conditions for washing the hybridized product can be 0.2 or 2 × SSC, 0.1% SDS, and a temperature of 20 to 68 ° C. Whether the conditions are stringent (high @ stringency) or mild (low @ stringency) can be determined depending on the salt concentration or temperature during Wash. When a difference in hybridization is provided depending on the salt concentration, the hybridization can be performed using 0.2 × SSC, 0.1% SDS as high stringency wash buffer, 2 × SSC as 0.1% SDS as low stringency wash buffer, and 0.1% SDS. In addition, when a difference in hybridization is provided by temperature, 68 ° C. in the case of high stringency, 42 ° C. in the case of moderate stringency, and room temperature (20-25 ° C.) in the case of low stringency. X SSC, 0.1% SDS.
[0016]
Flagellin is a protein widely distributed in flagellated bacteria. In addition to the above-mentioned flagellin derived from the Acidoborax aveane strain N1141, for example, Pseudomonas syringae pv taosauna, Pseudomonas osamudosa, Pseudomonas ギ モ モ ギ ｍ, ａｃ フ ラ ａｃ ａｃ ジ ェ. putida), Helicobacter mustelae, Campylobacter coli, Vibrio parahaemolyt, Serratia marcescens Proteus mirabilis (Proteus mirabilis), there is a Bacillus subtilis (Bacillus subtilis) derived from the flagellin protein. The use of the genes encoding these flagellin proteins in the method of the present invention is also within the scope of the present invention.
[0017]
In the present invention, the flagellin gene is introduced into a plant by connecting it to a promoter that functions in a plant cell. In the present invention, it has been shown for the first time that connecting a flagellin gene to a promoter that functions in a plant cell makes the plant resistant to disease. In the examples described below, a cauliflower mosaic virus 35S promoter that expresses constitutively was used as a promoter that functions in plant cells, but is not limited thereto. If a technique for imparting disease resistance by combining a constitutive promoter (promoter with low disease and stress response and low tissue / time specificity) and flagellin is disclosed, the cauliflower mosaic virus shown in Examples described below will be disclosed. Constitutive promoters other than the 35S promoter, for example, ubiquitin promoters (Cornejo et al. (1993) Plant Mol Biol 23: 567-581), actin promoters (McElroy et al. (1990) Plant {Cell} 2: 163-171), alpha Tubulin promoter (Carpenter et al. (1993) Plant Mol Biol 21: 937-942), Sc promoter (Schenk et al. (1) 99) Constitutive promoters such as Plant \ Mol \ Biol 39: 1221-1230, or PPDK (Matsuoka et al. (1993) Proc \ Natl \ Acad \ Sci \ USA \ 90: 9586-9590) and PEPC (Yanagzawa \ and \ Izumi106). : 982-987, Matsuoka et al. (1994) Plant @ J 6: 311-319), Rubisco (Matsuoka et al. (1994) Plant @ J 6: 311-319), etc., are highly expressed in green organs. It is expected that the combination of a promoter and a flagellin gene will impart disease resistance. Disease-inducible promoters, for example, PAL promoter (Zhu et al. (1995) Plant Mol Biol 29: 535-550), Prp1 promoter (Tokuhei 10-500312), hsr203J promoter (Pontier et al. (1994)) Plant J 5: 507-521), EAS4 promoter (Yin et al. (1997) Plant Physiol 115: 437-451), PR1b1 promoter (Tornero et al. (1997) Mol Plant Microbe Interact 10: 624-634), t Promoter (Mohan et al. (1993) Plant MoI Biol 22: 475-490), A PR1 promoter (Warner et al (1993) Plant J 3:. 191-201) and promoters such as disease resistance imparting are expected also in combination with flagellin gene. A method using such a combination is also included in the present invention.
[0018]
The method of the present invention includes the following steps.
(A) connecting the flagellin gene downstream of the aforementioned promoter and upstream of the terminator to construct an expression cassette,
(B) constructing a plasmid having the above expression cassette and a selectable marker gene,
(C) introducing this into the plant genome to transform the plant,
(D) screening the transformed plant cells with a selectable marker,
(E) Complete plants (T₀Generation) to obtain disease-resistant plants.
(F) T₀Seeds (T₁Generation) and cultivating the seed to obtain a disease resistant plant.
[0019]
Here, the flagellin gene is introduced into a plant genome by a transformation method known to those skilled in the art, for example, Agrobacterium method, electroporation method, PEG method, microinjection method, particle gun method, or the like. May be performed. Preferably, a transformation method using the Agrobacterium method is used.
[0020]
The selection marker gene used here may be a selection marker gene known to those skilled in the art, for example, a hygromycin resistance gene or a kanamycin resistance gene.
[0021]
The promoter and terminator are not particularly limited as long as they function in plant cells. Examples of the promoter for constitutive expression include actin and ubiquitin gene promoters in addition to the 35S promoter previously incorporated in the above vector. Is done. However, more preferably, an inducible promoter may be incorporated and used. It is preferable to introduce a start codon, a stop codon, a terminator, a replicable unit, and the like as required downstream of these promoters.
[0022]
When Agrobacterium is used for the transformation, the medium, the concentration of the bacterium, the infection time, the culture time, and the like described in WO94 / 00977, WO95 / 06722, and WO98 / 17813 can be used. However, the present invention is not limited to these.
[0023]
For the purpose of producing a disease-resistant plant using the DNA fragment of the present invention, a plant transformation vector is useful. The plant vector is not particularly limited as long as it has the ability to express the gene in a plant cell and produce the protein. For example, pBI221, pBI121 (all manufactured by Clontech), and derivatives derived therefrom Vector. In particular, for transformation of monocotyledonous plants, pIG121Hm, pTOK233 (Hiei et al., Plant J., 6, 271-282 (1994)), and pSB424 (Komari et al., Plant @ J., 10, 165-174 (1996)) ) Is exemplified.
[0024]
Furthermore, the plant that imparts disease resistance by the method of the present invention may be, for example, rice, but other plants include wheat, barley, rye, corn, sugarcane, sorghum, soybean, adzuki bean, onion, and garlic. Such as monocotyledonous plants, cotton, sunflower, peanuts, tomato, potato, sweet potato, pea, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, carrot, eggplant, pumpkin, cucumber, apple, pear, melon, strawberry, Includes but is not limited to dicotyledons such as grapes, tobacco, Arabidopsis, petunia, chrysanthemum, carnation, saintpaulia, and zinnia.
[0025]
In the present invention, the disease resistance imparted to a plant by the introduction of a flagellin gene is, for example, resistance to rice blast, but another disease is Rhizoctonia solani, AG-1. Fungal diseases such as foliage seedling disease (Gibberella fujikuroi), sesame leaf blight (Bipolarislesiae), and koji disease (Claviceps @ virens), blight blight (Xanthomonas b. Bacterial diseases such as Burkholderia glumae, bacterial wilt disease (Burkholderia plantarii), stripe blight (rice stripe), dwarf ( ice dwarf virus), and the like viral diseases such as yellow leaves disease (rice transitory yellowing virus), which is resistant to a variety of diseases.
[0026]
As one embodiment of the present invention, a construct 35S-1141 in which a 1141-fla1 gene (SEQ ID NO: 2) encoding a flagellin derived from a plant pathogenic bacterium (Acidoborax aveane N1141 strain) is connected to a cauliflower mosaic virus 35S promoter that expresses constitutively. fla1 is constructed, and the construct is introduced into rice cultivar Koshihikari by the Agrobacterium method which is a gene introduction technique known to those skilled in the art. Expression of flagellin in regenerated individuals can be confirmed by Western analysis using anti-flagellin serum. In the regenerated individual, an individual that accumulates flagellin in an amount of 0.005 to 0.15% based on the total soluble protein is obtained. In addition, among these regenerated individuals, most of the individuals expressing flagellin have normal seed fertility, and seeds can be obtained from the regenerated individuals.
[0027]
Conventionally, the introduction of a proteinaceous elicitor into the plant genome using a constitutively expressed promoter always leads to a hypersensitive response (HR) of the plant, which means that the plant cells It was thought that this would lead to serious problems in plant development as it would lead to programmed cell death. Therefore, it was thought that it would be difficult to produce a resistant plant by introducing a proteinaceous elicitor into the plant genome without using an inducible expression promoter. However, in the present invention, a rice expressing a flagellin protein was successfully produced using a promoter that constitutively expresses the flagellin gene. White spots appeared on the leaves of the flagellin-expressing individuals as they developed, and many plants showed some decrease in plant height, but the seed fertility was normal. Therefore, in the method of the present invention, a promoter that causes constitutive expression can be used without being limited to a promoter that causes inducible expression. That is, all promoters that work in plant cells can be used in the method of the present invention.
[0028]
In an individual that has accumulated flagellin at a high level, the expression level of the PAL gene is higher than in a recombinant that does not accumulate flagellin. The PAL gene is one of defense-related genes whose transcription is induced by a disorder or an elicitor derived from the cell wall of a pathogenic bacterium (Zhu et al., (1995) Plant \ Mol \ Biol \ 29: 535-550). Therefore, it is suggested that at least a part of the defense mechanism is activated in the transformed rice by flagellin introduction.
[0029]
T₀At the generation (transformation generation), a rice plant in which flagellin protein was accumulated was selected, and its self-pollinated body (T₁) May be obtained. T₁The transformants of the generation may be inoculated with a rice blast fungus (filamentous fungus) as one mode of the disease and tested for resistance. In most of the transgenic rice plants in which flagellin 1141-fla1 was expressed, the number of blast lesions or the area ratio of blast after inoculation of blast was compared with that of prototype varieties such as Koshihikari which did not express flagellin. Can be significantly reduced. On the other hand, T₀Such a decrease is not observed in one line in which only flagellin expression was not observed in the generation. Confirmation of the lesion after the inoculation of the pathogen may be performed within 6 days after the inoculation or within 16 days after the inoculation. T₁When Western analysis was performed for each individual generation, a positive correlation was found between the expression level of flagellin and resistance. Therefore, expression of flagellin can enhance rice disease resistance.
[0030]
【Example】
Embodiment 1 FIG. Gene construction and plant transformation
The XhoI site is added immediately upstream of the translation initiation codon, and the restriction enzyme HindIII site is added immediately downstream of the translation stop codon. The flagellin gene of the Acidovorax @ avenenae @ N1141 line (N1141-fla1, 1479 bp, Gene @ Bank Accession No .: AB040139, The plasmid vector (Che et al., J Biol Chem (2000) 275: 32347-32356) into which the sequence number 1 in the column list was incorporated was double-digested with restriction enzymes XhoI and HindIII (Takara Shuzo Co., Ltd.). The N1141-fla1 gene was excised and blunt-ended with Klenow @ fragment (Takara Shuzo). On the other hand, the super binary vector pSB21 (35S pro-GUS-NOS ter, Komari et al. (1996) Plant 10: 165-174) was digested with restriction enzymes XbaI and SacI to remove the GUS gene, and T4 DNA polymerase (Takara Shuzo) To blunt end. A CIAP (Boehringer) treatment was performed, and the terminal portion was dephosphorylated. The above-mentioned N1141-fla1 gene was incorporated therein to obtain p3511Fla.
[0031]
Based on pSB11 (Komari et al., supra), an intermediate vector pKY211 having a hygromycin resistance gene cassette was created. A nopaline synthase terminator (NOS @ ter) was connected to the corn ubiquitin promoter and the ubiquitin intron (Ubi @ pro-Ubi @ in). The hygromycin resistance gene (hptII) was inserted between Ubi in and NOSter of this construct (Ubi pro-Ubi in-NOS ter) to prepare Ubi pro-Ubi in-hptII-NOS ter. Further, this construct was inserted into HindIII and EcoRI sites of pSB11 to obtain pKY205. Finally, Transformer^TMUsing a site-directed mutagenesis kit (Clontech), a HindIII site was inserted immediately upstream of the EcoRI site of pKY205 to obtain pKY211.
[0032]
After p3511Fla was digested with HindIII and dephosphorylated, a 3.3 kb HindIII fragment (ubiquitin promoter-hptII-NOS) derived from pKY211 was ligated, and a selection marker gene (hptII, hygromycin resistance gene) was incorporated. By the above procedure, construct p3511FlaHpt (35S {pro-1141-fla1-NOSter} + Ubi \ pro-Ubi \ in-hptII-NOS @ ter) was constructed. The cauliflower mosaic virus 35S promoter is a constitutive high expression promoter, and rice transformed with this construct is expected to accumulate flagellin systemically. A mixture of the E. coli LB392 strain containing this construct, the Agrobacterium LBA4404 strain containing the acceptor vector pSB1 (Komari et al. (1996) Plant @ J. 10: 165-74.), And the E. coli HB101 strain containing the helper plasmid pRK2013 are mixed. Then, a construct containing flagellin was introduced into Agrobacterium by tribacterial conjugation. Transformation of rice is described in Hiei et al. According to the method of (1994) (Plant J. 1994 1994 6: 271-282), calli derived from immature embryos of rice cultivar Koshihikari were transformed using Agrobacterium.
[0033]
Embodiment 2. FIG. Analysis of transformants
a) Confirmation of flagellin expression by Western analysis
Fifty individual regenerated plants were obtained. Transformation generation (T₀) For Western analysis. Leaves (3-4 cm in length) of the transformed rice at the 5-6 leaf stage were ground in a 0.1 M HEPES-KOH pH 7.5 buffer using a mortar. The supernatant after centrifugation at 15000 × g for 10 minutes was used as a protein sample. Protein quantification was performed using Bio-Rad Protein Assay kit (BIO-RAD). Approximately 30 (g protein was fractionated by SDS-PAGE according to the method of Laemmni (1970) Nature # 227: 680-685. The gel used was 12.5% @PAGEL (ATTO). Was transferred to a PVDF membrane (millipore) for 30 minutes in a 1 × TBS buffer containing 0.5% skim milk, and then the 1141 flagellin antibody (Che et al., J Biol Chem (2000) 275: 32347-). 32356) was shaken overnight in the same buffer containing 1/1000 (v / v) at room temperature. Gate (B O-RAD) was used at a concentration of 1/1000 (v / v), and the coloring system was HRP Color Development Development Agent (BIO-RAD), and alkaline phosphorase kitrate II (Vector Laboratories). The expressed protein amount is determined by the color development degree of an E. coli-expressed flagellin protein sample whose concentration is known (Che et al., J Biol Chem (2000) 275: 32347-32356), and a densitometer (Model GS-670, BIO-RAD). It was calculated by comparison using T.₀The results of Western analysis of generations are summarized in Table 1, and specific examples are shown in FIG.
[0034]
[Table 1]

[0035]
Apparent accumulation of flagellin protein was detected in approximately 2/3 of the transformants. White spots appeared on the leaves of these flagellin-expressing individuals as they grew. In addition, many plants showed a slight decrease in plant height, but the seed fertility was normal.₁Seeds were obtained. For some individuals, the copy number of the introduced flagellin gene was estimated by genomic Southern analysis using the 1141-fla1 gene (XhoI, HindIII 1.5 kb fragment) as a probe.
[0036]
b) Expression analysis of defense-related genes by Northern analysis
In order to examine whether or not the expression of defense-related genes was induced in the transformants expressing flagellin, four transformants expressing flagellin (D / 11-20-2, D / 11-24-3) were used. , D / 11-27-1, D / 11-27-2), and pSB24 (35S pro-IGUS-NOS ter, Komari et al. (1996) Plant J. @ 10: 165-174). RNA was extracted from the leaves of the two lines (Q1, Q2) by the phenol-SDS method, and Northern analysis was performed using the PAL gene as a probe. The PAL gene was cloned as follows. Based on the sequence of OSPAL (Accession No. X16099), a primer was designed to extend the entire length of coding region, and Occultated cell (Oryza sativa L. C5924 (Baba, A. et al. (1986) Plant @ Cell. -472)), the PAL gene was cloned by RT-PCR. The nucleotide sequence was analyzed and it was confirmed that the gene was PAL. It has been reported that transcription of the PAL gene is induced by injury, elicitor derived from the cell wall of a pathogenic bacterium, etc. (Zhu et al. 1995 Plant Mol Biol 29: 535-550).
[0037]
Total RNA (15 (g)) extracted from the transformant and the control line was subjected to 1.5% agarose gel containing formaldehyde in 1 × MOPS buffer (20 mM MOPS-KOH, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA). After electrophoresis, a nylon membrane Hybond @ N⁺(Amersham). The PAL gene probe is obtained by purifying the above PCR product by microcon {YM-30} (Millipore),³²Those labeled with [P] -dCTP were used.
[0038]
As a result, in all the four lines examined, the accumulation amount of the transcript of the PAL gene was larger than that in the control (FIG. 2). This suggested that at least a part of the defense mechanism was activated in transformed rice by flagellin introduction.
[0039]
c) Blast resistance test of flagellin-expressing rice in T1 generation
T into which the 1141-fla1 gene has been introduced₀Four lines (D / 11-20-1, D / 11-20-2, D / 11-20-3, D / 11-24-3) in which flagellin accumulated among the transformants of the generation, and Western analysis Selected one strain (D / 11-31-1) whose flagellin accumulation was below the detection limit,₁The incidence of rice blast in generations was investigated. The seed fertility of the five selected high expression individuals was normal, and many self-fertilized seeds could be obtained. As controls, a rice cultivar Koshihikari with extremely low field resistance to blast (the original strain of the transformation experiment), Nipponbare with low to medium field resistance to blast, and a galaxy with extremely high field resistance to blast were used. Was. 8 seeds × 2 rows of seeds were sown in a seedling case containing cultivated soil, cultivated in a greenhouse, and subjected to a disease test during the 4.8 to 5.2 leaf stage. Race 007 was used for the rice blast fungus (Magnaporthe grisea). For the inoculation, blast fungi were cultured on oatmeal / sucrose agar medium (dark conditions at 28 ° C.), and after the growth of the flora, conidia formed by irradiation with near-ultraviolet light at 25 ° C. for 3 days were used. Inoculation of blast fungi is 2 × 10 with 0.02% @ Tween20.⁵This was performed by spraying and inoculating 30 ml of the suspension adjusted to the number of conidia / ml per three seedling cases. The rice subjected to spray inoculation was kept in a humidified incubator (SLPH-550-RDS, manufactured by Nippon Medical Chemical Co., Ltd.) at 25 ° C. and 100% humidity for 24 hours after inoculation, and then transferred to a greenhouse. The setting conditions of the greenhouse were a light condition of 25 ° C. for 16 hours and a dark condition of 22 ° C. for 8 hours. The disease resistance was evaluated by visually counting the number of progressive lesions on the uppermost developed leaf (fifth leaf) at the time of inoculation 6 days after the inoculation, and evaluating the symptom index (FIG. 3). Further, 16 days after the inoculation, the rate of the progressive lesion area of the uppermost developed leaf (fifth leaf) at the time of the inoculation was evaluated as a symptom level (FIG. 5). The results were subjected to a significant difference test with Koshihikari (comparison between all groups, Steel-Dass test).
The results 6 days after the inoculation are shown in FIG. The distribution of the symptom index of the line D / 11-31-1 in which flagellin was not expressed was very similar to that of the original line Koshihikari (weakly resistant). The median symptom index was 6 (the number of lesions per leaf was large and unmeasurable), and the range was 4 (7-3). The median symptom index was 5 (the number of lesions per leaf was 25-50) in Nipponbare, which had low blast resistance to medium, and flagellin accumulation lines D / 11-20-3 and D / 11-24. The median of 3 was also 5, but the range of Nipponbare's symptom index was 3, whereas that of these flagellin-accumulating lines was 4, indicating a low symptom level and enhanced resistance. The proportion of individuals that For galaxies with strong blast field resistance, the median symptom index is 4 (10-30 lesions per leaf) and 3 is the index (3-15 lesions per leaf). And 4 were the most resistant, showing the strongest resistance among the lines tested. The median symptom indices of the flagellin accumulation lines D / 11-20-1 and D / 11-20-2 are also 4, 3.5. The proportion was large. In particular, the distribution of D / 11-20-2 was very similar to that of galaxies. As a result of the non-parametric significant difference test of the distribution of the symptom index for Koshihikari, the galaxy and three of the four lines in which flagellin accumulated (D / 11-20-2, D / 11-20-3, D / 11 In -24-3), a significant difference in the symptom index distribution of Koshihikari was detected at a risk rate of 1%.
On the other hand, in the results 16 days after the inoculation (FIG. 5), all of the 31 tested individuals died in the original strain Koshihikari. Nipponbare, D / 11-31-1 and D / 11-20-1 also showed the same distribution as Koshihikari, and the symptom level of most individuals was 9 (lesion area ratio 100%). In contrast, in the galaxy, more than half of the individuals had a symptom level of 5 or less (lesion area ratio of 20% or less). In three lines D / 11-20-2, D / 11-20-3, and D / 11-24-3, which were significantly different from Koshihikari 6 days after inoculation, individuals with low symptom levels were found. In the distribution map of the symptom levels of these strains, a statistically significant difference was detected at a 1% risk rate from that of Koshihikari. From the above results, it was shown that disease resistance of rice can be enhanced by flagellin introduction.
Protein was extracted from each individual 16 days after inoculation, and the accumulated amount of flagellin protein was analyzed by Western analysis. Further, in order to examine the relationship between the expression level of flagellin and the strength of blast resistance, the relationship between the expression level of flagellin of each individual and the blast symptom index (FIG. 3) 6 days after inoculation of the individual is shown in FIG. Indicated. As a result of Wilcoxon's rank sum test, a significant difference was obtained at a 5% risk rate in the distribution of the blast symptom index between the expression (+) and the absence (-) of flagellin. This result indicates that the enhancement of blast resistance was brought about by the expression of flagellin.
[0040]
[Table 2]

[0041]
【The invention's effect】
According to the present invention, it has become clear for the first time that disease resistance can be imparted to a plant by connecting a gene encoding flagellin to a constitutive promoter and introducing the gene into a plant. This flagellin-introduced plant is thought to be useful for elucidating the mechanism of action of the proteinaceous elicitor flagellin and for elucidating the mechanism of local and systemic acquired resistance. We also show that the creation of resistant plants by introducing proteinaceous elicitors, which was considered to be difficult without an inducible promoter, is sufficiently applicable using a constitutive promoter. The expansion of the coverage could be shown. The DNA sequence encoding flagellin can be converted into a suitable constitutive, organ / period specific, or a promoter sequence induced by stress or pest, or a chemical substance capable of functioning in a plant cell, and a terminator sequence capable of functioning in a plant cell. The present invention has shown that the method of producing a disease-resistant plant by incorporating the gene into an expression cassette, introducing the gene into a plant cell, and obtaining a regenerated individual is a genetic engineering feasible and effective approach.
[0042]
[Sequence list]

[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG.₀It is a figure which shows the example of detection of the accumulation of flagellin protein by the western analysis in the transgenic rice of a generation. PC indicates control-expressed flagellin (30 ng) as a control, T indicates a transformant (30 μg of total soluble protein), and C indicates Koshihikari of the original strain (30 μg of total soluble protein). Evaluation of the expression level is as described in Table 1.
FIG. 2 shows T₀FIG. 3 is a view showing the expression of PAL gene by generation of northern blot in transformed rice. D / 11 indicates a transformant. As a control, a GUS transgenic individual was used.
FIG. 3 is a schematic diagram of a blast symptom index 6 to 7 days after inoculation.
FIG. 4 shows T₁It is the graph which showed the blast disease assay result 6 days after inoculation in the transformed rice of the generation. The evaluation of the symptom index is as described in FIG.
FIG. 5 shows T₁It is the graph which showed the blast disease assay result 16 days after inoculation in the transformed rice of the generation. The lesion area ratio at each symptom level is 0% for level 0, 1% for level 1, 2% for level 2, 5% for level 3, 10% for level 4, 20% for level 5, and 6 for level 6. 40%, Level 7 is 60%, Level 8 is 80%, and Level 9 is 100%.
FIG. 6 shows T₁FIG. 4 is a diagram showing the relationship between the degree of blast resistance and the level of flagellin expression 6 days after inoculation in transgenic rice of generations. The evaluation of the symptom index is shown in FIG. 3, and the evaluation of the expression level is shown in Table 1.

Claims (21)

By introducing a gene encoding a protein or peptide having an elicitor activity in a plant, the gene comprising an amino acid sequence having at least 70% homology with the amino acid sequence of SEQ ID NO: 1 or a part thereof into a plant genome, A method for producing a disease-resistant plant, which comprises imparting disease resistance. By introducing a gene encoding a protein or peptide having an elicitor activity in a plant, the gene comprising an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO: 1 or a part thereof into a plant genome, The method for producing a disease-resistant plant according to claim 1, comprising imparting disease resistance. A method comprising imparting disease resistance to a plant by introducing a gene encoding a protein or peptide having an elicitor activity in a plant, comprising the amino acid sequence of SEQ ID NO: 1 or a part thereof, into the plant genome. Item 3. The method for producing a disease-resistant plant according to Item 1 or 2. By introducing a gene encoding a protein or peptide having elicitor activity in a plant having the following amino acid sequence or a part thereof into a plant genome, a disease-resistant plant comprising conferring disease resistance on the plant is obtained. Production method:
(A) the amino acid sequence of SEQ ID NO: 1, or
(B) an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1. By introducing a gene comprising a DNA sequence having at least 70% or more homology with the DNA sequence of SEQ ID NO: 2 or a gene having a part thereof into a plant genome, thereby imparting disease resistance to the plant. , A method for producing a disease resistant plant. By introducing a gene consisting of a DNA sequence having at least 90% or more homology with the DNA sequence of SEQ ID NO: 2 or a gene having a part thereof into a plant genome, thereby imparting disease resistance to the plant. A method for producing a disease-resistant plant according to claim 5. The disease resistance according to claim 5 or 6, comprising imparting disease resistance to the plant by introducing a gene having the DNA sequence of SEQ ID NO: 2 or a gene having a part thereof into the plant genome. How to make sex plants. A method for producing a disease-resistant plant, comprising imparting disease resistance to a plant by introducing a gene comprising the following DNA sequence or a gene comprising a part thereof into the plant genome:
(A) the DNA sequence of SEQ ID NO: 2, or
(B) a DNA sequence in which one or more bases have been substituted, deleted, inserted, and / or added in the DNA sequence of SEQ ID NO: 2. A gene that hybridizes with a nucleic acid having the DNA sequence of SEQ ID NO: 2 under stringent or mild conditions or a gene having a part thereof is introduced into a plant genome to confer disease resistance on the plant. A method for producing a disease-resistant plant, comprising: A method for producing a disease-resistant plant, comprising imparting disease resistance to a plant by introducing a gene encoding a flagellin protein having a protein-like elicitor activity in the plant into the plant genome. 10. The method according to claim 9, wherein the flagellin protein is a flagellin protein derived from Acidoborax aveane N1141 strain. 7. The method according to claim 1, wherein a gene encoding a protein or a peptide having an elicitor activity in a plant is connected to a promoter that functions in the plant, introduced into the plant genome, and imparts disease resistance to the plant. A method for producing a disease-resistant plant according to claim 1. The method for producing a disease-resistant plant according to claim 7, wherein the promoter is a constitutive promoter. (A) preparing the promoter and the terminator according to claim 11 or 12,
(B) preparing a plasmid having an expression cassette in which the gene according to any one of claims 1 to 10 is connected to a region downstream of the promoter and upstream of the terminator,
(C) introducing the expression cassette into a plant genome by an Agrobacterium method, an electroporation method, a PEG method, a microinjection method, or a particle gun method,
(D) screening the transformed plant cells with a selectable marker,
(E) culturing in a regeneration medium to complete plants (T ₀ generation) to obtain disease resistant plants;
(F) obtaining from the T ₀ generation seeds (T ₁ generation), obtaining a disease-resistant plant by growing the seed;
A method for producing a disease-resistant plant, comprising: The method for producing a disease-resistant plant according to any one of claims 1 to 13, wherein the plant is a monocotyledonous plant. The method for producing a disease-resistant plant according to any one of claims 1 to 14, wherein the plant is rice. The method for producing a disease-resistant plant according to any one of claims 1 to 13, wherein the plant is a dicotyledonous plant. The method for producing a disease-resistant plant according to any one of claims 1 to 16, wherein the disease is a fungal disease. The method for producing a disease-resistant plant according to any one of claims 1 to 17, wherein the disease is blast. The method for producing a disease-resistant plant according to any one of claims 1 to 16, wherein the disease is a bacterial disease. The method for producing a disease-resistant plant according to any one of claims 1 to 16, wherein the disease is a viral disease.

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