WO2004013331A1

WO2004013331A1 - Pathogenic genes of xanthomonas oryzae pv. oryzae and utilization thereof

Info

Publication number: WO2004013331A1
Application number: PCT/JP2003/009922
Authority: WO
Inventors: Hisatoshi Kaku; Hirokazu Ochiai; Masaru Takeya; Yasuhiro Inoue
Original assignee: National Institute Of Agrobiological Sciences
Priority date: 2002-08-06
Filing date: 2003-08-05
Publication date: 2004-02-12
Also published as: JP2004065088A

Abstract

Pathogenic genes and DNAs around them are cloned from Xanthomonas oryzae pv. oryzae and thus 14 novel genes relating to the pathogenicity of X. oryzae pv. oryzae are found out. Infection with X. oryzae pv. oryzae of a rice sample can be conveniently and accurately diagnosed by using the presence/absence of the DNAs of these genes as an indication.

Description

Description Pathogenic genes of rice leaf blight fungus: ¾ and its use

The present invention relates to crop disease diagnosis and crop breeding. Background art

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RD et al. (1995) .Whole-genome random seauencing and assembly of Haemophilus influenzae Rd. Science

269: 496-512), the whole genome sequence of Escherichia coli and Bacillus subtilis, which is a model microorganism, and the whole genome sequences of various bacteria including animal pathogenic bacteria have been reported. Bacteria have a genome size much smaller than animals and plants, and their genome size is about 1/10 or less than that of filamentous fungi, which is the same microorganism. Genome analysis is progressing.

Genome analysis analyzes all the genes present in a genome by revealing the genome that is the source of genetic information at the nucleotide sequence level, and understands the whole life phenomenon such as gene networks and interactions. It is expected to provide valuable information, and is widely used worldwide with a wide variety of organisms. In particular, in the field of animal pathogenic bacteria, elucidation of the pathogenic mechanism of human infection and disease, and the knowledge obtained therefrom, various research developments and applications such as genomic drug discovery are expected. Currently, tuberculosis bacteria ( Cole, ST et al. (1998) .Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.Nature 393: 537-44) and E. coli 0-157 (Hayashi, T. et al. (2001). Co. Immediately lete genome seauence 'of Entero emorr agic Esc elichia col i 0157: H7 and genomic comparison with a laboratory strain K-12.DNA Research 8: 11-22) System arrangements are becoming apparent.

On the other hand, in the field of plant pathogenic bacteria, an emergency meeting on genome analysis was held at the 7th International Society for Plant Pathology in 1998, and discussions were held on future developments. For plant pathogenic bacteria, there is great expectation for genome analysis from the same viewpoint as animal pathogenic bacteria, and it is being recognized as an important future research direction. At that time, in the genome analysis of plant pathogenic bacteria, the Brazilian group had taken the lead in the analysis of Xylella / / '/ r, and the French group was planning to analyze Ralstonia solanacearua. In Japan, it has been proposed to perform a genome analysis on rice bacterial wilt as a candidate, and it is almost complete at present.

Bacterial leaf blight of rice is a globally important disease of rice that occurs not only in Japan, but also in rice cultivation areas in Asia, Africa, Australia, Latin America and the United States. It is.

The pathogenic bacterium that causes this disease is Xanthom as oryzae pv. Oryzae, a rice cultivar that is known to have a large number of races that differ in pathogenicity to rice varieties, and that is highly differentiated. .

Pathogenic bacteria that cause leaf wilt invade the conduits, mainly through the water holes distributed on the leaf margins, through conduits, and multiply. It is believed that the leaves grow wrinkled locally as the bacteria that grow out impede the rise in water, causing the phenomenon of leaf blight. Although there is a known toxin theory about the onset mechanism of Bacterial blight, the details are unknown, and it is one of the issues that should be elucidated in the future.

The genus to which this bacterium belongs is composed of bacteria that cause disease in various plants, and is known to be of a very large variety, and is a group of bacteria important in agriculture.

On the other hand, rice, the infection partner (host), is an important crop, and at the same time, is a vigorous global model as a major model for molecular biology research on various plants, including breeding and disease resistance mechanisms. This is a model plant that has been studied. In general, the process by which phytopathogenic bacteria cause disease in plants involves two processes, host recognition / infection establishment and pathogen release / pathogenesis, and many genes are thought to be involved in these processes. . However, details are still unknown.

Until now, several pathogenicity-related genes have been cloned in the bacterial blight of rice, and their functional analysis has been performed. However, comprehensive analyzes have been conducted with the aim of establishing a molecular biological basis, such as comprehensively understanding the entire picture of the pathogenicity-related genes and identifying their distribution and location on the genome. Absent.

To date, rice blight disease bacterium has no effective materials such as pesticides. As a control measure, rice blast blight resistance genes are mainly introduced by breeding to control rice blast blight resistant varieties. It has been raised and cultivated. However, it has been reported that cultivation of disease-resistant varieties into which a resistance gene has been introduced becomes ill with the emergence of pathogenic bacteria exhibiting a new pathogenic type (race), so-called resistance crushing.

In addition, as a method for detecting pathogens, a method of predicting the outbreak by detecting phages specifically infecting Bacterial Blight Pathogen of rice is known. However, a simple technique for diagnosing rice blight disease by detecting DNA is not known. Disclosure of the invention

The present invention has been made in view of such a situation, and an object of the present invention is to identify a gene involved in the pathogenicity of a rice bacterial wilt disease which is an important pathogenic bacterium of rice. More specifically, it is an object of the present invention to provide a method for easily diagnosing rice leaf blight on a test rice by setting the presence or absence of a gene related to the virulence of rice leaf blight fungus as an indicator. .

In general, it is known that pathogenic bacteria, both plants and animals, exist in the form of large clusters (pathogenic islands) on genomes or plasmids, causing genes that cause pathogens. It was predicted that the bacterial pathogen of the present invention may form such a virulence gene cluster. Therefore, the present invention In particular, we found that the hrp gene class encoding a pathogenic secretory device (type III secretion device) that is important for expressing pathogenicity in plant pathogenic bacteria such as Bacterial Blight Fungus of Rice, and race differentiation ^ Pathogenic genes involved in diversification of pathogenicity such as

(avrilucence gene) analysis.

First, the present inventors set out to establish a molecular biological basis for elucidating the mechanism of pathogenicity expression and response mechanism on the microorganism side in plant-microbe interaction. In detail, through the analysis of the disease resistance gene obtained in the rice genome research, with the view that it will be applied to the research on plant-microorganism interaction, the present inventors have analyzed the genome of rice white leaf blight fungus. Was done. The strain used for genome analysis was Xal (Yoshimura, S. et al. (1998), a rice leaf blight resistance gene in rice genome research.

Expression of Xal, a bacterial blight-resistance gene in rice, is induced by bacterial inoculat ion.Pro Natl.Acad.Sci. USA 95: 1663-8) and its structural analysis was performed. In other words, in elucidating the interaction, it is considered that strains having a non-pathogenic gene (avrXal) with respect to the resistance gene (Xal) may have more non-pathogenic genes (pathogenicity). As a result of considering the narrow spectrum, we tested MAFF311018 (T7174), a representative strain of Race I in Japan.

For a quick and simple diagnosis of pathogenic bacteria, PCR-based detection that amplifies a gene region specific to Bacterial Blight Leaf Blight is effective. In addition, it is effective to introduce a virulence gene derived from a pathogenic bacterium into a plant body so that the plant can always induce resistance to the plant side. The present inventors cloned the DNA of the virulence gene group and the DNA in the vicinity thereof from Japanese rice leaf blight fungus stored at the National Institute for Agricultural Resources, and determined the nucleotide sequence. We succeeded in identifying 14 novel virulence genes unique to Bacterial Blight Pathogen that do not show significant homosexuality. Since these genes are not found in the rice genome, the use of these novel pathogenic genes in test rice as an index makes it easy and accurate Can be diagnosed. Therefore, the novel gene discovered by the present inventors can be said to be extremely useful. The present inventors have actually hybridized to the novel virulence gene and succeeded in specifically detecting rice Bacterial Blight on test rice using a PCR primer set capable of spreading the gene. In addition, the information on the genome analysis of Bacterial Leaf blight obtained by the present invention will be a powerful 'I blueprint' for elucidating the pathogenic mechanism, as well as genetic information including the diversity of pathogenicity of XanthomoMS bacteria. It is expected to be useful knowledge in the analysis of genetic diversity and classification research.

That is, the present invention relates to a novel virulence gene group of rice bacterial wilt and its use.

[1] Any one of the following (a) to (d) derived from the bacterial wilt of rice

(a) SEQ ID NO: DNA encoding a protein consisting of the amino acid sequence of any one of the even numbers of any one of 28, et al.

(b) SEQ ID NO: DNA containing the coding region of the nucleotide sequence described in any of the odd-numbered amino acids of SEQ ID NO: 27.

(c) SEQ ID NO: Encodes a protein having an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the even-numbered amino acid sequence of any one of SEQ ID NOS: 2 to 28 DNA.

(d) a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 27;

[2] DNA according to any one of the following (a) or (b):

(a) DNA encoding an antisense RNA complementary to the transcript of the DNA of [1].

(b) DNA encoding NA having lipozyme activity that specifically cleaves the transcription product of the DNA according to [1]. [3] A vector containing the DNA of [1] or [2].

[4] A transformed cell carrying the DNA of [1] or [2] or the vector of [3].

[5] the transformed cell of [4], which is a plant cell;

[6] A transformed plant comprising the transformed cell according to [5].

[7] A transformed plant, which is a progeny or clone of the transformed plant of [6].

[8] A material for propagation of the transformed plant according to [6] or [7].

[9] An oligonucleotide comprising at least 15 consecutive nucleotide sequences in the arrangement (J number: any one of the odd-numbered numbers from 1 to 27, or its complementary arrangement [J].

[10] SEQ ID NO: 1. An oligonucleotide which specifically hybridizes with DNA consisting of the nucleotide sequence of any one of odd numbers of SEQ ID NO: 27 and has a chain length of at least 15 bases.

[11] A method for diagnosing whether or not rice is infected with the bacterial wilt disease,

(a) preparing a nucleic acid sample from a rice plant or a part thereof,

(b) detecting the presence of the DNA or the expression product thereof according to (1) in the nucleic acid sample,

A method for determining that the rice to be diagnosed is infected with Bacterial Leaf blight, if the presence of the DNA or its expression product according to [1] is detected in step (b). Things.

The present inventors have identified 14 novel virulence genes of the bacterial leaf blight fungus. First, the present invention provides DNA derived from the bacterial wilt of rice.

The nucleotide sequence of the virulence gene of Bacterial Leaf blight of Rice isolated by the present inventors, which is included in the present invention, is encoded by an odd number of SEQ ID NO: 1 and the like by each of the nucleotide sequences. The amino acid sequence of the protein is shown in SEQ ID NO: 2 Shown in the numbers. That is, the present invention relates to a DNA encoding a protein consisting of the amino acid sequence described in any one of SEQ ID NOS: 2 to 28 derived from Bacterial Leaf blight of rice, and any one of SEQ ID NOs: 1 to 27. A DNA comprising the coding region of the nucleotide sequence described in the odd number.

The present invention also provides a DNA encoding a protein structurally similar to the protein described in any of SEQ ID NOs: 2 to 28. Examples of such MA include a DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted or inserted in the protein.

Methods well known to those skilled in the art for preparing the above-mentioned DNA include / \ technology (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR). In addition to technology (Saiki, RK. Et al., Science, 1985, 230, 1350 ·, Saiki, RK. Et al., Science, 1988, 239, 487.), for example, for the DNA, si te—directed mu tagenes is method (Kramer,. & Fritz, HJ., Methods

Enzymol, 1987, 154, 350.). 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. Further, even when the nucleotide sequence is mutated, the mutation may not accompany the amino acid mutation in the protein (degenerate mutation), and such a degenerate mutant DNA is also included in the present invention.

The DNA of the present invention includes natural or isolated / purified genomic DNA, cDNA, and chemically synthesized DNA. Preparation of genomic DNA and cDNA can be performed by a person skilled in the art using conventional means. For example, genomic DNA is extracted from an organism having a gene encoding a protein represented by any one of SEQ ID NOs: 2 to 28, and a genomic library (plasmid, phage , Cosmids, BACs, PACs, etc. can be used), developed and developed using a probe prepared based on the DNA encoding the protein. It can be prepared by performing knee hybridization or plaque hybridization. It can also be prepared by preparing a primer specific to the DNA encoding the protein described in any of the even-numbered SEQ ID NOs: 2 to 28, and performing PCR using the primer. For cDNA, for example, a cDNA is synthesized based on mRNA extracted from an organism having a gene encoding the protein, inserted into a vector such as λ ZAP to prepare a cDNA library, and developed. Then, it can be prepared by performing colony hybridization or plaque hybridization as described above, or by performing PCR. Thus, DNA that can be isolated by the hybridization technique or the PCR technique and that hybridizes with the DNA consisting of the base sequence described in any of the odd-numbered SEQ ID NO: 27 or 27 is also used. It is included in the DNA of the present invention.

To isolate such DNA, a hybridization reaction is preferably performed under stringent conditions. In the present invention, the stringent hybridization conditions refer to conditions of 6 M urea, 0.4% SDS, 0.5 XSSC or a hybridization condition of a stringency equivalent thereto. Under conditions of higher stringency, for example, 6M urea, 0.4% SDS, and 0.1 × SSC, it is expected that more homologous DNA can be isolated. The DNA thus isolated is considered to have high homology at the amino acid level to the amino acid sequence described in any of the even-numbered SEQ ID NOs: 2 to 28. High homology refers to sequence identity of at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of the entire amino acid sequence.

The amino acid sequence and nucleotide sequence identity can be determined by the algorithm BLAST (Proc. Natl. Acad. Sei. USA, 1990, 87, 2264-2268. Karl in, S. & Al tscliul, SF. Natl. Acad. Sei. USA, 1993, 90, 5873.). A program called BLASTN or BLASTX based on the BLAST algorithm. A program has been developed (Altschul, SF. Et al., J Mol Biol, 1990, 215, 403.). When a nucleotide sequence is analyzed using BLASTN, the parameters are, for example, score = 100 and wordlength = 12. Also, when analyzing amino acid sequences using BLASTX, the parameters should be, for example, score = 50 and wordlength = 3.

When using BLAST and Gapped BLAST programs, use the default parameter of each program. Specific methods of these analysis methods are known.

(ht tp: //www.ncbi.nlm.nih.gov/) ₀

The present invention also provides a DNA encoding an antisense RNA complementary to a transcript of the DNA of the present invention, and a DNA encoding an RNA having a lipozyme activity that specifically cleaves the transcript of the DNA of the present invention. .

In a preferred embodiment of the present invention, the above-mentioned DNA can be used, for example, to produce a plant resistant to bacterial blight of rice with suppressed expression of the DNA of the present invention.

When producing a transformed plant in which the expression of the DNA of the present invention is suppressed, a DNA for suppressing the expression of the DNA of the present invention is inserted into an appropriate vector and introduced into a plant cell. Then, the transformed plant cells thus obtained are regenerated. "Suppression of expression of the DNA of the present invention" includes suppression of gene transcription and suppression of translation into Z or protein. It also includes a decrease in expression as well as a complete cessation of DNA expression.

As a method for suppressing the expression of a specific gene in a plant, a method utilizing antisense technology is most often used by those skilled in the art. The antisense effect in plant cells was first demonstrated by Ecker et al. Showing that antisense RNA introduced by electroporation exerts an antisense effect in plants (Ecker JR & Davis RW: Proc. Natl. Acad. Sci. USA 83: 5372, 1986). Since then, it has been reported that the expression of the target gene was reduced in tobacco and petunia due to the expression of antisense RNA (van der Krol AR, et al: Nature 333: 866, 1988). As a means to suppress gene expression in plants -1 o-Established.

The action of the antisense nucleic acid to suppress the expression of the target gene has several factors as follows. In other words, inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally formed by RNA polymerase, inhibition of transcription by formation of a hybrid with RNA that is undergoing synthesis Inhibition of splicing by hybridization at the junction of intron and exon; inhibition of splicing by hybridization with spliceosome-forming sites; inhibition of nuclear-to-cytoplasmic translocation by hybridization with mRNA; capping; (A) Splicing inhibition by the formation of a hybrid with the additional site, translation initiation inhibition by the formation of a hybrid with the translation initiation factor binding site, translation inhibition by the formation of a hybrid with the liposomal binding site near the start codon, translation region of mRNA And hybrids with polysome binding sites Outgrowth inhibitory peptide chain by forming, and the like gene expression inhibition by Haipuriddo forming the interaction site with our and nucleic acids and proteins. In this way, antisense nucleic acids suppress target gene expression by inhibiting various processes such as transcription, splicing, and translation. (Hirashima and Inoue: Shinsei Kagaku Kenkyusho Lecture Nucleic acid IV gene replication and expression (Japan Biochemical Society, Tokyo Chemical Dojin) p.319-347, 1993).

The antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, designing an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of a gene is considered to be effective in inhibiting translation of the gene. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, the antisense DNA used in the present invention includes not only the translated region of the gene but also the DNA containing the antisense sequence of the untranslated region. The antisense DNA to be used is ligated downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is ligated on the 3 'side. The DNA thus prepared can be transformed into a desired plant by using a known method. Wear. The sequence of the antisense DNA is preferably a sequence that is complementary to the pathogenic gene of rice leaf blight of the present invention or a part thereof, but is completely complementary as long as gene expression can be effectively suppressed. It does not have to be. The transcribed RNA has preferably 90% or more, and most preferably 95% or more complementarity to the transcript of the target gene. To effectively suppress the expression of a target gene using an antisense sequence, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more. The length of commonly used antisense DNA is shorter than 5 kb, preferably shorter than 2.5 kb.

Suppression of gene expression can also be performed using DNA encoding lipozyme. Liposomes refer to RNA molecules that have catalytic activity. There are various types of liposomes that have various activities.Research focused on lipozymes as enzymes that cleave RNA has made it possible to design liposomes that cleave RNA in a site-specific manner. Was. Lipozymes include those with a size of 400 nucleotides or more, such as Daphlepe I intron type and Ml RNA contained in RNase P.Hammerhead type ゃ Hairpin type has an active domain of about 40 nucleotides There are also others (Makoto Koizumi and Eiko Otsuka: Protein Nucleic Acid Enzyme, 35: 2191, 1990).

For example, the self-cleaving domain of the hammerhead lipozyme cleaves the 3 'side of C15 in the sequence G13U14C15, but its activity requires base pairing between U14 and A9, and A15 instead of C15. Alternatively, it has been shown that U15 can also be cleaved (Koizumi M, et al: FEBS Lett 228: 228, 1988). By designing a lipozyme in which the substrate binding site is complementary to the RNA sequence near the target site, it is possible to create a restriction-enzymatic RNA-cleaving ribozyme that recognizes 、;, UU or UA in the target RNA. (Koizumi M, et al: FEBS Lett 239: 285, 1988, Makoto Koizumi and Eiko Otsuka: Protein Nucleic Acid Enzyme 35: 2191, 1990, Koizumi M, et al: Nucl Acids Res 17: 7059, 1989). For example, the DNA (SEQ ID NO: 1 to 2) It is presumed that there are a plurality of potential target sites in some of the odd numbers in 7).

Hairpin type liposomes are also useful for the purpose of the present invention. This lipozyme is found, for example, in the minus strand of satellite RNA of evening bacillus spot virus (Buzayan JM: Nature 323: 349, 1986). It has been shown that a hairpin-type lipozyme can also produce a target-specific NA-cleaved lipozyme (Kikuchi Y & Sasaki N: Nucl Acids Res 19: 6751, 1991; Kikuchi, Hiroshi: Chemistry and biology 30: 112, 1992) .

The lipozyme designed to cleave the target is ligated to a promoter and transcription termination sequence, such as the cauliflower mosaic virus 35S promoter, so that it is transcribed in plant cells. At this time, if extra sequences are added to the 5'-end or 3'-end of the transcribed RNA, lipozyme activity may be lost.In such cases, the transcripted ribozyme contains In order to accurately cut out only the lipozyme portion from A, it is also possible to arrange another trimming ribozyme that acts on cis on the 5 'or 3' side of the lipozyme portion (Taira K, et al: Protein Eng 3: 733) , 1990, Dzianott AM & Bujarski JJ: Proc Natl Acad Sc i USA 86: 4823, 1989, Gross ans CA & Cech T: Nucl Acids Res 19: 3875, 1991, Taira K, et al: Nucl Acids Res 19: 5125, 1991). In addition, by arranging such structural units in tandem so that multiple sites in the target gene can be cleaved, the effect can be further enhanced (Yuyama N, et al: Biochem Biop ys Res Commun 186: 1271, 1992). Thus, by specifically cleaving the transcript of the target gene of the present invention using lipozyme, expression of the gene can be suppressed. It is also effective to introduce the DNA of the present invention into a plant so that the plant always induces resistance.

The DNA of the present invention can be used, for example, for preparing a recombinant protein. When preparing a recombinant protein, usually, the DNA of the present invention is inserted into an appropriate expression vector, the vector is introduced into appropriate cells, and the expressed protein is purified by culturing the transformed cells. I do. The recombinant protein can be expressed as a fusion protein with another protein, for example, to facilitate purification. For example, a method for preparing a fusion protein with maltose binding protein using E. coli as a host (vector pMAL series released by New Engl and BioLabs, USA), a method for preparing a fusion protein with glutathione-S-transferase (GST) (Amersham It is possible to use the vector pGEX series released by Pharmacia Biotech) or the method of adding and preparing a histidine tag (Novagen's pET series). The host cell is not particularly limited as long as it is a cell suitable for the expression of the recombinant protein. Thus, for example, yeast, various animal and plant cells, insect cells, and the like can be used. Various methods known to those skilled in the art can be used for introducing a vector into a host cell. For example, a method using calcium ions for introduction into E. coli (Mandel, M. & Higa, A., Journal of Molecular Biology, 1970, 53, 158-162., Hanahan, D., Journal of Molecule ar Biology, 1983, 166, 557-580.). The recombinant protein expressed in the host cell can be purified and recovered from the host cell or a culture supernatant thereof by a method known to those skilled in the art. When the recombinant protein is expressed as a fusion protein with the above-mentioned maltose binding protein or the like, affinity purification can be easily performed. The protein encoded by the DNA of the present invention thus produced is also included in the present invention.

By using the obtained recombinant protein, an antibody that binds to the protein can be prepared. The antibody can be used for a method for diagnosing whether or not the rice is infected with the bacterial leaf blight described below.

The polyclonal antibody is, for example, a purified protein of the present invention or one thereof. A part of the peptide can be immunized to immunized animals such as egrets, and after a certain period of time, blood can be collected and prepared from serum from which blood bladders have been removed. In addition, the monoclonal antibody is obtained by fusing antibody-producing cells of the animal immunized with the above-mentioned protein or peptide with bone tumor cells to produce a single-clonal cell (hybrid-ma) that produces an antibody that is used as an eye-opening stump. It can be prepared by isolating and obtaining an antibody from the cells. The antibody thus obtained can be used for purification and detection of the protein of the present invention. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and the fragmentability of these antibodies.

Further, the present invention provides a vector comprising the above DNA, a transformed meniscus which carries the vector, a transformed cell which is a plant cell, a transformed plant comprising the transformed cell, a transformed plant comprising the transformed cell, Provided are a transformed plant that is a progeny or a clone, and a propagation material of the transformed plant.

The introduction of the DNA or nucleic acid of the present invention into plant cells can be carried out by those skilled in the art by known methods, for example, the agrobacterium method, the electroporation method (elect-portation method), and the particle gun method. it can.

When the agrobacterium method is used, for example, the method of Nagel et al. (Microbiol. Lett., 1990, 67, 325.) is used. According to this method, the recombinant vector is transformed into bacterium agrobacterium, and the transformed agrobacterium is then introduced into plant cells by a known method such as a leaf disk method. The vector contains an expression promoter so that the DNA of the present invention is expressed in the plant, for example, after introduction into the plant. In general, the DNA of the present invention is located downstream of the promoter, and furthermore, one minute and one minute is located downstream of the DNA. The recombinant vector used for this purpose is appropriately selected by those skilled in the art according to the method of introduction into the plant or the type of the plant. Examples of the above promoter include CaMV35S derived from cauliflower mosaic virus, corn ubiquitin promoter (Japanese Patent Publication No. 2-79983), and the like. In addition, the above-mentioned one-minute-one-one-one-one can be exemplified by a terminator derived from a cauliflower mosaic virus or a terminator-derived one derived from a nopaline synthase gene. If it is one, it is not limited to these.

The plant into which the DNA or nucleic acid of the present invention is introduced may be an explant, or cultured cells may be prepared from these plants and introduced into the obtained cultured cells. The "plant cell" of the present invention includes, for example, plant cells such as leaves, roots, stems, flowers, and scutellum in seeds, virulent, suspension cultured cells, and the like.

Further, in order to efficiently select plant cells transformed by introducing the DNA or nucleic acid of the present invention, the above-mentioned recombinant vector contains an appropriate selection marker gene and a selection marker marker gene. It is preferably introduced into a plant cell together with the containing plasmid vector. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene, which is resistant to the antibiotic hygromycin, the neomycin phosphotransferase, which is resistant to kinamicin or genyumycin, and the herbicide phosphinothricin And an acetyltransferase gene that is resistant to acetyl.

The plant cells into which the recombinant vector has been introduced are placed on a known selection medium containing an appropriate selection agent and cultured according to the type of the introduced selection marker gene. As a result, transformed plant culture cells can be obtained.

Next, a plant is regenerated from the transformed cell into which the DNA or nucleic acid of the present invention has been introduced. Plant regeneration can be performed by a method known to those skilled in the art depending on the type of plant cell (Toki. Et al., Plant Physiol, 1995, 100, 1503-1507.). For example, for rice, a method for producing a transformed plant is to introduce a gene into protoplasts using polyethylene glycol to regenerate the plant (Indian rice varieties are suitable) (Dat ta, S K. et al., In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.), 1995, 66-74. Gene transfer into toplasts to regenerate plants (suitable for Japanese rice varieties) (Mi. et al., Plant Physiol, 1992, 100, 1503-1507.). Genes can be directly introduced and the plant can be regenerated (Christou, et al., Bio / technology, 1991, 9, 957-962.). Some techniques such as a method for regenerating a plant (Hiei. Et al., Plant J, 1994, 6, 271-282.) Have already been established and are widely used in the technical field of the present invention. In the present invention, these methods can be suitably used.

The plant regenerated from the transformed cells is then cultured in a conditioned medium. Thereafter, when the regenerated acclimated plant is cultivated under normal cultivation conditions, a plant is obtained, which can be matured and ripened to obtain a seed.

The presence of the introduced foreign DNA or nucleic acid in the transformed and cultivated transformed plant is determined by a known PCR method or Southern hybridization method, or by the base of the nucleic acid in the plant. It can be confirmed by analyzing the sequence. In this case, extraction of DNA or nucleic acid from the transformed plant can be performed according to the known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989).

When a foreign gene consisting of the DNA of the present invention present in a regenerated plant is analyzed by PCR, an amplification reaction is performed using the nucleic acid extracted from the regenerated plant as type III as described above. . When the nucleic acid of the present invention is DNA, a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the DNA is used as a primer, and the resulting mixture is mixed in a reaction solution. To perform amplification reactions. In the amplification reaction, DNA denaturation, annealing, and extension reactions are repeated several tens of times to obtain an amplification product of a DNA fragment containing the MA sequence of the present invention. When the reaction solution containing the amplified product is subjected to, for example, agarose electrophoresis, each amplified DNA fragment is fractionated, and it is confirmed that the DNA fragments correspond to the DNA of the present invention. It is possible to do.

Once a transformed plant into which the DNA of the present invention has been introduced into the chromosome is obtained, progeny can be obtained from the plant by sexual or asexual reproduction. In addition, it is also possible to obtain a propagation material (eg, seeds, fruits, cuttings, tubers, tubers, strains, calli, protoplasts, etc.) from the plant, its progeny or clones, and mass-produce the plant based on them. It is.

The present invention also provides an oligonucleotide comprising at least 15 contiguous base sequences in any of the odd-numbered base sequences of SEQ ID NOs: 1 to 27 or a complementary sequence thereof. As used herein, the term “acquisition sequence” refers to the other sequence for one strand of a double-stranded DNA consisting of A: T, G: C base pairs. The nucleotide sequence is not limited to a completely complementary sequence in at least 15 contiguous nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95% or more. Such DNA is useful as a probe for detecting or isolating the DNA of the present invention, and as a primer for performing amplification.

Furthermore, the present invention provides an oligonucleotide having a chain length of at least 15 bases, which specifically hybridizes with a DNA consisting of the base sequence represented by any of the odd-numbered SEQ ID NOs: 1 to 27. The U-nucleotide specifically hybridizes to the DNA of the present invention. As used herein, "specifically hybridizes" means under ordinary hybridization conditions, preferably under stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York). , USA, 2nd edition 1989), does not significantly cause cross-hybridization with DNA encoding other proteins. The oligonucleotide need not be completely complementary to the DNA of the present invention, if specific hybridization is possible. Oligonucleotides that hybridize to the DNA of the present invention and have a chain length of at least 15 nucleotides can be used as probes and primers for detecting the DNA of the present invention. When the oligonucleotide is used as a primer, its length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the DNA of the present invention.

When the above oligonucleotide is used as a probe, the probe is not particularly limited as long as it specifically hybridizes to the MA of the present invention. The probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp or more.

When the oligonucleotide of the present invention is used as a probe, it is preferable that the oligonucleotide is appropriately labeled before use. Labeling can be performed by using T4 polynucleotide kinase to phosphorylate the 5 'end of the oligonucleotide with ³² P, or by random DNA oligonucleotide using a DNA polymerase such as Klenow enzyme. A method of incorporating a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin or the like using the primers as a primer (random prime method, etc.) can be exemplified.

Further, the present invention provides a method for diagnosing whether or not rice is infected with the bacterial wilt of rice.

In the diagnostic method of the present invention, first, (a) a nucleic acid sample is prepared from a rice plant or a part thereof. Next, (b) detecting the presence of MA of the present invention or its expression product in the nucleic acid sample. In (b), when the expression product is detected, it is determined that the rice to be diagnosed is infected with the bacterial wilt of rice. The “part thereof” refers to, for example, a plant tissue, a cell, a cell extract, or the like.

One embodiment of the diagnostic method of the present invention uses a primer or a probe to detect a DNA encoding a rice bacterial wilt disease protein. Such a probe group As the primer, the above oligonucleotide of the present invention can be used. The present inventors have actually hybridized to the gene of the present invention, and succeeded in detecting the bacterial leaf blight of a test rice in a specific white stump using a PCR primer set capable of amplifying the gene. Therefore, the above-mentioned oligonucleotide of the present invention can be suitably used for the diagnostic method of the present invention. As an example of the oligonucleotide used in the above-described diagnostic method of the present invention, a set of primers described in Example 5 described below can be shown, but is not limited thereto. Those skilled in the art can appropriately design a set of primers capable of amplifying the DNA of the present invention based on the base sequence described in any one of the odd numbers of SEQ ID NOS: 1 to 28. Similarly, it is easy for those skilled in the art to design a probe capable of detecting the DNA of the present invention based on the base sequence described in any of the odd numbers of SEQ ID NO: 1 and 28. That power S is possible. Primers and probes may be labeled as necessary. Examples of the label include a radiolabel. Primers or probes that can be used in the diagnostic method of the present invention are also included in the present invention.

The diagnostic method of the present invention includes, for example, a method of preparing a nucleic acid sample from a rice plant or a part thereof suspected of being infected with the bacterial leaf blight fungus, and a polymerase chain reaction (PCR) method using the above primer, or It can be carried out by a Northern plotting method or the like using the above-mentioned probe.

Another aspect of the diagnostic method of the present invention is a method for diagnosing a test rice using an antibody as an index based on the presence or absence of a protein encoded by the pathogenic gene of rice leaf blight of the present invention. It is. The antibody used for this diagnosis can be prepared, for example, as described above. The antibody may be labeled if necessary. Examples of the label include an enzyme label. Instead of directly labeling the antibody itself, the target protein may be detected by labeling with a substance that binds to the antibody, such as protein A. In this diagnosis, for example, a test sample is prepared from a rice plant or a part thereof suspected of having been infected with the bacterial leaf blight fungus, It can be performed by ELISA or Western blotting using antibodies. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a gene map of hrp and peripheral regions.

FIG. 2 shows the sequences of SEQ ID NO: 19 and SEQ ID NO: 21 used for preparing a set of PCR primers. The portion where the sequence is indicated by a black frame indicates the sequence of each primer. A: PCR primer in SEQ ID NO: 19; B: PCR primer in SEQ ID NO: 21.

FIG. 3 is a photograph showing the results of PCR detection of various 属 7 ^ iM¾7 bacteria using a primer set designed based on the nucleotide sequence of SEQ ID NO: 19. Bacterial names in each lane are shown below the photograph.

FIG. 3 is a diagram comparing the structures of the hpaB-hrpF regions between X. oryzae pv. Oryzae, I. axo lord odis pv. Citri, and yohi campestris pv. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. [Example 1] Preparation of one BAC library

BAC (bacterial art if icial chromosome) can handle large DNA fragments stably and generate less chimeric DNA, so it is possible to cover the entire genome with a few clones, One of the effective tools. Therefore, the present inventors prepared a BAG library for about 16 genomes with an average insert length of about 100 to 120 kb (Ocliiai, H., Inoue, Υ., Hasebe, A. and Kaku, H. (2001). Cons truct ion and character izat ion of a Xan thomonas oryzae pv. oryzae bacterial artificial chromosome library. FEMS Microbiol. Lett. 200: 59-65).

[Example 2] Analysis of the hr gene cluster and surrounding regions

There are two major processes by which phytopathogenic bacteria can cause disease in plants: the establishment of host infection by Shinkanzin Z and the release / development of pathogens. The genes included in the hrp gene class are not only those encoding the pathogenic factor secretory mechanism (typelll), which plays an important role in the process of establishing host recognition infection, but also those that are not. It is also known to be the most important pathogenic genes in plant pathogenic bacteria (Bonas, U., Schulte, R., Fenselau, S., Minsavage, GV , Staskawicz, BJ, and Stall, RE (1991). Isolation of a gene cluster from Xanthomonas campestris pv.vesicatoria that determines pathogenicty and the hypersensitive response on pepper and tomato. Mol. Plant-Microbe Interact.

Therefore, the nucleotide sequence of the BAC clone having the hrp gene cluster isolated by PCR screening was determined by the shotgun method. The determined region is about 220 kb including about 30 kb of the hrp gene cluster. Figure 1 shows the results of gene prediction in this region. A total of 171 genes were predicted, including all 24 genes that make up the known hrp gene cluster in the XantJw genus.

What is unique in this region is that the transposase homologue (insert sequence: IS) was inserted in multiple regions and in tandem, including inside the hrp cluster. About 20% of the predicted 171 genes, 36 transposase-homologs were found. In addition, we found genes related to several transporters and clusters of genes related to grecan. On the other hand, as a result of a blast search of the predicted gene, 14 homologous genes with other organisms at the present time were found; Sequence the base sequence of the gene Numbers: The odd-numbered numbers from 1 to 27 are described, and the amino acid sequences predicted from the respective nucleotide sequences are described in the even-numbered numbers of SEQ ID NO: 28 to 28, respectively.

[Example 3] Specific detection of bacterial bacterial wilt of rice by PCR

PCR was carried out using the primer set shown in FIG. 2A on the rice 白 and various Xantho nasae bacteria belonging to the same genus as the rice wilt fungus under the following conditions. As the PCR primer set, primer sets of 20 sense nucleotides and antisense nucleotides were prepared from SEQ ID NOs: 19 and 21, respectively.

(Figure 2 ) .

PCR primer in SEQ ID NO: 19

Sense primer: 5'-ATGATCTTGGAATCGCACAA (SEQ ID NO: 29)

Antisense primer: 5'-TCATGATGCCACCTCCTGCG (SEQ ID NO: 30) PCR primer in SEQ ID NO: 21

Sense primer: 5'-ATGAAACTCTCCGGCGGTAT (SEQ ID NO: 31)

Antisense primer: 5'-TCATGCTCGCCCGCTTTGCC (SEQ ID NO: 32)

The PCR reaction conditions were as follows: initial denaturation 94 ° C for 2 minutes, denaturation 94 15 seconds, annealing 55 ° C 30 seconds, extension reaction 72 2 minutes 30 cycles, and final extension reaction 72 ° C 7 minutes .

The PCR amplification product was detected by agarose dull electrophoresis and detected after ethidium bromide staining. As a result, it was detected only in lanes A13, A16, B15, B16 and C15 on which the PCR amplification product of rice bacterial blight fungus DNA type II was placed (FIG. 3).

[Example 4] Comparative analysis of genome among bacteria belonging to the genus Xan tho

In May 2002, a Brazilian research group reported a comparative genomic analysis of the fungus U¾? Axonopodis v. Citri) and cabbage black i disease (Janthom as campesiris) in May 2002 (da Silva, ACR et al. (2002). Compar i son of the genomes of two Xant omonas pathogens without dif fering host spec if ici ties. Nature 417: 459-463). Genome rearrangement was observed in some regions, but the overall homology was clearly high. However, it has also been shown that 15 to 18% (650 to 800) of genes in each genome are species-specific and non-existent. Therefore, genomic information obtained from Bacterial white blight fungus was compared with three species by genomic Southern analysis.

First, the genomic structure of the hrp gene cluster was compared. As a result, there was a slight difference in the homology between the genes, but they were the same except for the hpaB-hrpF region (Fig. 4). One of the features of the hpaB-hrpF region is that the homolog of four tranpososes is inserted into rice Bacterial blight, and its length is almost doubled. Was not found. In cabbage black rot fungi, hrpW was present downstream of hpaB, but was not found in the other two species. Furthermore, in cabbage black rot fungi, tipaF downstream of hrpF was deleted and the transcription direction of hrpF was reversed. Industrial potential

Since the pathogenic gene of Bacterial blight of the rice plant identified by the present invention has no significant homology with the known nucleotide sequence and amino acid sequence, a specific probe or PCR primer set using the nucleotide sequence is prepared. Then, it can be used for simple detection of rice leaf blight by PCR.

In addition, by introducing this gene into plants, the development of new disease-resistant plants is expected.

Claims

The scope of the claims

1. Encodes a protein derived from the bacterial wilt of rice and comprising the amino acid sequence of any one of (a) SEQ ID NO: 2 to 28 described in any of (a) to (d) below DNA.

(b) SEQ ID NO: DNA containing the coding region of the nucleotide sequence described in any one of odd numbers 1 to 27.

(c) a protein having an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, or added or deleted in the even-numbered amino acid sequence of any of SEQ ID NOs: 2 to 28; DNA to be loaded.

(d) a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 27.

2. DNA described in either (a) or (b) below.

(a) DNA encoding an antisense RNA complementary to the DNA transcript according to claim 1.

(b) DNA encoding RNA having lipozyme activity that specifically cleaves the transcription product of the DNA according to claim 1.

3. A vector comprising the DNA according to claim 1 or 2.

4. A transformed cell carrying the DNA of claim 1 or 2 or the vector of claim 3.

5. The transformed cell according to claim 4, which is a plant cell.

6. A transformed plant comprising the transformed cell according to claim 5.

7. A transformed plant, which is a progeny or a clone of the transformed plant according to claim 6.

8. A propagation material for the transformed plant according to claim 6 or 7.

9. SEQ ID NO: 1. An oligonucleotide comprising at least 15 contiguous nucleotide sequences in the odd-numbered nucleotide sequence of any one of 27 and the complementary sequence thereof.

10. SEQ ID NO: 1 An oligonucleotide which specifically hybridizes with DNA consisting of the base sequence described in any of the odd-numbered numbers of SEQ ID NO: 27 and has a chain length of at least 15 bases.

1 1. A method for diagnosing whether or not rice is infected with the bacterial leaf blight fungus,

(a) preparing a nucleic acid sample from a rice plant or a part thereof,

(b) detecting the presence of the DNA or its expression product according to claim 1 in the nucleic acid sample,

Step. A method in which, in (b), the presence of the DNA or the expression product thereof according to claim 1 is detected, whereby it is determined that the rice to be diagnosed is infected with the bacterial wilt of rice.