JP5812387B2 - Plant tan genes and uses thereof - Google Patents

Description

The present invention relates to a tan gene involved in a pigment reaction to an external disorder caused by a plant pest, etc., a method for determining the susceptibility or resistance of a plant to a pathogenic filamentous fungus targeting the tan gene, and a pathogenic filamentous form using the tan gene The present invention relates to a method for producing a plant resistant to bacteria.

As a response reaction to pests, sorghum generally synthesizes anthocyanins, and the area around the part that was afflicted by the disease and the part that was damaged by the sorghum generally changes from magenta to orange. On the other hand, in the so-called tan trait, anthocyanins are not synthesized in these parts and a yellowish brown color is exhibited (FIG. 1).

From previous studies, it has been elucidated that the tan trait of sorghum is dominated by a single recessive gene (Non-patent Document 1). In addition, it is known that sorghum having a tan trait increases field resistance against coat blight (Non-patent Document 2). Furthermore, it is known that sorghum having a tan character (Non-patent Document 3) and sorghum that stores flavan-4-ol, which is a precursor of anthocyanin, increase the overall resistance to mold (Non-patent Document 4). The tan trait is a trait that is widely used in sorghum in tropical regions such as India, and is considered to be an advantageous trait in terms of disease resistance under such environmental conditions.

  On the other hand, it is known that sorghum contains a large amount of 3-deoxyanthocyanin, which is an anthocyanin that is rarely found in nature. 3-Deoxyanthocyanin, which is abundant in the sorghum peel, is apigeninidin (Apigeninidin (red)) and 3'OH luteolinidin (luteolinidin (black purple)) (Non-patent Document 5) and is widely present in the plant kingdom. Like other anthocyanidins, these 3-deoxyanthocyanidins are thought to accumulate in intracellular vacuoles in the form of glycosides (Non-Patent Document 6). Based on the knowledge thus far, the synthesis route of 3-deoxyanthocyanin is thought to be branched from the anthocyanin biosynthesis route. Furthermore, it has been suggested that 3-deoxyanthocyanin biosynthesis branches from naringenin by flavanone-4-reductase (DFR) (Non-patent Document 7). The flavan-4-ol thus produced is considered to be polymerized to flobaphenes and also to 3-deoxyanthocyanidins. The synthesis reaction to 3-deoxyanthocyanidin was previously thought to be due to anthocyanidin synthase (ANS), but it has been shown that the enzyme responsible for the final reaction is unlikely to be ANS. (Non-patent document 7).

  Furthermore, sorghum has been mainly cultivated as a livestock feed in Japan until now, but in recent years, it has been attracting attention as a raw material for biofuel (ethanol). For this reason, preventing sorghum revenues due to disease is an important issue not only for agricultural policy but also for energy policy. However, in the past, sorghum has been rarely used to control diseases in general because of its main use being livestock feed.

Therefore, although it is expected to identify a gene that causes a tan trait and has a relationship with resistance to 3-deoxyanthocyanin synthesis pathway and blight disease, which is related to the overall resistance to mold, it is still clear It is not.

Doggett, H., Sorghum second edition, John Wiley & Sons, Inc, New York, 1987, pp. 171-172 Shigemitsu Kasuga et al., “Effect of combined UV-A / B irradiation and wind stimulation on resistance to blight of sorghum,” Hokuriku Crop Science Society, 2005, 40, 110-112. Murty, D.S., `` Development of early and mold free grain '', ICRISAT's Sorghum program: A review by international consultants InternationalCrops Research Institute for the Semi-arid Tropics, Patancheru, India, 1975, pp. 26-30 JAMBUNATHAN, R. et al., `` Flavan-4-ol concentration in leaf tissues of grain mold susceptible and resistant sorghum plants at differentstages of leaf development. '', Journal of Agricultural and Food Chemistry, 1991, 39, 1163-1165. SIAME, B.A., et al., `` Role of pigments and tannins in thereaction of tan and red near isogenic sorghum lines to leaf diseases. '', American Crop Science journal, 1993, pp. 123-130. GROTEWOLD, E., "THE GENETICS AND BIOCHEMISTRY OF FLORAL PIGMENTS.", Annual Review of Plant Biology, 2006, 57, 761-780 LIU, H. et al., "Molecular dissection of the pathogen-inducible 3-deoxyanthocyanidin biosynthesis pathway in sorghum.", Plant Cell Physiol, 2010, 51, 1173-1185.

The present invention has been made in view of such a situation, and its purpose is to identify a gene that causes a tan trait having a relationship with 3-deoxyanthocyanin synthesis pathway in sorghum, resistance to blight, etc. There is to do. Furthermore, the present invention aims to identify genes in other plants that correspond to genes that cause tan traits in sorghum. Moreover, an object of this invention is to provide the method of determining easily the tan character of a plant based on the identified gene. A further object of the present invention is based on the development of mechanisms elucidated tan trait is to provide a method for efficiently producing a plant introduced with tan trait.

In order to solve the above-mentioned problems, the present inventors have established a sorghum in a large-scale crossing population (F3, F5) of a variety of sorghum that shows purple due to disease, a cultivar of MS-3B and tan trait Greenleaf. The tan gene was mapped using the SSR marker and insertion / deletion marker (Figs. 1 and 2).

Specifically, first, F5 RIL individuals (recombinant inbred lines (Recombinat
Inbred Line) and 143 individuals), the results of rough mapping using SSR markers revealed that the tan gene sits on chromosome 6. Next, the terrorist groups individuals (1142 individuals) to F3 part, were selected individuals (63 individuals) with a recombination between SSR markers SB3751 of SB3764, performs a tan traits of investigation in the field, the existence region of the tan gene Was narrowed down to a region of about 150 kbp. Furthermore, mapping using the CAPS marker and SNP marker found based on the base sequence information of sorghum variety BTx623 was performed, and the tan gene existing region was finally narrowed down to a region of about 29 kbp (FIG. 3).

When the present inventors investigated the annotation of the gene in the narrowed-down area | region in the database, it turned out that the gene which has the homology to the leucoanthocyanidin reductase of a corn has made the cluster in this area | region. Within this narrowed region were four candidate genes with homology to corn leucoanthocyanidin reductase. When the expression of these four genes was investigated by RT-PCR, only the expression pattern of a specific candidate gene (Sb06g029550, hereinafter also referred to as “ tan gene” or “sensitive tan gene”) among candidate genes However, it was found that gene expression increases when sorghum turns purple due to injury (FIG. 4). Moreover, the expression of the remaining genes was not observed by RT-PCR. From this, it was speculated that this gene is a causative gene of coloring.

When the nucleotide sequence around the tan gene was determined and the amino acid sequences encoded were compared between varieties, the genome sequence of the gene region encoding the protein was already clarified in the purple MS-3B variety. The cultivar was BTx623 (Fig. 5). However, in the amino acid sequence encoded by Greenleaf's tan gene (hereinafter also referred to as “resistant tan gene”), two sites were found that differed in sequence from the amino acid sequence encoded by the sensitive tan gene. As a result, it was speculated that the normal protein encoded by the tan gene was not synthesized in Greenleaf of the tan trait (FIG. 5).

In addition, in sorghum exhibiting a tan trait that had undergone external damage, it was revealed that luteolin and the like were accumulated instead of 3-deoxyanthocyanidins (apigenidin and luteolinidine) (FIGS. 6 to 8). Furthermore, a correlation between resistance type tan gene and resistance to soot disease was revealed (FIG. 10).

From these facts, the tan gene is a gene involved in sorghum coloration, and due to its expression suppression and mutation, the original function is not exhibited, and 3-deoxyanthocyanidin is not synthesized, indicating that the individual exhibits the tan trait There was found. Furthermore, in the tan trait, it was also found that resistance to pathogenic filamentous fungi is exhibited because luteolin or the like having antibacterial activity accumulates when receiving external damage.

Based on the above findings, the present inventors can determine the susceptibility or resistance to pathogenic filamentous fungi in plants by targeting the identified tan gene. -Deoxyanthocyanidin is an enzyme that performs the final stage of the synthesis reaction, and it has been found that by suppressing the expression and function of the tan gene, it is possible to confer resistance derived from the tan trait to plants. The present invention has been completed.

More specifically, the present invention provides the following.
<1> The DNA according to any one of the following (a) to (c), which encodes a protein that suppresses 3-deoxyanthocyanidin synthesis in a plant.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 6
(B) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 4 or 5
(C) DNA encoding a protein comprising an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 6
<2> The DNA according to any one of the following (a) to (c), which encodes a protein having an activity of imparting resistance to a pathogenic filamentous fungus to a plant.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 6
(B) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 4 or 5
(C) DNA encoding a protein comprising an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 6
<3> A vector comprising the DNA according to <1> or <2>.
<4> A plant cell into which the DNA according to <1> or <2> has been introduced.
<5> A plant comprising the cell according to <4>.
<6> A plant that is a descendant or clone of the plant according to <5>.
<7> Plant propagation material according to <5> or <6>.
<8> A method for imparting resistance to pathogenic filamentous fungi to a plant, which suppresses the expression or function of the DNA according to any one of the following (a) to (c) in the plant.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(B) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(C) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein
<9> A method for imparting resistance to pathogenic filamentous fungi to a plant, comprising the step of introducing the DNA according to <2> into the plant and converting both of the tan alleles in the plant into the DNA.
<10> A method for imparting resistance to a pathogenic filamentous fungus to a plant, comprising a step of introducing a DNA encoding the RNA according to any one of (a) to (c) below into the plant.
(A) a double-stranded RNA having an activity of imparting resistance to a pathogenic filamentous fungus to a plant and complementary to the transcription product of DNA according to any one of (i) to (iii) below
(B) An antisense RNA having an activity to impart resistance to pathogenic filamentous fungi to a plant and complementary to the transcription product of DNA according to any of (i) to (iii) below
(C) RNA having an activity to confer resistance to pathogenic filamentous fungi on a plant and a ribozyme activity that specifically cleaves a transcription product of DNA according to any one of (i) to (iii) below
(I) a DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(Ii) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(Iii) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein
<11> A drug for imparting resistance to pathogenic filamentous fungi, comprising the DNA according to <2> or a vector into which the DNA is inserted, and using both of the tan alleles in plants as the DNA. .
<12> A drug for imparting resistance to pathogenic filamentous fungi, comprising DNA encoding the RNA according to any one of the following (a) to (c), or a vector into which the DNA is inserted.
(A) a double-stranded RNA having an activity of imparting resistance to a pathogenic filamentous fungus to a plant and complementary to the transcription product of DNA according to any one of (i) to (iii) below
(B) An antisense RNA having an activity to impart resistance to pathogenic filamentous fungi to a plant and complementary to the transcription product of DNA according to any of (i) to (iii) below
(C) RNA having an activity to confer resistance to pathogenic filamentous fungi on a plant and a ribozyme activity that specifically cleaves a transcription product of DNA according to any one of (i) to (iii) below
(I) a DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(Ii) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(Iii) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein
<13> A method for determining sensitivity or resistance to pathogenic filamentous fungi in a plant, wherein the base sequence of a tan gene in a test plant is analyzed, and the base sequence is any of the following (a) to (c): If the base sequence of the DNA according to claim 2 is determined, it is determined that the test plant has a high probability of having a susceptibility to pathogenic filamentous fungi. A method for determining that a plant has a high probability of having resistance to pathogenic filamentous fungi.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(B) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(C) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein
<14> A method for determining sensitivity or resistance to pathogenic filamentous fungi in a plant, wherein the DNA according to any one of the following (a) to (c) in the test plant or the DNA according to <2> A method comprising detecting expression.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(B) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(C) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein
<15> A method for breeding a plant resistant to pathogenic filamentous fungi,
(A) crossing a plant variety resistant to pathogenic filamentous fungi with any plant variety,
(B) determining the susceptibility or resistance to pathogenic filamentous fungi in the individual obtained by mating in step (a) by the method according to <13> or <14>; and (c) pathogenic filamentous fungi Selecting a variety that has been determined to have resistance to.
<16> A plant bred by the method according to <15>.
<17> A plant that is a descendant or clone of the plant according to <16>.
<18> The plant propagation material according to <16> or <17>.

In the present invention, the “pathogenic filamentous fungus” to which resistance is imparted by the tan trait may be a filamentous fungus that causes disease to plants, such as Thanatephorus cucumeris that causes blight, and soot disease. Setosphaeria
turcica , Colletotrichum graminicola which causes anthrax.

  In the present invention, “susceptibility to pathogenic filamentous fungi” means a property that a plant is infected with pathogenic filamentous fungi. In the present invention, “resistance to pathogenic filamentous fungi” means resistance of a plant to infection by pathogenic filamentous fungi. Due to this resistance, the onset of infection symptoms of pathogenic filamentous fungi in plants or the degree of symptoms (for example, the extent of lesion formation) is suppressed.

According to the present invention, a gene tan gene (susceptibility-type tan gene) that is involved in a pigment reaction to external damage caused by plant pests and the like and brings about susceptibility to pathogenic filamentous fungi is identified, and the position and structure of the gene on the chromosome, and The mechanism of tan trait, and thus the mechanism of 3-deoxyanthocyanin synthesis and the resistance to pathogenic filamentous fungi were elucidated. This makes it possible to easily determine the susceptibility and resistance of a plant to pathogenic filamentous fungi using the tan gene and markers in the vicinity thereof. Moreover, it became possible to efficiently produce plant varieties resistant to pathogenic filamentous fungi by suppressing the expression and function of the sensitive tan gene. The present invention greatly contributes to prevention of a decrease in plant yield due to pathogenic filamentous fungi.

It is a photograph which shows the leaf color of the sorghum at the time of receiving a pest damage. In the figure, the leaves of the sensitive sorghum variety MS-3B are purple, while the leaves of the sorghum resistant variety Greenleaf are tan. It is a graph which shows the result of having performed the chromosomal mapping of the tan character (pp) in the large-scale mating population (sorghum F5 RIL population) of Na line MS-3B and Greenleaf. The region where the tan gene exists is narrowed down to about 150 kbp, and mapping is performed using the CAPS and SNP markers found based on the nucleotide sequence information of the sorghum variety BTx623, and the region where the tan gene exists is finally reduced to about It is the schematic which shows having narrowed down to the 29kbp area | region. In addition, “t” shown in the lower part of the figure indicates that the leaf exhibits an tan color when subjected to pest or the like, and “p” indicates that the leaf is purple when subjected to pest or the like. Indicates that the individual has (purple). "B" indicates that the gene or region is derived from Greenleaf for both alleles, "A" indicates that the gene or region is derived from both MS-3B for both alleles, and "H" is heterozygous It shows that. Four genes homologous to maize leucoanthocyanidin reductase, which constitutes a cluster within the region of about 29 kbp shown in FIG. 3, only a portion of homology to maize leucoanthocyanidin reductase outside the region of about 29 kbp. It is the photograph of electrophoresis which shows the result of having investigated the expression of the gene (Sb06g029520) which has, and actin (SbActin) by RT-PCR. In the figure, “0d” indicates the expression result of each gene in the sorghum leaf on day 0 after cutting, and “3d” in the figure indicates the sorghum leaf maintained on the agar medium for 3 days after cutting. The expression result of each gene in is shown. In addition, “N” indicates the expression result of each gene in the leaves of the naso MS-3B, and “G” indicates the expression result of each gene in the leaves of Greenleaf. It is a figure which shows the amino acid sequence (upper part) of the tan protein (sequence number: 3) derived from the sensitive cultivar Japanese MS-3B and the amino acid sequence (lower part) of the tan protein derived from Greenleaf (sequence number: 6). In addition, the alphabetic character which is shown by the bold type and underlined in the figure shows the amino acid (C252Y, I268V) which is different in Na-type MS-3B and Greenleaf. It is a graph which shows the light absorbency (wavelength 493nm) of the liquid extracted from the leaf | leaf of MS MS-3B and Greenleaf which shredded and incubated on water agar. In the figure, the horizontal axis represents the incubation time of shredded leaves on water agar. It is a photograph which shows the result of having analyzed the pigment | dye extracted from the leaf of BTx623 (BTx) which showed a disease spot, Na-type MS-3B (Na), and Greenleaf (GL) by thin layer chromatography (TLC). The figure also shows the results obtained by developing the standard products (Luteolinidine (Lute) and Apigeninidine (Api)) with (TLC). It is a mass-spectrum figure which shows the result of having analyzed the pigment | dye produced | generated in the leaf of green leaf which gave external damage, and an untreated leaf by HPLC-MS. It is a schematic diagram which shows the anthocyanin synthetic pathway of sorghum and the enzyme which functions at each reaction step. It is a figure which shows the result of having conducted QTL analysis about the gene area | region which shows resistance to soot disease in a tan character. In the figure, the vertical axis indicates the RIL individuals subjected to the analysis, and the horizontal axis indicates the analyzed SSR marker and “tan pheno (tan character)”. "B" indicates that the gene or region is derived from Greenleaf for both alleles, "A" indicates that the gene or region is derived from both MS-3B for both alleles, and "H" is heterozygous It shows that. Furthermore, the figures below “tan pheno” indicate the degree of morbidity when the susceptibility test for soot disease was conducted in 2010. The “morbidity” is a value calculated by the formula “morbidity = Σ (index x number of leaves belonging to each index) / (5 × total number of leaves) × 100”, where “index” was investigated. It is a value indicating the degree of soot disease in the leaves.

<Synthesis-inducing DNA, synthesis-inhibiting DNA, sensitive DNA, resistant DNA>
The present invention provides a DNA encoding a protein that induces 3-deoxyanthocyanidin synthesis in plants (hereinafter also referred to as “synthesis-inducing DNA”). The present invention also provides a DNA encoding a protein having an activity of imparting susceptibility to pathogenic filamentous fungi to plants (hereinafter also referred to as “sensitive DNA”). The base sequence of tan cDNA derived from Na-type MS-3B, which is identified by the present inventors, is a cultivar showing purple in the sorghum disease-affected site, and is susceptible to soot disease etc. SEQ ID NO: 1 The base sequence of tan genomic DNA is shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by these DNAs is shown in SEQ ID NO: 3. One embodiment of the synthetic inducible DNA or sensitive DNA of the present invention (hereinafter also referred to as “sensitive DNA etc.”) is a DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 (typically DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2.

In the current state of the art, if a person skilled in the art obtains base sequence information such as sensitive DNA in a specific sensitive sorghum variety (for example, Na-type MS-3B), the base sequence is modified, Although the encoded amino acid sequence is different, it is also possible to obtain DNA that is also synthetically induced or sensitive. Also in nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence. Therefore, the present invention comprises an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence (SEQ ID NO: 3) of the tan protein in Na-type MS-3B. It also includes DNA that encodes a protein that induces 3-deoxyanthocyanidin synthesis or DNA that encodes a protein having an activity to confer susceptibility to pathogenic filamentous fungi on plants. The term “plurality” as used herein refers to the number of amino acid modifications within a range in which the modified tan protein can induce 3-deoxyanthocyanidin synthesis in the plant, or the range in which the activity of imparting susceptibility to pathogenic filamentous fungi to the plant is maintained. The number of amino acid modifications, usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (eg, within 5 amino acids, within 3 amino acids, 2 amino acids).

Furthermore, in the current state of the art, if a person skilled in the art obtains sensitive DNA from a specific sensitive sorghum variety (for example, Na MS MS-3B), the base sequence information of the sensitive DNA, etc. It is possible to obtain DNA encoding a homologous gene that is also a synthetic induction type or a DNA encoding a homologous gene that is a sensitive type from other sorghum varieties and other plants (for example, rice, maize). It is. Accordingly, the present invention is a DNA that hybridizes under stringent conditions with tan DNA (SEQ ID NO: 1 or 2) in Na-type MS-3B, and encodes a protein that induces 3-deoxyanthocyanidin synthesis in plants. It also includes DNA or DNA encoding a protein having an activity to confer susceptibility to pathogenic filamentous fungi on plants.

Whether the mutant DNA or homologous DNA thus obtained encodes a protein that induces 3-deoxyanthocyanidin synthesis in plants, for example, the tan trait in which the sites affected by pests show a yellowish brown color. Whether or not the cultivar is sprayed or inoculated with pathogenic filamentous fungi, or adult leaves of the cultivar are subjected to cutting or punching, and then the infection site of the pathogenic filamentous fungus, or whether the cutting or punching site is purple Can be determined by testing. (See Figure 1).

In addition, whether or not the mutant DNA or homologous DNA thus obtained encodes a protein having an activity that confers susceptibility to pathogenic filamentous fungi on plants, for example, to resistant cultivars introduced with these DNAs, It can be determined by spraying or inoculating a filamentous fungus and then examining whether or not it exhibits infection symptoms or the degree of infection symptoms. Examples of infectious symptoms include the formation of lesions. In addition, as described in Non-Patent Document 2, inoculation with blight fungus on tan traits varieties, and then using the ratio of diseased individuals (morbidity rate) and disease height rate as indicators Can do. Note that the lesion height ratio (RLH) is calculated by, for example, measuring the leaf sheath height (FH) and the lesion height (LH) for each individual and calculating the equation “RLH (%) = LH / FH × 100”. Obtained by.

The present invention also provides a DNA encoding a protein that suppresses 3-deoxyanthocyanidin synthesis in plants (hereinafter also referred to as “synthesis-inhibiting DNA”). Furthermore, the present invention provides a DNA encoding a protein having an activity to confer resistance to pathogenic filamentous fungi on plants (hereinafter referred to as “resistant DNA”). The base sequence of Greenleaf-derived tan cDNA, which shows the tan trait of sorghum identified by the present inventors and is a resistant cultivar against soot disease, is shown in SEQ ID NO: 4, and the base sequence of tan genomic DNA The amino acid sequence of the protein encoded by these DNAs is shown in SEQ ID NO: 6 in SEQ ID NO: 5. One embodiment of the synthesis-suppressing DNA or resistance-type DNA of the present invention (hereinafter also referred to as “resistance-type DNA”) is a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 (typically Is a DNA comprising the coding region of the base sequence described in SEQ ID NO: 4 or 5.

As shown in this Example, the base sequence of tan cDNA derived from Greenleaf (SEQ ID NO: 4) is compared with the base sequence of tan cDNA derived from sensitive cultivar MS-3B (SEQ ID NO: 1). The bases at positions 755 and 805 are different. Therefore, the Greenleaf-derived tan protein (SEQ ID NO: 6) has two amino acid substitutions, C252Y and I268V, compared to the tan protein derived from the sensitive cultivar MS3B (SEQ ID NO: 3) (FIG. 5). ) The function of the original tan protein is suppressed. It is considered that suppression of this function suppresses 3-deoxyanthocyanidin synthesis and imparts resistance to pathogenic filamentous fungi. In the current state of the art, those skilled in the art can suppress the 3-deoxyanthocyanidin synthesis of the protein encoded by the tan DNA base sequence of a specific resistant sorghum variety (for example, Greenleaf), or pathogenicity. Modifications can be made that maintain resistance to filamentous fungi. Also in nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence. Accordingly, the present invention comprises 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 (SEQ ID NO: 6) of tan protein in Greenleaf. -DNA which codes the protein which suppresses deoxyanthocyanidin synthesis, or DNA which codes the protein which has the activity which provides the resistance with respect to a pathogenic filamentous fungus to a plant is also included. The term “plurality” as used herein means that the modified tan protein has a modified number of amino acids within the range that can suppress 3-deoxyanthocyanidin synthesis in the plant, or a range in which the plant has an activity to impart resistance to pathogenic filamentous fungi. The number of amino acid modifications. If the tan protein in sorghum does not exhibit its original function, the synthesis of 3-deoxyanthocyanidins is suppressed and resistance to pathogenic filamentous fungi is considered. Therefore, the number of amino acid modifications is essentially not limited. The modification is, for example, within 100 amino acids, within 50 amino acids, within 30 amino acids (eg, within 10 amino acids, within 5 amino acids, within 3 amino acids, 2 amino acids, 1 amino acid).

Further, in the current state of the art, if a person skilled in the art obtains tan DNA from a specific sorghum variety, using the nucleotide sequence information of the DNA, other sorghum varieties and other plants (for example, From rice and maize), DNA encoding a homologous gene that is a synthetic inhibitory type or a resistant type can be obtained. Accordingly, the present invention is a DNA that hybridizes with tan DNA (SEQ ID NO: 4 or 5) in Greenleaf under stringent conditions, and that encodes a protein that suppresses 3-deoxyanthocyanidin synthesis in a plant, or a plant DNA encoding a protein having an activity of imparting resistance to pathogenic filamentous fungi is included.

The gene of rice which corresponds to tan gene sorghum, for example, Os04g0630300 in SALAD database, Os04g0630400, Os04g0630600, Os04g0630800, Os04g0631000. Examples of a gene for maize that correspond to the tan gene sorghum, e.g., GenBank
ACCESSION No. EU956420 and EU966048 are listed.

Whether the mutant DNA or homologous DNA thus obtained encodes a protein that suppresses 3-deoxyanthocyanidin synthesis in plants is, for example, recombining the tan gene of a susceptible variety against pathogenic filamentous fungi, It can be determined by producing a plant that retains the DNA homozygously, spraying or inoculating with pathogenic filamentous fungi, and then examining whether or not it exhibits an infectious symptom or the degree of the infectious symptom. As an infectious symptom, for example, the site of infection exhibits yellowish brown color by suppressing the synthesis of 3-deoxyanthocyanidin at the site of infection (see FIG. 1). Alternatively, with the DNA, recombine the tan gene of a susceptible variety against pathogenic filamentous fungi, create a plant that retains the DNA homozygously, cut or punch the adult leaf of the variety, and then cut or punch the site It can be determined by testing whether or not the color of the material exhibits yellowish brown color.

In addition, whether the mutant DNA or homologous DNA thus obtained encodes a protein having an activity to confer resistance to pathogenic filamentous fungi on the plant, for example, whether the DNA is a cultivar susceptible to pathogenic filamentous fungi By recombining the tan gene, creating a plant that retains the DNA homozygously, spraying or inoculating pathogenic filamentous fungi, and then examining whether it exhibits infection symptoms or the degree of infection symptoms, Can be determined. Examples of infectious symptoms include the formation of lesions. In addition, as described in Non-Patent Document 2, inoculation with blight fungus on tan traits varieties, and then using the ratio of diseased individuals (morbidity rate) and disease height rate as indicators You can also. Note that the lesion height ratio (RLH) is calculated by, for example, measuring the leaf sheath height (FH) and the lesion height (LH) for each individual and calculating the equation “RLH (%) = LH / FH × 100”. Obtained by.

  Further, the synthesis-inducing DNA of the present invention is a drug for inducing 3-deoxyanthocyanidin synthesis in plants in the sense that introduction thereof can induce 3-deoxyanthocyanidin synthesis in plants. On the other hand, synthesis-inhibited DNA is a drug for suppressing 3-deoxyanthocyanidin synthesis in plants in the sense that introduction thereof can suppress 3-deoxyanthocyanidin synthesis in plants.

  Furthermore, the sensitive DNA of the present invention is a drug for imparting susceptibility to pathogenic filamentous fungi to plants in the sense that introduction thereof can impart susceptibility to pathogenic filamentous fungi to plants. On the other hand, the resistance-type DNA of the present invention is intended to impart resistance to pathogenic filamentous fungi to plants in the sense that introduction thereof can impart resistance to pathogenic filamentous fungi to plants. It is a drug.

  In addition, artificial mutations introduced into DNA to produce the above-mentioned mutant DNA are, for example, site-directed mutagenesis method (Kramer, W. & Fritz, HJ., Methods Enzymol). 154: 350-367, 1987).

  Moreover, as a method for isolating the above-mentioned homologous gene, for example, hybridization technology (Southern, EM, Journal of Molecular Biology, 98: 503, 1975) or polymerase chain reaction (PCR) technology (Saiki, RK, et al. Science, 230: 1350-1354, 1985, Saiki, RK et al. Science, 239: 487-491, 1988). In order to isolate DNA encoding a homologous gene, a hybridization reaction is usually carried out under stringent conditions. Examples of stringent hybridization conditions include 6M urea, 0.4% SDS, 0.5xSSC conditions, or equivalent stringency hybridization conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1xSSC. The isolated DNA is at least 50% or more, preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) at the nucleic acid level or amino acid sequence level. ) Sequence identity. Sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. When analyzing an amino acid sequence by BLASTX, parameters are set to, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

The DNA encoding the tan protein of the present invention is not particularly limited in its form, and includes cDNA, genomic DNA, and chemically synthesized DNA. Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For genomic DNA, for example, genomic DNA is extracted from sorghum to produce a genomic library (as vectors, plasmids, phages, cosmids, BACs, PACs, etc. can be used), expanded, and tan genes (for example, The DNA can be prepared by performing colony hybridization or plaque hybridization using a probe prepared based on the base sequence of DNA (SEQ ID NO: 1, 2, 4 or 5). It is also possible to prepare a primer specific for the tan gene and perform PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from sorghum, inserted into a vector such as λZAP to create a cDNA library, developed, and colony hybridization as described above. Alternatively, it can be prepared by performing plaque hybridization or by performing PCR.

<DNA used to suppress the expression of sensitive tan genes>
The present invention also provides DNA used to suppress the expression of plant sensitive tan genes (such as sensitive DNA). By introducing these DNAs, it is possible to suppress the synthesis of 3-deoxyanthocyanidins in plants and to impart resistance to pathogenic filamentous fungi to plants. In this sense, the DNA used to suppress the expression of sensitive tan genes in plants is a drug for suppressing 3-deoxyanthocyanidin synthesis in plants, and imparts resistance to pathogenic filamentous fungi to plants. It is a drug. Here, “suppression of tan gene expression” includes both suppression of gene transcription and suppression of protein translation. Further, “suppression of expression” includes not only complete cessation of expression but also decrease of expression.

One embodiment of the DNA used to suppress the expression of the sensitive tan gene in plants is a DNA encoding a dsRNA (double-stranded RNA) complementary to the transcription product of the above-described sensitive DNA of the present invention. . A phenomenon called RNAi (RNA interference), in which the expression of the introduced foreign gene and target endogenous gene is both suppressed by introducing dsRNA having the same or similar sequence to the target gene into the cell. Can cause. When dsRNA of about 40 to several hundred base pairs is introduced into a cell, an RNase III-like nuclease called Dicer having a helicase domain is converted to about 21 to 23 base pairs from the 3 ′ end in the presence of ATP. Each one is cut out to generate siRNA (short interference RNA). A specific protein binds to this siRNA to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the transcript (mRNA) of the target gene at the center of the siRNA by RNaseIII-like enzyme activity. In addition to this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. There is also a possibility that this dsRNA becomes Dicer's substrate again to generate new siRNA and amplify the action.

  The DNA encoding the dsRNA of the present invention includes an antisense DNA encoding an antisense RNA for any region of a target gene transcription product (mRNA), and a sense DNA encoding a sense RNA for any region of the mRNA And antisense RNA and sense RNA can be expressed from the antisense DNA and the sense DNA, respectively. Moreover, dsRNA can be produced from these antisense RNA and sense RNA.

In the case where the dsRNA expression system of the present invention is held in a vector or the like, there are a case where antisense RNA and sense RNA are expressed from the same vector, and a case where antisense RNA and sense RNA are expressed from different vectors, respectively. is there. As a configuration for expressing antisense RNA and sense RNA from the same vector, for example, pol upstream of antisense DNA and sense DNA, respectively.
In this configuration, an antisense RNA expression cassette and a sense RNA expression cassette linked with a promoter capable of expressing a short RNA such as the III system are constructed, and these cassettes are inserted into the vector in the same direction or in the opposite direction.

  It is also possible to construct an expression system in which antisense DNA and sense DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA are separated from each strand on both sides. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 'end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. Further, in this palindromic style expression system, the two promoter types are preferably different.

  In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense RNA expression in which a promoter capable of expressing a short RNA such as polIII is linked upstream of antisense DNA and sense DNA, respectively. A cassette and a sense RNA expression cassette are constructed, and these cassettes are held in different vectors.

The dsRNA used in the present invention is preferably siRNA. “SiRNA” means a double-stranded RNA consisting of short strands in a range that is not toxic in cells. The chain length is not particularly limited as long as the expression of the target tan gene can be suppressed and it does not show toxicity. The dsRNA has a chain length of, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs.

  As the DNA encoding the dsRNA of the present invention, an appropriate sequence (preferably an intron sequence) is inserted between inverted repeats of the target sequence, and a double-stranded RNA having a hairpin structure (self-complementary 'hairpin' RNA (hpRNA )) (Smith, NA, et al. Nature, 407: 319, 2000, Wesley, SV et al. Plant J. 27: 581, 2001, Piccin, A. et al. Nucleic Acids Res. 29 : E55, 2001) can also be used.

The DNA encoding the dsRNA of the present invention need not be completely identical to the base sequence of the target tan gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more). Sequence identity can be determined by the technique described above (BLAST program).

  The part of the double-stranded RNA in which the RNAs in dsRNA are paired is not limited to a perfect pair, but mismatch (corresponding base is not complementary), bulge (no base corresponding to one strand) ) Or the like may include an unpaired portion. In the present invention, both bulges and mismatches may be included in the double-stranded RNA region where RNAs in dsRNA pair with each other.

Another embodiment of the DNA used to suppress the expression of the sensitive tan gene in plants is a DNA (antisense DNA) encoding an antisense RNA complementary to the above-mentioned sensitive DNA transcription product of the present invention. . Antisense DNA suppresses target gene expression by inhibiting transcription initiation by triplex formation, suppressing transcription by hybridization with a site where an open loop structure is locally created by RNA polymerase, Inhibition of transcription by hybridization with certain RNA, suppression of splicing by hybridization at the junction of intron and exon, suppression of splicing by hybridization with spliceosome formation site, suppression of transition from nucleus to cytoplasm by hybridization with mRNA , Splicing suppression by hybridization with capping site and poly (A) addition site, translation initiation suppression by hybridization with translation initiation factor binding site, translation suppression by hybridization with ribosome binding site near initiation codon, translation of mRNA Area or poly Outgrowth inhibitory peptide chains by the formation of a hybrid with over arm binding sites, and the like gene silencing and the like by hybridization with interaction site between a nucleic acid and protein. They inhibit transcription, splicing, or translation processes and suppress target gene expression (Hirashima and Inoue, "Neurochemistry Experiment Course 2, Nucleic Acid IV Gene Replication and Expression", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin , pp.319-347, 1993). The antisense DNA used in the present invention may suppress the expression of the target tan gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the target gene is designed, it will be effective for inhibiting translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.

Antisense DNA is a phosphorothioate method (Stein, Nucleic Acids Res., 16: 3209-) based on the sequence information of the sensitive DNA of the present invention (for example, the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1). 3221, 1988). The prepared DNA can be introduced into plants by a known method described later. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous sensitive tan gene transcript of the plant, but may not be completely complementary as long as the gene expression can be effectively inhibited. . The transcribed RNA preferably has a complementarity of 90% or more (eg, 95%, 96%, 97%, 98%, 99% or more) to the transcript of the target gene. In order to effectively inhibit the expression of the target gene, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.

Another embodiment of the DNA used for suppressing the expression of the sensitive tan gene in plants is a DNA encoding an RNA having a ribozyme activity that specifically cleaves a transcript such as the sensitive DNA of the present invention. Some ribozymes have a group I intron type and a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 35: 2191, 1990).

  For example, the self-cleaving domain of hammerhead ribozyme cleaves 3 ′ of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, it has been shown to be cleaved by A or U (Koizumi et. Al., FEBS Lett. 228: 225, 1988). If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. (Koizumi et.al., FEBS Lett. 239: 285, 1988, Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 35: 2191, 1990, Koizumi et.al., Nucleic. Acids. Res. 17: 7059, 1989).

Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751, 1992, Hiroshi Kikuchi, Chemistry and Biology 30: 112, 1992). A ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in plant cells. Such structural units can be arranged in tandem so that multiple sites in the target gene can be cleaved to further increase the effect (Yuyama et al., Biochem. Biophys. Res. Commun. 186: 1271, 1992 ). Using such a ribozyme, the transcription product of the target tan gene can be specifically cleaved to suppress the expression of the gene.

<Vector, transformed plant cell, transformed plant body>
The present invention also provides a vector comprising the above-described DNA of the present invention (synthesis-inducing DNA, synthesis-suppressing DNA, sensitive DNA, resistant DNA, DNA for suppressing the expression of sensitive tan gene), Plant cells (for example, sorghum cells, rice cells, corn cells) into which the DNA of the invention or a vector containing the same has been introduced, plants (for example, sorghum, rice, corn) containing the plant cells, progeny of the plants or Provided are plants that are clones, and propagation materials for these plants.

  As the vector of the present invention, for example, an autonomously replicable vector or a vector capable of homologous recombination in a chromosome can be used. The vector of the present invention usually contains a suitable expression promoter so that the DNA of the present invention is expressed after introduction into a plant cell. Examples of promoters used in the present invention include cauliflower mosaic virus-derived 35S promoter and corn-derived ubiquitin promoter. The vector can appropriately include a selection marker, a replication origin, a terminator, a polylinker, an enhancer, a ribosome binding site, and the like. In general, the DNA of the present invention is located downstream of the promoter, and a terminator is located downstream of the DNA. Examples of the terminator include a terminator derived from a cauliflower mosaic virus and a terminator derived from a nopaline synthase gene.

  The form of the plant cell into which the vector is introduced is not particularly limited, and examples thereof include suspension culture cells, protoplasts, leaf sections, callus, immature embryos, pollen and the like. As a method for introducing the vector into a plant cell and regenerating the plant body, a conventional method in this technical field can be used.

  In sorghum, for example, a method of regenerating plants by introducing genes into immature embryos or callus by the Agrobacterium method or particle gun method, and a method of pollination using pollen that has been gene-transferred by ultrasound are preferably used. (JA Able et al., In Vitro Cell. Dev. Biol. 37: 341-348, 2001, AM Casas et al., Proc. Natl. Acad. Sci. USA 90: 11212-11216, 1993, V. Girijashankar et al., Plant Cell Rep 24: 513-522, 2005, JM JEOUNG et al., Hereditas 137: 20-28, 2002, V Girijashankar et al., Plant Cell Rep 24 (9): 513-522, 2005, Zuo -yu Zhao et al., Plant Molecular Biology 44: 789-798, 2000, S. Gurel et al., Plant Cell Rep 28 (3): 429-444, 2009, ZY Zhao, Methods Mol Biol, 343: 233- 244, 2006, AK Shrawat and H Lorz, Plant Biotechnol J, 4 (6): 575-603, 2006, D Syamala and P Devi Indian J Exp Biol, 41 (12): 1482-1486, 2003, Z Gao et al . Plant Biotechnol J, 3 (6): 591-599, 2005).

  In rice, for example, gene transfer to protoplasts using polyethylene glycol to regenerate plants (Datta, SK In Gene Transfer To Plants (Potrykus I and Spanenberg Eds.) Pp 66-74, 1995), electric pulse A method of introducing a gene into a protoplast by regenerating a plant body (Toki et al. Plant Physiol. 100, 1503-1507, 1992), a method of directly introducing a gene into a cell by a particle gun method and regenerating a plant body (Christou) et al., Bio / technology, 9: 957-962, 1991) and Agrobacterium-mediated methods for regenerating plants (H . Ei et al Plant J.6: 271-282,1994), etc. Several techniques already established and widely used in the technical field of the present invention.

  Examples of corn include the method described in Shillito et al. (Bio / Technology, 7: 581, 1989) and the method described in Gorden-Kamm et al. (Plant Cell 2: 603, 1990).

  The above DNA of the present invention can be introduced into a plant as exogenous DNA, but can also be introduced into a plant by crossing with a variety having the DNA of the present invention. “Introduction” in the present invention includes both forms.

  Once a plant body in which the DNA of the present invention is introduced into the chromosome is obtained, offspring can be obtained from the plant body by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, callus, protoplast, pollen, seed, cut ear, etc.) from the plant body, its progeny or clone, and mass-produce the plant body based on them. The present invention includes a plant cell into which the DNA of the present invention is introduced, a plant containing the cell, a progeny and clone of the plant, and a propagation material for the plant, its progeny and clone.

Plants into which the sensitive DNA of the present invention has been introduced can be used, for example, for the development (screening) of drugs to confer resistance to pathogenic filamentous fungi on plants and for the elucidation of the mechanism of onset of susceptibility to pathogenic filamentous fungi. It can be used as an experimental plant. On the other hand, the plant into which the resistance type DNA of the present invention or the DNA for suppressing the expression of the sensitive type tan gene of the present invention is introduced is compared with the sensitive plant body into which these DNAs are not introduced. Increase in yield can be expected, and it can be used as a more useful crop or biomass.

<Method for producing a plant with resistance to pathogenic filamentous fungi>
The present invention also provides a method for producing a plant imparted with resistance to pathogenic filamentous fungi, characterized by suppressing the expression or function of the sensitive DNA (sensitive tan gene) of the present invention in the plant. provide. In the present invention, “to impart” resistance to pathogenic filamentous fungi to plants is not only to give resistance to pathogenic filamentous fungi to varieties that have no resistance to pathogenic filamentous fungi. It is intended to include further increasing the resistance to pathogenic filamentous fungi in varieties having resistance to certain pathogenic filamentous fungi.

One embodiment for suppressing the expression or function of the sensitive DNA of the present invention in a plant is to introduce the above-described resistant DNA of the present invention into the plant. Because resistance to pathogenic filamentous fungi in plants is dominated by a single recessive gene, in order to confer a trait of resistance to pathogenic filamentous fungi on plants, it is usually resistant to both tan alleles in individuals It needs to be a type DNA. As a result, only resistant DNA is expressed in the individual, and the plant can be given resistance to pathogenic filamentous fungi. Introduction of the resistant DNA of the present invention into a plant chromosome can be performed by, for example, mating or homologous recombination. Instead of introducing resistant DNA, a specific DNA sequence may be introduced into sensitive DNA on plant chromosomes to destroy its function.

Another embodiment for suppressing the expression or function of the sensitive DNA of the present invention in a plant is to introduce the DNA for suppressing the expression of the tan gene of the present invention into the plant. As a result, no sensitive translation product is produced from the sensitive DNA in the individual, so that resistance to pathogenic filamentous fungi can be imparted to the plant.

  Other embodiments for suppressing the expression or function of the sensitive DNA of the present invention in plants include, for example, a drug that suppresses the expression of sensitive DNA or a drug that binds to a sensitive translation product and suppresses its function. The use of is also considered.

<Method for determining sensitivity or resistance to pathogenic filamentous fungi in plants>
The present invention also provides a method for determining susceptibility or resistance to pathogenic filamentous fungi in plants. One embodiment of the determination method of the present invention is a method characterized by analyzing a base sequence of a tan gene in a plant and comparing it with a base sequence of a control.

In analyzing the base sequence of the tan gene, an amplification product obtained by amplifying the tan gene by PCR can be used. In case of carrying out the PCR, primers used are limited as long as it can specifically amplify the tan gene is no sequence information of tan gene (e.g., SEQ ID NO: 1, 2, 4 or 5) based on Can be designed as appropriate. A suitable primer includes a primer having the base sequence described in SEQ ID NO: 17, 18, 25 or 26 described in Table 2 below. A specific base sequence of the tan gene can be amplified by appropriately combining these primers.

The “control nucleotide sequence” to be compared with the nucleotide sequence of the tan gene in the test plant is typically the nucleotide sequence of the tan gene in the sensitive or resistant variety. By comparing the determined nucleotide sequence of the tan gene with the nucleotide sequence of the susceptible variety (for example, SEQ ID NO: 1, 2) or the resistant sequence (for example, SEQ ID NO: 4, 5), It can be evaluated whether the tan gene in the plant test is resistant or sensitive. For example, when there is a mutation (for example, C252Y, I268V in sorghum) that loses the function of the protein compared to the base sequence (eg, SEQ ID NO: 1, 2) in the sensitive variety, The tan gene is determined to have a high probability of being resistant.

Whether or not the base sequence of the tan gene in the test plant is different from the base sequence of the control can be indirectly analyzed by various methods other than the determination of the direct base sequence described above. Examples of such methods include PCR-SSCP (single-strand conformation polymorphism) method, RFLP method using restriction fragment length polymorphism (RFLP), Examples include PCR-RFLP, denaturant gradient gel electrophoresis (DGGE), allele specific oligonucleotide (ASO) hybridization, and ribonuclease A mismatch cleavage.

  In the determination method of the present invention, DNA from a test plant can be prepared using a conventional method, for example, the CTAB method. As a plant for preparing DNA, not only a grown plant body but also a plant seed or a young plant body can be used.

  The base sequence can be determined by a conventional method such as the dideoxy method or the Maxam-Gilbert method. In determining the base sequence, a commercially available sequence kit and sequencer can be used.

Another embodiment of the determination method of the present invention is a method characterized by detecting the expression of a tan gene in a plant or the molecular weight of an expression product. Here, “detection of gene expression” includes both detection at the transcription level and detection at the translation level. In addition, “detection of expression” is intended to include not only detection of the presence or absence of expression but also detection of the degree of expression.

Detection at the transcription level can be carried out by a conventional method such as RT-PCR (Reverse transcribed-Polymerase chain reaction) or Northern blotting. Primers used in the case of carrying out the PCR are limited as long as it can specifically amplify the tan gene is no sequence information of tan gene (e.g., SEQ ID NO: 1, 2, 4 or 5) on the basis It can be designed as appropriate. A preferred example of the primer is a combination of the primer consisting of the base sequence described in SEQ ID NO: 17 and the primer consisting of the base sequence described in SEQ ID NO: 18 described in Table 2 above.

  On the other hand, detection at the translation level can be performed by a conventional method, for example, Western blotting. The antibody used for Western blotting may be a polyclonal antibody or a monoclonal antibody, and methods for preparing these antibodies are well known to those skilled in the art.

Result of the detection of gene expression in a subject, susceptible type tan gene expression level sensitive variety (e.g.,那系MS-3B in sorghum) if significantly lower than the expression level of, also, of susceptibility type tan gene If the molecular weight of the expression product is significantly different from the molecular weight in a sensitive variety (for example, Na-type MS-3B in sorghum), it is considered that the expression of the function of the sensitive tan gene is suppressed. Therefore, in this case, it is determined that the test plant has a high probability of having resistance to pathogenic filamentous fungi.

Another embodiment of the determination method of the present invention is a method characterized by analyzing a base sequence of a molecular marker linked to a tan gene in a test plant and comparing it with a base sequence of a control. Here, the “molecular marker” refers to a DNA region that is genetically linked to the tan gene and can be distinguished from other DNA regions. Molecular markers, as located in the vicinity of the tan gene, for simultaneously easy genetic and tan gene, is highly useful in the determination method of the present invention. The highly useful molecular marker of the present invention is usually present within 50 kbp, more preferably within 20 kbp, and even more preferably within 10 kbp from both terminal bases of the coding region of the tan gene.

Preferred embodiments of the molecular marker of the present invention are insertion / deletion markers, SSR (simple repetitive sequence) markers, CAPS (amplified cleavage polymorphism) markers and SNP (single nucleotide polymorphism) markers. An insertion / deletion marker is a DNA polymorphism caused by insertion and / or deletion of a base. The SSR marker is a unit of 2 or 3 bases (for example, “CA”, “CG”, “TA”, “TC”, “AGG”, “CTT”, “CGC”, “GAG”, etc.) several times Is a repetitive sequence that repeats several hundred times. Since the number of repetitions varies depending on the individual or strain, this difference in the number of repetitions can be used as a DNA polymorphism. The CAPS marker is a DNA polymorphism caused by insertion, deletion, substitution, etc. of a base in a restriction enzyme recognition sequence. The SNP marker is a DNA polymorphism caused by substitution of one base in the DNA base sequence. A person skilled in the art can appropriately extract a base sequence serving as a molecular marker based on, for example, comparison of base sequences of tan genes between varieties.

  In addition to the method of directly determining the nucleotide sequence, the nucleotide sequence of the insertion / deletion marker is subjected to PCR using a primer containing the nucleotide sequence containing the marker region, and the obtained amplification product is electrophoresed. The method can be carried out by detecting the difference in the position of the DNA band on the gel. In addition, the analysis of the base sequence in the SSR marker is not limited to the method of directly determining the base sequence and detecting it as a difference in the repeat sequence, but also using PCR using a primer capable of amplifying the base sequence containing the repeat portion. The amplification product thus obtained can be electrophoresed and detected as a difference in the position of the DNA band on the gel. The analysis of the nucleotide sequence in the SNP marker is performed, for example, by performing PCR using a primer that can amplify the nucleotide sequence including the single nucleotide polymorphism part, and the nucleotide sequence incorporated into the single nucleotide polymorphism part in the obtained amplification product. The method can be carried out by specifying the type with a polarization fluorescence analyzer.

As the “control base sequence”, a base sequence of a known molecular marker linked to the tan gene can be used. For example, it sits on a region derived from sorghum chromosome 6 amplified by a primer consisting of the base sequence shown in SEQ ID NO: 7 and a primer consisting of the base sequence shown in SEQ ID NO: 8 shown in Table 1 below. SPS marker (SB25792), CAPS seated in a region derived from sorghum chromosome 6 amplified by a primer consisting of the base sequence set forth in SEQ ID NO: 9 and a primer consisting of the base sequence set forth in SEQ ID NO: 10 SNP markers located on a region derived from sorghum chromosome 6 amplified by a marker, a primer consisting of the base sequence shown in SEQ ID NO: 11 and a primer consisting of the base sequence shown in SEQ ID NO: 12.

  Therefore, the SSR marker amplified with the primer consisting of the base sequence set forth in SEQ ID NO: 7 and the primer consisting of the base sequence set forth in SEQ ID NO: 8 is sensitive depending on the sequence or chain length of the amplified DNA fragment. It is possible to determine whether it is a resistant type or not. In addition, the CAPS marker amplified with the primer consisting of the base sequence shown in SEQ ID NO: 9 and the primer consisting of the base sequence shown in SEQ ID NO: 10 is used when the amplified DNA fragment is cleaved by the restriction enzyme DraI. Can be determined to be resistant if the amplified DNA fragment is not cleaved by the restriction enzyme DraI. Furthermore, the SNP marker amplified with the primer consisting of the base sequence set forth in SEQ ID NO: 11 and the primer consisting of the base sequence set forth in SEQ ID NO: 12 is described later by decoding the base sequence of the amplified DNA fragment. As shown in the examples, it is possible to determine whether it is a sensitive type or a resistant type.

  As described above, the comparison between the base sequence of the molecular marker in the test plant and the base sequence of the control is not limited to the direct base sequence comparison, but other indicators that can evaluate the difference in the base sequence (for example, the PCR described above). By comparison of the molecular weight etc. of the amplification products by

  As a result of comparison, if the base sequence of the molecular marker in the test plant is the same type as the sensitive molecular marker, the test plant is determined to have a high probability of sensitivity, and the same type as the resistant molecular marker The test plant is determined to have a high probability of resistance.

<Method of breeding plants resistant to pathogenic filamentous fungi>
The present invention also provides a method for breeding plants that are resistant to pathogenic filamentous fungi. The breeding method of the present invention comprises (a) a step of crossing a plant variety resistant to pathogenic filamentous fungi with an arbitrary plant variety, (b) sensitivity or resistance to pathogenic filamentous fungi in an individual obtained by the crossing. And (c) selecting a variety that has been determined to have resistance to pathogenic filamentous fungi.

  Examples of “arbitrary plant varieties” to be crossed with plant varieties resistant to pathogenic filamentous fungi include, but are not limited to, susceptible varieties and individuals obtained by crossing susceptibility varieties and resistant varieties. . By using the breeding method of the present invention, it becomes possible to select plants resistant to pathogenic filamentous fungi at the stage of seeds and seedlings, and breeding of varieties having a trait resistant to pathogenic filamentous fungi. It becomes possible to carry out in a shorter period than before.

  Accordingly, the present invention relates to a plant (eg, sorghum, rice, corn) bred by the method for breeding a plant resistant to pathogenic filamentous fungi, a plant that is a descendant or clone of the plant, And propagation materials (eg, callus, protoplast, pollen, seeds, cuttings, etc.) of these plants.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example. Further, the following examples were tested and analyzed as follows.

[Example 1]
<Narrowing down the sorghum tan gene region>
In large breeding population from the product shows a purple by illness disorders sorghum那系MS-3B and tan trait varieties Greenleaf (F3-F5), by using the SSR markers and insertion and deletion markers sorghum, tan Gene mapping was performed (FIGS. 1, 2, and 3). The experimental method used for the mapping is as follows.

(Test of tan character)
The tan trait was tested by changing the leaf color due to punching in the greenhouse. In addition, the test was performed based on changes in leaf color due to lesions, damage, and insect damage that occur spontaneously by cultivating the sorghum of the varieties in the field. Moreover, since the seed cocoon color is deep purple and greenleaf is reddish brown, the tan character was tested using this point as an index.

(PCR)
Extraction of DNA from sorghum leaves was performed using general c-TAB method or extraction method by crushing. In PCR to amplify a nucleotide sequence containing a polymorphic marker, 5 μl of PCR reagent (GoTaq (registered trademark) Green Master Mix), 4.4 μl of distilled water, 0.1 μl of DNA (20 ng / μl), primers A total of 10 μl of a reaction solution containing 0.5 μl of (10p) was prepared. Then, 9.5 μl is used per reaction, 94 ° C for 2 minutes, 94 ° C for 1 minute → 55 ° C for 1 minute → 72 ° C for 2 minutes, 35 cycles, 72 ° C for 10 minutes. PCR reaction was performed. In addition, Table 1 shows information on primers for analyzing the CAPS marker and SNP marker used for mapping described below. In Table 1, “SB25792” is the name of the SSR marker to be analyzed, and “Sb06g029510.1” and “Sb06g029530.1” are the genes of the CAPS marker and SNP marker that are to be analyzed. It is a name.

(Analysis by each marker)
For the rough mapping described later, the SSR marker described in Yonemaru J, et al., DNA Res, 2009, Vol. 16, pp. 187-193 was used. Further, as described above, CAPS markers and SNP markers were analyzed using the primers shown in Table 1.

  That is, in the analysis using the CAPS marker, 5 ul of the product amplified by the PCR reaction was treated with the restriction enzyme DraI and electrophoresed on a 3% agarose gel. And since the amplification product derived from Na-type MS-3B genome is digested with DraI, the polymorphism was discriminated from the band size.

  In addition, regarding the analysis using the SNP marker, the product amplified by the PCR reaction was subjected to a direct sequence, and the base sequence was decoded. As a result, in the decoded base sequence, it is found that there are two SNPs that are T (thymine) in Na-based MS-3B and C (cytosine) and G (guanine) in Greenleaf, The mapping described below was performed using these SNPs as indices.

First, R5 individuals (143 individuals) of F5 were subjected to rough mapping using SSR markers, and it was clarified that the tan gene was located on chromosome 6. Next, the terrorist groups individuals (1142 individuals) to F3 part, were selected individuals (63 individuals) with a recombination between SSR markers SB3751 of SB3764, it performs a tan traits of the test (research) in the field, tan gene The existence region was narrowed down to a region of about 150 kbp.

Furthermore, mapping using the CAPS marker and SNP marker found based on the base sequence information of sorghum variety BTx623 was performed, and the tan gene existing region was finally narrowed down to a region of about 29 kbp (FIG. 3).

[Example 2]
<Comparison of tan genes between Greenleaf and Nasal MS-3B>
Next, the present inventors investigated the gene annotations in the narrowed region in a database, and found that genes having homology to maize leucoanthocyanidin reductase form a cluster in this region. That is, four candidate genes having homology to maize leucoanthocyanidin reductase existed in this narrowed region. Then, when the expression of these four genes was investigated by RT-PCR, only the expression pattern of a specific candidate gene (hereinafter referred to as “ tan gene”) among the candidate genes became purple due to disease disorder. It was found that gene expression increases when present (FIG. 4). The expression of the remaining genes was not confirmed by RT-PCR. From this, it was speculated that this gene is a causative gene of coloring. RT-PCR was performed using Kikuchi R.K. Et al., Plant Physiol, 2009, 149, 1341-1353, and Shimada S. et al. Et al., Plant J. et al. 2009, Vol. 58, pages 668-681. In addition, Table 2 shows the sequences of primers (primers consisting of the nucleotide sequences described in SEQ ID NOs: 13 to 24) used for RT-PCR.

Furthermore, when the nucleotide sequence around the tan gene was determined and the amino acid sequences encoded were compared between varieties, the varieties of purple-colored cultivar MS-3B already had the genomic nucleotide sequence of the gene region encoding the protein. The cultivar was BTx623 (Fig. 5). On the other hand, in Greenleaf, it was found that there are two amino acid sequences in the region that are different from those of Na-type MS-3B. From these results, it was speculated that the protein encoded by the tan gene, that is, the leucoanthocyanidin reductase-like protein having a normal function, was not synthesized in Greenleaf of the tan trait (FIG. 5). The alignment and comparison of the base sequence and amino acid sequence was performed using software GENETYX, CrastalW (http://clustalw.ddbj.nig.ac.jp/top-j.html).

[Example 3]
<Analysis of the induction of anthocyanin production in Greenleaf and naso MS-3B leaves>
Nasal MS-3B and Greenleaf leaves were shredded to 5 mm width and incubated at 26 ° C. on water agar. At the start of incubation, the leaves were collected on the second, fourth and sixth days after the start of incubation. Next, an extract was obtained from the obtained leaves (fresh weight 0.01 g) with 1 ml of methanol at 4 ° C. for 24 hours. Then, the absorbance (wavelength 493 nm) of the extract was measured. The obtained result is shown in FIG.

  As is clear from the results shown in FIG. 6, the leaves of the nacelle MS-3B were shredded and the production of anthocyanins was induced, whereas the greenleaf leaves were not.

[Example 4]
<Analysis of 3-deoxyanthocyanidin production in the leaves of Greenleaf, Naseki MS-3B and BTx623>
The pigments were extracted from 1 g of leaves of BTx623, Naskei MS-3B and Greenleaf showing lesions with 0.01% hydrochloric acid methanol. The obtained dye was hydrolyzed with 2N hydrochloric acid at 100 ° C. for 1 hour, lipids were removed with ethyl acetate, and the dye was extracted with isoamyl alcohol. The extracted pigment was air-dried, dissolved in methanol, and spotted on a TLC sheet. Thin layer chromatography (TLC) was developed using an acetic acid / water / hydrochloric acid solvent (acetic acid: water: salt = 30: 10: 3 v / v (volume ratio)). The TLC sheet used was Merck and TLCcellulose F, and the standard anthocyanin (apigenidin and luteolinidine) was Fluka. The obtained results are shown in FIG.

  As is clear from the results shown in FIG. 7, both apigeninidine and luteolinidine were produced in the leaves of Naseki MS-3B that showed lesions, but neither pigment was produced in the leaves of Greenleaf. . However, although not shown in the figure, spots were observed by applying UV to the TLC lane where the leaf extract of Greenleaf was developed. From this, it was speculated that flavonoids and the like, which are pigments that can behave together with anthocyanins, were generated even in Greenleaf, although the presence could not be confirmed with visible light.

[Example 5]
<Identification of pigments accumulated in leaves of externally damaged Greenleaf>
Attempts were made to identify the pigments produced in externally impaired Greenleaf leaves suggested in Example 4. That is, as described above, the cut Greenleaf leaves were incubated on water agar. The dyes induced in the 3-day incubation were extracted with methanol and analyzed using HPLC-MS. The obtained result is shown in FIG.

The HPLC conditions were maintained for 10 minutes after changing the mobile phase concentration (3% acetic acid: methanol) from 70:30 to 50:50 in a gradient over 20 minutes. In the analysis, Cosmo Seal 5C ₁₈ -AR-II (manufactured by Nacalai Tesque Co., Ltd.) was used as the column. Furthermore, MS was detected in API-ES positive mode. As a standard product, a standard product manufactured by CAYMAN CHEMICAL was used.

  As shown in FIG. 8, in the extract from Greenleaf leaves that had been cut and incubated, a peak was observed at about 18.2 minutes. This peak was derived from luteolin from the results of comparison with a standard product and MS analysis. It became clear that.

The other peaks obtained by MS analysis are unknown. Further, in Non-Patent Document 4, it was described that apigenin was accumulated in sorghum showing tan character , but this was not confirmed in this analysis.

  Although not shown in the figure, expression of a gene encoding an enzyme acting in each reaction step in the sorghum anthocyanin synthesis pathway (see FIG. 9) was detected by RT-PCR. Together with Greenleaf, it was revealed that all genes encoding enzymes that catalyze reactions at each stage were expressed.

  From these results, in the Na-based MS-3B, 3deoxyanthocyanidin reductase (3-DANS), which is the final stage of anthocyanin synthesis, retains normal activity, so apigeninidine and luteonidin are synthesized, and they are outside the leaves. The part that has received a physical disorder is colored purple or the like. On the other hand, since 3-DANS does not function in Greenleaf, it is considered that this final reaction does not occur. Instead, it is presumed that luteolin accumulates by the action of the FNSII enzyme.

Furthermore, it was revealed that at least luteolin was accumulated instead of 3-deoxyanthocyanidin in Greenleaf leaves that were externally damaged as described above. In addition, since luteolin has antioxidant and antibacterial activities, the resistance to pathogenic filamentous fungi and the like is exhibited by the accumulation of this pigment in sorghum leaves exhibiting the tan character. Guessed.

[Example 6]
<Verification of correlation between tan traits and resistance to pathogenic fungi>
As described above, QTL analysis was performed to confirm the correlation between the tan trait having a mutant tan gene and resistance to pathogenic filamentous fungi and the like. That is, out of F5 RIL obtained by crossing Nasal MS-3B and Greenleaf, RIL 112 with chromosome 6 being partially heterozygous is selected, and DNA is extracted from each individual plant as described above. This region was then typed using SSR markers.

As for soot disease, we observed the extent of the soot disease that occurs in the second grass at the Ina Campus field in Shinshu University in 2010. As a result, although not shown in the figure, it was confirmed that the individual with the tan trait showed resistance to soot disease.

From these results, as shown in FIG. 10, the QTL region indicating the gene region resistant to soot disease is narrowed down to 2 MB, and this region contains the tan gene (or its locus). This strongly suggested that the tan trait may increase resistance to soot disease.

From the above results, since the tan gene is mutated in sorghum exhibiting the tan character , the function of the leucoanthocyanidin reductase-like protein encoded by this gene, ie, 3-deoxyanthocyanidin reductase (3-DANS) is inactivated. Yes. Therefore, in the portion receiving the external fault, 3- deoxy anthocyanidin (Apigeninijin and Ruteonijin) is not synthesized by luteolin like to have antimicrobial activity and the like is stored in place, the tan trait, pathogenic It became clear that the resistance to the filamentous fungus was exhibited.

According to the present invention, the tan gene involved in the susceptibility and resistance of plants to pathogenic filamentous fungi has been identified, and the mechanism of the onset of tan traits has been clarified. In order to determine the susceptibility and resistance of a plant to pathogenic filamentous fungi, it was conventionally necessary to conduct an infection test for the pathogenic fungus. However, if the tan gene is used as a marker, the inoculation test for the pathogenic fungus is not performed. Even at the seed or seedling stage, it is possible to easily determine, and thus it is possible to efficiently breed resistant varieties. Resistant varieties that are bred contribute to the reduction of damage caused by pathogenic filamentous fungi, and are expected to improve biomass production and produce high-quality feed. The tan gene may be used in the synthesis of 3-deoxyanthocyanidins.

SEQ ID NOs: 7-26
<223> Base sequence of artificially synthesized primer

Claims (18)

The DNA according to any one of the following (a) to (c), which encodes a protein that suppresses 3-deoxyanthocyanidin synthesis in plants.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 6
(B) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 4 or 5
(C) DNA encoding a protein comprising an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 6 The DNA according to any one of the following (a) to (c), which encodes a protein having an activity of imparting resistance to a pathogenic filamentous fungus to a plant.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 6
(B) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 4 or 5
(C) DNA encoding a protein comprising an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 6     A vector comprising the DNA according to claim 1 or 2.     A plant cell into which the DNA according to claim 1 or 2 is introduced.     A plant comprising the cell according to claim 4.     A plant that is a descendant or clone of the plant according to claim 5.     The plant propagation material according to claim 5 or 6. A method for imparting resistance to pathogenic filamentous fungi to a plant, comprising suppressing the expression or function of the DNA according to any one of the following (a) to (c) in the plant.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(B) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(C) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein     A method for imparting resistance to a pathogenic filamentous fungus to a plant, comprising the step of introducing the DNA according to claim 2 into the plant and converting both of the tan alleles in the plant into the DNA. A method for imparting resistance to pathogenic filamentous fungi to a plant, comprising a step of introducing a DNA encoding the RNA according to any one of (a) to (c) below to the plant.
(A) a double-stranded RNA having an activity of imparting resistance to a pathogenic filamentous fungus to a plant and complementary to the transcription product of DNA according to any one of (i) to (iii) below
(B) An antisense RNA having an activity to impart resistance to pathogenic filamentous fungi to a plant and complementary to the transcription product of DNA according to any of (i) to (iii) below
(C) RNA having an activity to confer resistance to pathogenic filamentous fungi on a plant and a ribozyme activity that specifically cleaves a transcription product of DNA according to any one of (i) to (iii) below
(I) a DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(Ii) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(Iii) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein     A drug for imparting resistance to pathogenic filamentous fungi, comprising the DNA according to claim 2 or a vector into which the DNA is inserted, and using both tan alleles in plants as the DNA. A drug for imparting resistance to pathogenic filamentous fungi, comprising DNA encoding the RNA according to any one of the following (a) to (c), or a vector into which the DNA is inserted.
(A) a double-stranded RNA having an activity of imparting resistance to a pathogenic filamentous fungus to a plant and complementary to the transcription product of DNA according to any one of (i) to (iii) below
(B) An antisense RNA having an activity to impart resistance to pathogenic filamentous fungi to a plant and complementary to the transcription product of DNA according to any of (i) to (iii) below
(C) RNA having an activity to confer resistance to pathogenic filamentous fungi on a plant and a ribozyme activity that specifically cleaves a transcription product of DNA according to any one of (i) to (iii) below
(I) a DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(Ii) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(Iii) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein A method for determining sensitivity or resistance to pathogenic filamentous fungi in a plant, wherein the base sequence of a tan gene in a test plant is analyzed, and the base sequence is any one of the following (a) to (c): If it is a DNA base sequence, it is determined that the test plant has a high probability of being susceptible to pathogenic filamentous fungi. If the DNA base sequence is the DNA base sequence according to claim 2, the test plant is a pathogen. A method of determining that there is a high probability of having resistance to sexual filamentous fungi.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(B) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(C) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein A method for determining sensitivity or resistance to pathogenic filamentous fungi in a plant, wherein the expression of the DNA according to any one of (a) to (c) below or the DNA according to claim 2 is detected in a test plant. A method characterized by:
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(B) DNA encoding a protein comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting susceptibility to pathogenic filamentous fungi to plants
(C) It consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 3, and has an activity of imparting susceptibility to pathogenic filamentous fungi to plants. DNA encoding protein A method for breeding a plant resistant to pathogenic filamentous fungi, comprising:
(A) crossing a plant variety resistant to pathogenic filamentous fungi with any plant variety,
(B) a step of determining the sensitivity or resistance to pathogenic filamentous fungi in the individual obtained by the mating in step (a) by the method according to claim 13 or 14, and (c) resistance to pathogenic filamentous fungi. Selecting a variety determined to have sex.     A plant bred by the method according to claim 15.     A plant that is a descendant or clone of the plant according to claim 16.     The propagation material of the plant body of Claim 16 or 17.