rice blast susceptibility gene pi21, resistance gene pi21 and uses thereof

Abstract

The present inventors succeeded in isolating the rice field resistance gene pi21 by linkage analysis, and found that field resistance to blast in plants could be modified by introducing or controlling the expression of the gene. Thus, it became possible to efficiently confer plants with field resistance. It also became possible to select, at an early stage, rice plants having field resistance to blast. Moreover, by changing the tissue specificity of expression and the expression level of the gene involved in field resistance, varieties having resistance as well as high practical use can be grown.

1 . A DNA of any one of the following (a) to (h):
 (a) a DNA that encodes a protein comprising the amino acid sequence of SEQ ID NO: 3 or 22;   (b) a DNA comprising a coding region of the nucleotide sequence of SEQ ID NO: 1, 2, 20, or 21;   (c) a DNA encoding a protein which comprises an amino acid sequence with a substitution, deletion, addition, and/or insertion of one or more amino acids in the amino acid sequence of SEQ ID NO: 3 or 22, and which has a function equivalent to that of a protein comprising the amino acid sequence of SEQ ID NO: 3 or 22;   (d) a DNA which hybridizes under stringent conditions to a DNA comprising the nucleotide sequence of SEQ ID NO: 1, 2, 20, or 21, and which encodes a protein having a function equivalent to that of a protein comprising the amino acid sequence of SEQ ID NO: 3 or 22;   (e) a DNA that encodes a protein comprising the amino acid sequence of SEQ ID NO: 6;   (f) a DNA comprising a coding region of the nucleotide sequence of SEQ ID NO: 4 or 5;   (g) a DNA encoding a protein which comprises an amino acid sequence with a substitution, deletion, addition, and/or insertion of one or more amino acids in the amino acid sequence of SEQ ID NO: 6, and which has a function equivalent to that of a protein comprising the amino acid sequence of SEQ ID NO: 6; and   (h) a DNA which hybridizes under stringent conditions to a DNA comprising the nucleotide sequence of SEQ ID NO: 4 or 5, and which encodes a protein having a function equivalent to that of a protein comprising the amino acid sequence of SEQ ID NO: 6.   
     
     
         2 . A DNA of any one of the following (i) to (iv), having an ability to confer plants with field resistance to blast:
 (i) a DNA that encodes an RNA complementary to a transcription product of the DNA of any one of (a) to (d) in  claim 1 ;   (ii) a DNA that encodes an RNA having the ribozyme activity to specifically cleave a transcription product of the DNA of any one of (a) to (d) in  claim 1 ;   (iii) a DNA that encodes an RNA which inhibits expression of the DNA of any one of (a) to (d) in  claim 1  by a co-suppression effect; and   (iv) a DNA that encodes an RNA having RNAi activity to specifically cleave a transcription product of the DNA of any one of (a) to (d) in  claim 1 .   
     
     
         3 . The DNA of  claim 2 , wherein the plant is rice, wheat, barley, oat, corn, Job's tears, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, foxtail millet, or sugarcane. 
     
     
         4 . A vector comprising the DNA of any one of  claims 1  to  3 . 
     
     
         5 . A transformed cell that maintains the DNA of any one of  claims 1  to  3  in an expressible state. 
     
     
         6 . A transformed plant cell into which the DNA of any one of (a) to (d) in  claim 1  has been introduced. 
     
     
         7 . A transformed plant cell into which the DNA of  claim 2  has been introduced. 
     
     
         8 . The transformed plant cell of  claim 6  or  7 , wherein the plant is rice, wheat, barley, oat, corn, Job's tears, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, foxtail millet, or sugarcane. 
     
     
         9 . A transformed plant comprising the transformed cell of  claim 6  or  7 . 
     
     
         10 . A transformed plant that is a progeny or clone of the transformed plant of  claim 8 . 
     
     
         11 . A propagation material of the transformed plant of  claim 9 . 
     
     
         12 . A method for producing the transformed plant comprising a transformed plant cell into which the DNA of any one of (a) to (d) in  claim 1 , or the DNA of  claim 2  or  3  has been introduced, which comprises the step of introducing into a plant cell the DNA of any one of (a) to (d) in  claim 1  or the DNA of  claim 2  or  3 , and then regenerating a plant from the plant cell. 
     
     
         13 . A method for conferring a plant with field resistance to blast, which comprises the step of expressing the DNA of  claim 2  or  3  in a cell of the plant. 
     
     
         14 . The method of  claim 13 , wherein the plant is rice, wheat, barley, oat, corn, Job's tears, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, foxtail millet, or sugarcane. 
     
     
         15 . A protein encoded by the DNA of any one of (a) to (d) in  claim 1 . 
     
     
         16 . A method for producing the protein of  claim 15 , which comprises the step of culturing a transformed cell comprising a vector that comprises the DNA of any one of (a) to (d) in  claim 1 , and then collecting a recombinant protein from the cell or its culture supernatant. 
     
     
         17 . An antibody that binds to the protein of  claim 15 . 
     
     
         18 . A DNA comprising at least 15 consecutive nucleotides complementary to the DNA of  claim 1  or a complementary sequence thereof. 
     
     
         19 . An agent that increases field resistance to blast in a plant, which comprises any one of the DNA of  claim 2  or  3  or the vector comprising the DNA. 
     
     
         20 . A primer set that amplifies all or a part of the nucleotide sequence of SEQ ID NO: 1, 4, or 20. 
     
     
         21 . A primer set, that is at least any one of the following (a) to (c):
 (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 8, and a DNA comprising the nucleotide sequence of SEQ ID NO: 9;   (b) a DNA comprising the nucleotide sequence of SEQ ID NO: 16, and a DNA comprising the nucleotide sequence of SEQ ID NO: 17; and   (c) a DNA comprising the nucleotide sequence of SEQ ID NO: 26, and a DNA comprising the nucleotide sequence of SEQ ID NO: 27.   
     
     
         22 . A DNA comprising the nucleotide sequence of SEQ ID NO: 7, 10, 18, 19, 23, or 25. 
     
     
         23 . A method comprising the following steps (a) to (c):
 (a) preparing a DNA sample from a test plant;   (b) amplifying the DNA region described in  claim 1  from the DNA sample; and   (c) comparing the molecular weight or the nucleotide sequence of the amplified DNA fragment with that of the DNA of (e) or (f) in  claim 1 ,   which is a method that judges the test plant to have field resistance to blast when the molecular weight or nucleotide sequence is consistent with that of the DNA of (e) or (f) in  claim 1 .   
     
     
         24 . A method comprising the following steps (a) to (d):
 (a) preparing a DNA sample from a test plant;   (b) amplifying the DNA region described in  claim 1  from the DNA sample;   (c) separating the amplified double-stranded DNA on a non-denaturating gel; and   (d) comparing the mobility of the separated double-stranded DNA on the gel with that of the DNA of (e) or (f) in  claim 1 ,   which is a method that judges the test plant to have field resistance to blast when the mobility on the gel is consistent with that of the DNA of (e) or (f) in  claim 1 .   
     
     
         25 . A method comprising the following steps (a) to (e):
 (a) preparing a DNA sample from a test plant;   (b) amplifying the DNA region described in  claim 1  from the DNA sample;   (c) dissociating the amplified DNA into single-stranded DNAs;   (d) separating the dissociated single-stranded DNAs on a non-denaturating gel; and   (e) comparing the mobility of the separated single-stranded DNAs on the gel with that of the DNA of (e) or (f) in  claim 1 ,   which is a method that judges the test plant to have field resistance to blast when the mobility on the gel is consistent with the DNA of (e) or (f) in  claim 1 .   
     
     
         26 . A method comprising the following steps (a) to (d):
 (a) preparing a DNA sample from a test plant;   (b) amplifying the DNA region described in  claim 1  from the DNA sample;   (c) separating the amplified DNA on a gel with a gradually increasing concentration of a DNA denaturant; and   (d) comparing the mobility of the separated DNA on the gel, with that of the DNA of (e) or (f) in  claim 1 ,   which is the method that judges the test plant to have field resistance to blast when the mobility on the gel is consistent with that of the DNA of (e) or (f) in  claim 1 .   
     
     
         27 . A method for selecting a plant having field resistance to blast, which comprises the following steps (a) and (b):
 (a) producing a hybrid variety by crossing a plant having field resistance to blast with a plant having an arbitrary function; and   (b) judging whether the plant produced in step (a) has field resistance to blast by the method of any one of  claims 23  to  26 .   
     
     
         28 . A method for judging a test rice plant to have field resistance to blast when a molecular marker linked to the DNA of  claim 1  shows the same genotype as that in a rice plant having field resistance to blast. 
     
     
         29 . The method of  claim 28 , wherein the molecular marker comprises the DNA of SEQ ID NO: 10. 
     
     
         30 . A method for selecting a rice plant having field resistance to blast, wherein the method comprises the following steps (a) and (b):
 (a) producing a hybrid variety by crossing a rice plant having field resistance to blast with a rice plant having an arbitrary function; and   (b) judging whether the rice plant produced in step (a) has field resistance to blast using the method of  claim 28  or  29 .   
     
     
         31 . A method of screening for an agent that prevents or ameliorates blast in a plant, wherein the method comprises the following steps (a) to (c):
 (a) contacting a test compound with a transcription product of the DNA of any one of (a) to (d) in  claim 1 ;   (b) detecting the binding of the transcription product of the DNA of any one of (a) to (d) in  claim 1  to the test compound; and   (c) selecting a test compound that binds to the transcription product of the DNA of any one of (a) to (d) in  claim 1 .   
     
     
         32 . A method of screening for an agent that prevents or ameliorates blast in a plant, wherein the method comprises the following steps (a) to (c):
 (a) contacting a test compound with a cell collected from a plant;   (b) measuring the expression level of a transcription product of the DNA of any one of (a) to (d) in  claim 1 ; and   (c) selecting a test compound that decreases the expression level of the transcription product as compared to when the test compound is not contacted.   
     
     
         33 . A method of screening for an agent that prevents or ameliorates blast in a plant, which comprises the following steps (a) to (d):
 (a) providing a cell or cell extract comprising a DNA in which a reporter gene is operably linked downstream of a promoter region of the DNA of any one of (a) to (d) in  claim 1 ;   (b) contacting a test compound with the cell or cell extract;   (c) measuring the expression level of the reporter gene in the cell or cell extract; and   (d) selecting a test compound that decreases the expression level of the reporter gene as compared to when the test compound is not contacted.   
     
     
         34 . A method of screening for an agent that prevents or ameliorates blast in a plant, which comprises the following steps (a) to (d):
 (a) regenerating a transformed plant from the transformed plant cell of  claim 6 ;   (b) contacting the blast fungus and a test compound with the transformed plant; and   (c) selecting a test compound that suppresses blast in the transformed plant as compared to when the test compound is not contacted.   
     
     
         35 . A kit for use in the screening method of any one of  claims 31  to  34 .