Rice gene resistant to blast disease

Details Rice gene resistant to blast disease Rice gene resistant to blast disease

1999-06-11

2001-08-14

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C800S278000, C800S295000, C800S298000, C800S320200, C435S069100, C435S320100, C435S419000, C435S468000, C435S430000, C536S023100, C536S023600

Reexamination Certificate

active

06274789

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a gene controlling resistance to blast disease in plants, a protein encoded by said gene, and their use.
BACKGROUND OF THE INVENTION
Blast disease is a serious disease in plants such as rice and is caused by the rice blast fungi,
Magnaporthe grisea.
The disease has substantially damaged the rice yields in Japan and many other rice-breeding countries. The damage is particularly severe at low temperatures and in high humidity. The disease has been obviated by breeding resistant varieties as well as applying agricultural chemicals. Originally, there were rice strains resistant to the disease. These strains and varieties carry genes resistant to a specific race of the blast fungi, and these genes have been analyzed for a long time. Presently, about 30 genes have been identified as being blast-disease resistant (Kinoshita, Rice Genet. Newsl. 7:16-57 (1990), Iwata, Rice Genet. Newsl. 13:12-35 (1996), Iwata, Rice Genet. Newsl. 14:7-22 (1997)). These genes have been utilized to breed highly resistant varieties, and in consequence, a number of resistant varieties have been bred. However, the introduced resistance genes are becoming ineffective due to the emergence of novel races of the blast fungi (collapse of resistant varieties). Furthermore, the molecular mechanisms of expression of the blast disease resistance and the interaction between the rice blast fungi and resistance genes remain unknown.
The resistance gene Pi-b is located at the end of the long arm of rice chromosome
2
and displays resistance to all races of blast fungi identified in Japan except for 033b (Table 1).
TABLE 1
Fungal strain
Ine
Cho
2101
Ine
THB9
Cho
F67-
Ine
P-2b
Ai74 -
Variety
Gene
#003
#007
#013
#031
#033 b+
#035
#047
#101
#303
#477
Shin 2
—
S
S
S
S
S
S
S
S
S
S
Aichiasahi
Pi-a
S
S
S
R
S
R
S
R
S
S
Inabawase
Pi-i
R
S
R
R
R
S
S
R
R
S
Kanto 51
Pi-k
R
R
S
S
S
S
R
R
R
S
Tsuyuake
Pi-km
R
R
R
S
S
S
R
R
R
S
Fukunishiki
Pi-z
R
R
R
R
R
R
S
R
R
S
Yashiromochi
Pi-ta
R
R
R
R
R
R
R
R
S
R
Pi No.4
Pi-ta2
R
R
R
R
R
R
R
R
S
R
Toride 1
Pi-zt
R
R
R
R
R
R
R
R
R
S
Ouu 316
Pi-b
R
R
R
R
S
R
R
R
R
R
R: resistant
S: susceptible
The gene has been carried in Indica varieties such as Engkatek, Milek Kuning, Tjina, and Tjahaja in Indonesia and Malaysia. In Japan, TohokuIL9, a strain homozygous for the Pi-b and having a genetic background of the sensitive variety Sasanishiki, has been bred at the Miyagi Prefectural Furukawa Agriculture Experimental Station. However, the mechanism of the resistance expression has not been clarified, nor has the Pi-b gene been isolated.
SUMMARY OF THE INVENTION
An objective of the present invention is to provide Pi-b, a resistance gene to the blast disease, a functionally equivalent gene, and proteins encoded by the genes. Another objective is to create a plant resistant to the blast disease by utilizing the gene.
The present inventors have succeeded in isolating the rice blast disease resistance gene by using map-based cloning to isolate the gene Pi-b from a large chromosomal region. Specifically, the inventors performed linkage analysis using molecular markers. First, the Pi-b locus was assigned to a chromosomal region between specific markers. Next, a physical map was constructed by aligning cosmid clones near the assigned region. The nucleotide sequences of the clones were then determined to find the region of the Pi-b candidate gene containing the nucleotide binding site (NBS) that is commonly found in the resistance genes of several plants. A cDNA library was then constructed from a variety resistant to the blast disease. The library was screened using the above candidate genomic region as a probe, and a cDNA corresponding to said genomic region was isolated. Using oligonucleotide primers prepared based on the nucleotide sequence of the isolated cDNA, RT-PCR was performed on each MRNA fraction prepared from varieties sensitive and resistant to the blast disease to analyze the expression pattern of the isolated Pi-b candidate cDNA. The cDNA was specifically amplified in the resistant variety. The present inventors thus found that the isolated cDNA clone is the Pi-b gene. The present inventors also found that plants resistant to the blast disease can be created by utilizing the isolated gene or genes homologous thereto because there is a close relationship between the isolated gene and the resistance to the blast disease.
The present invention relates to the rice blast disease resistance gene Pi-b, homologous genes, and proteins encoded by the genes. The invention also relates to a method of producing a plant resistant to the blast disease by using the genes. More specifically, the present invention relates to the following:
(1) A protein that confers on plants resistance to the blast disease, wherein the protein comprises the amino acid sequence of SEQ ID NO: 1, or its modified sequence in which one or more amino acids are substituted, deleted, and/or added,
(2) A protein that confers on plants resistance to the blast disease, wherein the protein is encoded by a DNA that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 2 and/or No: 3,
(3) A DNA encoding the protein of (1) or (2),
(4) A vector comprising the DNA of (3),
(5) A host cell carrying the vector of (4),
(6) The host cell of (5), wherein said host cell is a plant cell,
(7) A method of producing the protein of (1) or (2), wherein the method comprises cultivating the host cell of (5),
(8) A transformed plant comprising the host cell of (6),
(9) The plant of (8), wherein said plant is the
Poaceae,
(10) The plant of (8), wherein said plant is
P. oryza
, (11) The plant of any one of (8), (9), or (10), wherein said plant displays resistance to the blast disease,
(12) An antibody that binds to the protein of (1) or (2), and
(13) A DNA comprising at least 15 nucleotides, wherein the DNA hybridizes specifically to the DNA of (3).


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