JP2008212101A - Defense response-controlling transgenic plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein involving resistant response control by chitin oligosaccharide other than CEBiP. <P>SOLUTION: The present invention provides a transgenic plant in which a gene encoding a protein controlling chitin oligosaccharide inducing defense response is introduced. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a transgenic plant into which a gene encoding a protein that controls a defense response of a plant induced by a chitin oligosaccharide has been introduced, and a method for producing the same.

  Higher plants such as rice have the ability to recognize pathogenic microorganisms and develop resistance reactions. It has been known for a long time that such a resistance reaction also occurs when a plant recognizes a cell component of a pathogenic bacterium, and the resistance thus induced is particularly called basic resistance. Basic resistance is an agriculturally useful trait in that it does not select many types of pathogenic bacteria. By enhancing this, it is considered possible to enhance resistance against a wide range of diseases. What is attracting attention as a substance that induces such basic resistance is a substance called an elicitor composed of bacterial cell components of pathogenic microorganisms. Many types of elicitors are known, including chitin oligosaccharides, but to date, little has been known about the receptors that plants recognize and cause resistance reactions.

  The cell walls of phytopathogenic fungi (molds) contain chitin almost in common, and the hydrolyzate chitin oligosaccharides can produce active oxygen in plants such as rice, barley and carrots even at very low concentrations. Causes disease-resistant reactions such as gene expression changes. That is, chitin oligosaccharide has a strong elicitor action. On the other hand, if the molecular structure of the chitin oligosaccharide is changed even slightly, such a resistance reaction does not occur. Therefore, the plant has a protein (receptor) that can distinguish this chitin oligosaccharide elicitor very sensitively and strictly. This elicitor is thought to induce a resistance response to rice through this receptor.

  The present inventors purified a protein (named CEBiP (Chitin Elicitor Binding Protein)) that binds specifically and with high affinity to chitin oligosaccharides from rice cell membranes and determined the amino acid sequence thereof (Non-patent Document 1). . This CEBiP is composed of 328 amino acids and contains two sequences called “LysM domains” that are presumed to be important for binding to chitin oligosaccharides. When the expression of the CEBiP gene is suppressed by the RNAi method, chitin oligosaccharides are not bound to the cell membrane, and at the same time, resistance reactions such as active oxygen generation and gene expression changes are remarkably suppressed even when the chitin oligosaccharide is treated. These results indicate that CEBiP is a receptor that recognizes and binds chitin oligosaccharide elicitors on the cell membrane and plays an important function in chitin oligosaccharide signal transduction. However, since CEBiP itself is thought not to have an intracellular domain, it was not considered that this molecule alone was used for signal transduction.

International Publication No. WO2005 / 085444 A1 Brochure

As described above, since CEBiP does not have an intracellular domain, it is unlikely that this protein alone will transduce signals into the cell. Moreover, even when the CEBiP gene is knocked down, the resistance reaction is not completely lost. From these facts, the existence of a protein other than CEBiP involved in resistance response control by chitin oligosaccharides was presumed.
An object of the present invention is to provide a protein involved in resistance response control by chitin oligosaccharides different from CEBiP.

As a result of intensive studies to solve the above problems, the present inventors have found that the protein encoded by the At3g21630 gene is a protein involved in resistance response control by chitin oligosaccharides different from CEBiP. Based on this, the present invention has been completed.
That is, the present invention provides the following (1) to (8).

(1) A transgenic plant into which a gene encoding a protein that controls a chitin oligosaccharide-induced defense response is introduced.
(2) The transgenic plant according to (1), wherein a gene encoding a protein that controls a chitin oligosaccharide-induced defense response is overexpressed in the plant.
(3) A gene encoding a protein that controls a chitin oligosaccharide-induced defense response is a gene encoding the following protein (a), (b), or (c): Or the transgenic plant according to (2),
(a) a protein represented by the amino acid sequence set forth in SEQ ID NO: 2,
(b) a protein that controls the chitin oligosaccharide-induced defense response, represented by an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 2;
(c) a plant-derived protein encoded by a DNA that is hybridized under stringent conditions with a DNA represented by the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA complementary thereto, and a chitin oligosaccharide-induced defense response Protein to control.

(4) The transgenic plant according to any one of (1) to (3), wherein the chitin oligosaccharide-induced defense response is production of active oxygen by chitin oligosaccharide or expression of an elicitor-responsive gene How to make
(5) A method for producing a transgenic plant, wherein a gene encoding a protein that controls a chitin oligosaccharide-induced defense response is introduced into a plant and expressed in the plant.
(6) The method for producing a transgenic plant according to (5), wherein a gene encoding a protein that controls a chitin oligosaccharide-induced defense response is overexpressed in the plant.

(7) The gene encoding a protein that controls a chitin oligosaccharide-induced defense response is a gene encoding the following protein (a), (b), or (c): (5) Or the production method of the transgenic plant as described in (6).
(a) a protein represented by the amino acid sequence set forth in SEQ ID NO: 2,
(b) a protein that controls the chitin oligosaccharide-induced defense response, represented by an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 2;
(c) a plant-derived protein encoded by a DNA that is hybridized under stringent conditions with a DNA represented by the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA complementary thereto, and a chitin oligosaccharide-induced defense response Protein to control.
(8) The transgenic plant according to any one of (5) to (7), wherein the chitin oligosaccharide-induced defense response is generation of active oxygen by chitin oligosaccharide or expression of an elicitor responsive gene. How to make

  Since the transgenic plant introduced with the chitin oligosaccharide-induced defense response control protein gene provided by the present invention contains more of the protein related to the defense response than the wild type plant, it is against disease (especially fungal disease). It is considered to show resistance.

Hereinafter, the present invention will be described in detail.
The transgenic plant of the present invention is characterized in that a gene encoding a protein that controls a chitin oligosaccharide-induced defense response (hereinafter, this gene may be referred to as “the present gene”) is introduced. It is.
Examples of this gene include genes encoding the following proteins (a), (b), and (c).

(a) a protein represented by the amino acid sequence of SEQ ID NO: 2
(b) a protein that controls a chitin oligosaccharide-induced defense response, represented by an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence described in SEQ ID NO: 2
(c) a plant-derived protein encoded by a DNA that is hybridized under stringent conditions with a DNA represented by the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA complementary thereto, and a chitin oligosaccharide-induced defense response Protein that regulates
The protein (a) is a protein encoded by a gene derived from Arabidopsis thaliana whose MIPS code is “At3g21630” (the base sequence is as shown in SEQ ID NO: 1).

  The protein (b) is a protein in which a mutation is introduced into the protein (a) within a range that does not affect the function of controlling the chitin oligosaccharide-induced defense response. Here, “controlling the chitin oligosaccharide-induced defense response” refers to the function of the protein (a), more specifically, the defense response against plant diseases in the presence of chitin oligosaccharide. A function that induces As used herein, “protective response to plant diseases” refers to, for example, generation of active oxygen, elicitor responsive genes (eg, PAL gene, Nit4 gene, Rboh D gene, Rboh F gene, PR-2 gene, PR-5 gene) , Expression of chitinase gene, glucanase gene). In addition, the “chitin oligosaccharide” referred to here usually means a chitin oligosaccharide having a degree of polymerization of about 4-8. The mutation in the protein (b) includes not only a mutation that occurs in nature but also an artificial mutation. Examples of means for causing artificial mutation include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982), but are not limited thereto. The number of mutated amino acids is not limited as long as the aforementioned function is not lost, but is usually within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, most preferably Preferably it is within 5 amino acids. Whether a protein with a mutation has a function to control the chitin oligosaccharide-induced defense response is determined by coding the protein with the mutation into a plant that has lost the function of the protein (a) and has changed the phenotype. It can be determined by introducing a gene to be examined and thereby examining whether the phenotype is restored.

  The protein (c) is a protein having the same function as (a) obtained by utilizing hybridization between DNAs. The “stringent conditions” in the protein (c) refer to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. Such conditions are usually conditions such as hybridization at 37 ° C. and washing treatment at 37 ° C. with a buffer containing 1 × SSC and 0.1% SDS, preferably hybridization at 42 ° C. and 0.5 × Conditions such as washing at 42 ° C. with a buffer containing SSC and 0.1% SDS, more preferably hybridization at 65 ° C. and washing at 65 ° C. with a buffer containing 0.2 × SSC and 0.1% SDS This is the condition.

The method for introducing this gene into plants is not particularly limited, and can be performed by a known method such as the Agrobacterium method, the electroporation method, the particle Kurgan method, etc., and particularly by the Agrobacterium method. preferable.
In order to express this gene in a plant, a sequence necessary for the expression of this gene such as a promoter may be introduced into the plant together with this gene. Examples of the promoter used include, in addition to the promoter of this gene itself, inducible promoters such as ubiquitin promoter, promoter for constant expression such as cauliflower mosaic virus 35S promoter, estrogen inducible promoter, injury / disease inducible promoter and the like. be able to. Among these, it is particularly preferable to use an inducible promoter.

It is preferable that this gene introduced into a plant is overexpressed at the time when a defense response is required. In order to overexpress this gene, the above-described constant expression promoter and inducible promoter can be used, and the use of a ubiquitin promoter and an inducible promoter is particularly preferable.
The kind of the transgenic plant of the present invention is not particularly limited, and examples thereof include useful crops such as cereals, vegetables and fruit trees, and ornamental plants such as foliage plants. Specifically, as the plant, grasses such as rice, corn, wheat, barley, Arabidopsis, Chinese cabbage, cabbage, rapeseed, etc., legumes such as soybean, eggplant, potato, tomato, tobacco, etc. Can be exemplified, such as Rosaceae plants, chrysanthemums, roses, pears and the like, among which Brassicaceae plants and Gramineae plants are preferable.

  It is possible to confer resistance to various pathogens by introducing a receptor kinase gene or protein kinase gene, which has been shown to play an important role in inducing a defense response, into a plant and overexpressing it (1 ) To (4).

(1) Wang, GL, Song, WY, Ruan, DL, Sideris, S., Ronald, PC; The cloned gene, Xa21, confers resistance to multiple Xanthomonas oryzae pv. Oryzae isolates in transgenic plants. Mol. Plant Microbe Interact. , 9, 850-855 (1996).
(2) Tang, X., Xie, M., Kim, YJ, Zhou, J., Klessig, DF, Martin, GB; Overexpression of Pto activates defense responses and confers broad resistance. Plant Cell, 11, 15-29 ( 1999).
(3) Xiao, F., Tang, X., Zhou, JM; Expression of 35S :: Pto globally activates defense-related genes in tomato plants. Plant Physiol., 126, 1637-1645 (2001).
(4) Chen, X., Shang, J., Chen, D., Lei, C., Zou, Y., Zhai, W., Liu, G., Xu, J., Ling, Z., Cao, G., Ma, B., Wang, Y., Zhao, X., Li, S., Zhu, L .; A B-lectin receptor kinase gene conferring rice blast resistance.Plant J., 46, 794-804 ( 2006).
From this, it is presumed that the transgenic plant of the present invention into which a gene encoding a protein that regulates chitin oligosaccharide-induced defense response is also resistant to pathogenic bacteria, similar to the plant described in the above document. Is done.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1]
Arabidopsis seeds were surface sterilized and then sown in MGRL medium containing 1% Sucrose and 0.1% Agarose. After low temperature treatment for 4 days, it was grown under conditions of 22 ° C., 16 hours light period, 15 ° C., 8 hours dark period. Young plantlets grown for 8-10 days were used for analysis of elicitor responsiveness.
For the analysis of the active oxygen response, young plants grown under the above conditions were transferred one by one to a 48-well plate containing a new MGRL medium and used for experiments. In order to stabilize the young plant body under the experimental conditions, it was left for 2 hours and then subjected to an elicitor treatment. For the elicitor treatment, chitin oligosaccharides having different degrees of polymerization were used, and the final concentration of each was 50 μg / ml, followed by treatment for a predetermined time. After elicitor treatment, 10 μl of medium was used for active oxygen measurement. Luminol 50 μl and potassium ferricyanide 100 μl were added to the medium after the elicitor treatment, and the intensity of light generated by chemiluminescence was quantified with a luminometer to obtain the amount of active oxygen. The result is shown in FIG.
As shown in the figure, generation of active oxygen was observed when treated with chitin oligosaccharide hexamer, heptamer and octamer.

[Example 2]
At3g21630 is an Arabidopsis gene encoding a receptor like protein kinase having a LysM domain. At3g21630 mutant Arabidopsis thaliana (homozygote and heterozygote) in which a Ds transposon was inserted into the ninth intron of this gene was obtained from RIKEN and used in the following experiments.
At3g21630 mutant Arabidopsis thaliana (homozygotes and heterozygotes) was treated with an elicitor, and the amount of active oxygen produced 0, 30, 60, and 120 minutes after the treatment was measured. The elicitor was used in the same manner as in Example 1 except that chitin oligosaccharide octamer having a final concentration of 100 μg / ml was used. The production amount of active oxygen was measured in the same manner as in Example 1. For comparison, the amount of active oxygen produced in wild-type Arabidopsis treated with elicitor and wild-type Arabidopsis untreated with elicitor was also measured. The result is shown in FIG.
As shown in the figure, no active oxygen was generated in the At3g21630 mutant homozygote treated with elicitor. On the other hand, the At3g21630 mutant heterozygote produced active oxygen, but the amount was almost half that of the wild type.

Example 3
Ten At3g21630 mutant homozygotes were treated with an elicitor, the amount of active oxygen produced after the treatment was measured, and the average value of 10 individuals was calculated. The elicitor treatment and the measurement of the amount of active oxygen produced were performed in the same manner as in Example 2. For comparison, the amount of reactive oxygen produced in wild-type Arabidopsis thaliana treated with elicitor, wild-type Arabidopsis thaliana not treated with elicitor, and At3g21630 mutant homozygote not treated with elicitor was also measured. The result is shown in FIG.
As shown in the figure, there was no difference in the amount of active oxygen produced by the At3g21630 mutant homozygote depending on the presence or absence of the elicitor treatment. From this, it is considered that the At3g21630 mutant homozygote has lost the chitin-induced defense response.

Example 4
The same concentration of Pseudomonas aeruginosa- derived lipopolysaccharide (LPS) was used in place of 100 μg / ml chitin oligosaccharide octamer, and the elicitor treatment was performed in the same manner as in Examples 2 and 3, and the amount of active oxygen produced was measured. did. The result is shown in FIG.
As shown in the figure, induction of reactive oxygen production by lipopolysaccharide is observed in both wild-type and At3g21630 mutant Arabidopsis thaliana, so mutation of At3g21630 gene specifically abolishes the response to chitin oligosaccharide elicitor it is conceivable that.

Example 5
In the same manner as in Examples 2 and 3, the elicitor treatment was performed on At3g21630 mutant type and wild type Arabidopsis thaliana. After 0 minutes, 30 minutes, 60 minutes, and 120 minutes from the treatment, RNA was extracted from about 12 plants using RNeasy Plant Mini Kit of Qiagen. 1 μg of this RNA was reverse transcribed, and PCR was performed using the obtained cDNA and primers corresponding to both sides of the transposon insertion site in the At3g21630 gene sequence. The result is shown in FIG.
As shown in the figure, in the At3g21630 mutant Arabidopsis thaliana, the gene fragment having the size observed in the wild type Arabidopsis thaliana was not amplified at all. This confirmed that normal mRNA transcription of At3g21630 gene did not occur in At3g21630 mutant Arabidopsis thaliana.

Example 6
In the same manner as in Example 5, the elicitor treatment was performed on At3g21630 mutant type and wild type Arabidopsis thaliana, and RNA was extracted from the treated plant body. Using this RNA, the expression of typical elicitor-responsive genes was examined. Expression analysis was performed by RT-PCR using primers specific to each gene. The result is shown in FIG.
As shown in the figure, the ACTIN gene that is not an elicitor responsive gene was expressed in both wild type and At3g21630 mutant Arabidopsis thaliana, but the elicitor responsive gene PAL gene, Nit4 gene, Rboh D gene, Rboh D The F gene, PR-2 gene, and PR-5 gene were not expressed in At3g21630 mutant Arabidopsis thaliana. This shows that the induction of the elicitor-responsive gene is lost in the At3g21630 mutant Arabidopsis thaliana.

Example 7
In the same manner as in Example 6, RNA prepared by subjecting At3g21630 mutant type and wild type Arabidopsis thaliana to 120 minutes elicitor treatment was used to examine the induction and suppression of elicitor-responsive gene expression using an Arabidopsis 44k array. The result is shown in FIG.
As shown in the figure, expression of the 1222 gene was induced more than twice in the wild type, and the expression of the 421 gene was suppressed to half or less. In contrast, in the At3g21630 mutant Arabidopsis thaliana, only 3 genes were induced and 2 genes were suppressed. This indicates that almost all of the genes controlled by the chitin oligosaccharide elicitor are unresponsive due to mutations in this gene.

Example 8
Agrobacterium was used to demonstrate that the loss of elicitor responsiveness seen in the At3g21630 gene mutant used in the experiments of Examples 2-7 was actually caused by this gene mutation. By transformation, the wild-type At3g21630 gene was introduced into this mutant, and its elicitor responsiveness was examined.
As shown in FIG. 8, the mutant (vector control) introduced with the vector not containing the At3g21630 gene does not show active oxygen induction by the chitin oligosaccharide elicitor, whereas the transformant introduced with the vector containing the At3g21630 gene. Then, the response to the elicitor is restored, and it is recognized that active oxygen is generated. This proves that the loss of responsiveness to chitin oligosaccharide elicitors in the mutants observed in Examples 2 to 7 is based on the mutation of this gene.

The figure which shows the relationship between the polymerization degree of chitin oligosaccharide, and the quantity of the active oxygen produced | generated from Arabidopsis thaliana seedling. Fig. 1 shows the amount of active oxygen produced from At3g21630 mutant Arabidopsis treated with chitin oligosaccharides (1). Figure (2) showing the amount of active oxygen produced from At3g21630 mutant Arabidopsis treated with chitin oligosaccharides. The figure which shows the amount of active oxygen produced | generated from At3g21630 mutant type Arabidopsis thaliana treated with LPS. The figure which shows the expression analysis result of At3g21630 gene using RT-PCR. The figure which shows the expression analysis result of the elicitor responsive gene using RT-PCR. The figure which shows the expression analysis result of the elicitor responsive gene using a microarray. The figure which shows the result of the elicitor responsiveness recovery experiment by introduction | transduction of At3g21630 gene.

Claims (8)

  A transgenic plant comprising a gene encoding a protein that controls a chitin oligosaccharide-induced defense response.   The transgenic plant according to claim 1, wherein a gene encoding a protein that controls a chitin oligosaccharide-induced defense response is overexpressed in the plant. The gene encoding a protein that controls a chitin oligosaccharide-induced defense response is a gene encoding a protein of the following (a), (b), or (c): The transgenic plant described,
(a) a protein represented by the amino acid sequence set forth in SEQ ID NO: 2,
(b) a protein that controls the chitin oligosaccharide-induced defense response, represented by an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 2;
(c) a plant-derived protein encoded by a DNA that is hybridized under stringent conditions with a DNA represented by the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA complementary thereto, and a chitin oligosaccharide-induced defense response Protein to control.   The method for producing a transgenic plant according to any one of claims 1 to 3, wherein the chitin oligosaccharide-induced defense response is generation of active oxygen by chitin oligosaccharide or expression of an elicitor-responsive gene. .   A method for producing a transgenic plant, comprising introducing a gene encoding a protein that controls a chitin oligosaccharide-induced defense response into a plant and expressing the gene in the plant.   The method for producing a transgenic plant according to claim 5, wherein a gene encoding a protein that controls a chitin oligosaccharide-induced defense response is overexpressed in the plant. 7. The gene encoding a protein that controls chitin oligosaccharide-induced defense response is a gene encoding a protein of the following (a), (b), or (c): A method for producing the described transgenic plant.
(a) a protein represented by the amino acid sequence set forth in SEQ ID NO: 2,
(b) a protein that controls the chitin oligosaccharide-induced defense response, represented by an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 2;
(c) a plant-derived protein encoded by a DNA that is hybridized under stringent conditions with a DNA represented by the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA complementary thereto, and a chitin oligosaccharide-induced defense response Protein to control.   The method for producing a transgenic plant according to any one of claims 5 to 7, wherein the chitin oligosaccharide-induced defense response is generation of active oxygen by chitin oligosaccharide or expression of an elicitor-responsive gene. .