Patent Application
20050034189
The present invention relates to plant elicitor- and gibberellin-responsive genes, and uses thereof.
DiLaurenzio et al. used chromosome walking to isolate and elucidate the structure of the SCARECROW gene, which controls the radial organization of Arabidopsis roots and stems (Laurenzio, L., D., Wysocka-Diller, J., Malamy, J. E., Pysh, L., Helariutta, Y., Freshour, G., Hahn, M. G., Feldmann, K. A., and Benfey, P. N., “The SCARECROW gene regulated an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root.” Cell, 1996, 86, 423-433). The SCARECROW gene was presumed from its structure to be a transcription factor. A number of genes with structures similar to the SCARECROW gene were identified in the Arabidopsis EST, showing clearly that SCARECROW formed a gene family (Pysh, L. D., Wysocka-Diller, J. W., Camilleri, C., Bouchez, D., and Benfey, P. N., “The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes” The Plant J., 1999, 18, 111-119).
Independently to this, two genes (GAI and RGA) that negatively regulate the signals of “gibberellin”, a plant hormone, were isolated in Arabidopsis. The predicted amino acid sequences displayed significant homology with SCARECROW, especially the two-thirds at the C-terminal end (Peng, J., Carol, P., Richards, D. E., King, K. E., Cowling, R. J., Murphy, G. P., and Harberd, N. P., “Genes and Development” 1997, 11, 3194-3205; Silverstone, A. L., Ciampaglio, C. N., and Sun, T-P., “The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway.” The Plant Cell, 1998, 10, 155-169).
Similar gibberellin signal regulatory factors were also isolated in corn, wheat (Peng, J., Richards, D. E., Hartley, N. M., Murphy, G. P., Devos, K. M., Flintham, J. E., Beales, J., Fish, L. J., Worland, A. J., Pelica, F., Sudhakar, D., Christou, P., Snape, J. W., Gale, M. D., and Harberd, N. P., “‘Green revolution’ genes encode mutant gibberellin response modulators.” Nature, 1999, 400, 256-261), and rice (Ogawa, M., Kusano, T., Katsumi, M., and Sano, H., “Rice gibberellin-insensitive gene homolog, OsGAI, encodes a nuclear-localized protein capable of gene activation at transcriptional level.” Gene, 2000, 245, 21-29; Ikeda, A., Ueguchi-Tanaka, M., Sonoda, Y., Kitano, H., Koshioka, M., Futsuhara, Y., Matsuoka, M., and Yamaguchi, J., “slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8.” The Plant Cell, 2001, 13, 999-1010), indicating structural conservation. In addition, the Lateral suppressor gene product in tomatoes was reported as belonging to the same family as SCARECROW (Schumacher, K., Schmitt, T., Rossberg, M., Schmitz, G., and Theres, K., “The Lateral suppressor(Ls) gene of tomato encodes a new member of the VHIID protein family.” Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 290-295).
Pysh et al. named this gene family GRAS, taking the capital letters of GAI, RGA and SCARECROW, which were found in Arabidopsis (Pysh, L. D., Wysocka-Diller, J. W., Camilleri, C., Bouchez, D., and Benfey, P. N., “The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes.” The Plant J., 1999, 18, 111-119). The amino acid sequences of the GRAS family are characterized by, for example, low homology in the N-terminal region, a leucine heptad structure, and VHIID regions. This type of structure is characteristic to plants, and is not known in animals or microorganisms.
Gibberellin signal regulatory factors share high homology upto the N-terminal. In particular, genetic research on GAI has revealed that the DELLA region plays a significant role in catching the gibberellin signal (Herbard, N. P., King, K., E., Carol, P., Cowling, R. J., Peng, J., and Richards, D. E. BioEssays, 1998, 20, 1001-1008).
The SCARECROW amino acid sequence includes the basic amino acid region found in leucine zipper-type transcription factors. Since 44% of the 267 amino acid residues at the N-terminal are glutamic acid, serine, threonine, proline and the like, SCARECROW was presumed to be a transcription factor (Laurenzio, L., D., Wysocka-Diller, J., Malamy, J. E., Pysh, L., Helariutta, Y., Freshour, G., Hahn, M. G., Feldmann, K. A., and Benfey, P. N., “The SCARECROW gene regulated an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root.” Cell, 1996, 86, 423-433).
Recently, the nuclear localization of fluorescence in recombinants introduced with RGA-GFP fusion proteins suggested that RGA was a transcription factor localized to nuclei (Silverstone, A. L., Ciampaglio, C. N., and Sun, T-P., “The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway.” The Plant Cell, 1998, 10, 155-169).
Chitin oligomers (N-acetylchitooligosaccharides), major components of the cell wall of rice blast disease fungus were found to induce various defense responses in cultured rice cells at low concentrations. In other words, these substances were found to act as powerful elicitors (substances that induce biological defense responses) (Yamada, A., Shibuya, N., Kodama, O., and Akatsuka, T., “Induction of phytoalexin formation in suspension-cultured rice cells by N-acetylchitooligosaccharides”, Biosci. Biotech. Biochem. (1993) 57, 405-409). In this process of chitin oligomer action, known genes of defense-related enzymes, PAL, chitinase, and glucanase, were shown to be expressed (He, D.-Y., Yazaki, Y., Nishizawa, Y., Takai, R., Yamada, K., Sakano, K., Shibuya, N., and Minami, E., “Gene activation by cytoplasmic acidification in suspension-cultured rice cells in response to the potent elicitor, N-acetylchitoheptaose”, Mol. Plant-Microbe Int. (1998) 12, 1167-1174; Nishizawa, Y., Kawakami, A., Hibi, T., He, D.-Y., Shibuya, N., and Minami, E., “Regulation of the chitinase gene expression in suspension-cultured rice cells by N-acetylchitooligosaccharides: differences in the signal transduction pathways leading to the activation of elicitor-responsive genes”, Plant Mol. Biol. (1999) 39, 907-914). In addition, three novel early genes, EL2, EL3, and EL5, have been found to be expressed with more rapid time-courses (Minami, E., Kuchitsu, K., He, D.-Y., Kouchi, H., Midoh, N., Ohtsuki, Y., and Shibuya, N., “Two novel genes rapidly and transiently activated in suspension-cultured rice cells by treatment with N-acetylchitoheptaose, a biotic elicitor for phytoalexin production”, Plant Cell Physiol. (1996) 37, 563-567; Takai, R., Hasegawa, K., Kaku, K., Shibuya, N., and Minami, E., “Isolation and analysis of expression mechanisms of a rice gene, EL5, which shows structural similarity to ATL family from Arabidopsis, in response to N-acetylchitooligosaccharide elicitor”, Plant Sci. (2001) 160, 577-583), and their structures and expression characteristics were revealed.
An object of the present invention is to identify novel elicitor-responsive genes in plants, and to provide these genes and plants in which these genes are regulated.
To achieve the above tasks, the present inventors conducted exhaustive studies. First, since the expression of a number of genes is thought to change in response to elicitors, the present inventors used a DNA microchip, containing 1265 varieties of rice EST, to screen for genes whose expression is induced in the early stages of elicitor treatment. A chitin oligomer (N-acetylchitooligosaccharide), an important component in the cell wall of rice blast fungus, was used as the elicitor. As a result, six types of novel elicitor-responsive ESTs, including two types with SCARECROW family cDNA, were identified. Of these, the products of the two SCARECROW-like genes (named CIGR1 and CIGR2 genes) had GRAS family-specific motifs, revealing for the first time that GRAS family members, other than Gibberellin signal transduction regulatory factors, also exist in rice. Interestingly, in a rice suspension cell culture and the absence of auxin, the expression of these two genes was induced in response to gibberellin in a very short period. Gibberellin is the generic name for compounds that comprise the ent-gibberellane backbone. However, only active forms of gibberellin are effective in inducing the expression of these genes. In addition, the signal transduction process leading to the expression of these genes suggests the involvement of protein phosphorylation and dephosphorylation. This kind of sudden gene expression in response to gibberellin was also observed in rice green leaves.
In plants, elicitors are known to induce various defense-related enzyme genes, and to bring about a defense response. Thus, it is expected that the elicitor-inducible CIGR1 and CIGR2 genes will be of value in conferring disease resistance to crops. Gibberellin is used as an agricultural hormone for governing essential crop characteristics, such as dormancy or budding. However, a gibberellin-inducible transcription factor has yet to be reported. Thus, it is anticipated that the genes of the present invention will also be useful in the production of recombinant crops whose valuable characteristics are regulated by gibberellin.
Thus, the present invention relates to elicitor- and gibberellin-responsive genes, and to uses thereof. Specifically, the present invention relates to:
The present inventors identified two novel elicitor-responsive genes in plants: the CIGR1 and CIGR2 genes. They also revealed that these genes display responsiveness to gibberellin.
The present invention provides DNAs coding for plant CIGR2 and CIGR1 proteins. Examples of the above-mentioned plant of the present invention include, without limitation, valuable agricultural products such as cereals, vegetables and fruit trees, and ornamental plants such as foliage plants, etc. Specifically, examples of these plants can include rice, corn, wheat, barley, rapeseed, soybeans, cotton, tomatoes, potatoes, chrysanthemums, roses, carnations, cyclamens, and such.
The above-mentioned plant of the present invention preferably includes rice. The nucleotide sequence of the cDNA of the rice CIGR1 gene is presented in SEQ ID NO: 1, and SEQ ID NO: 2 shows the amino acid sequence of the protein coded by this cDNA. Equally, the CIGR2 gene's cDNA sequence is shown in SEQ ID NO: 3, and the amino acid sequence of the protein coded by this cDNA is presented in SEQ ID NO: 4.
Where it occurs in plants other than rice, the DNA of the present invention can be isolated using methods typically known to one skilled in the art. These methods include, for example, methods using hybridization technique (Southern, EM., J. Mol. Biol., 1975, 98, 503) or Polymerase Chain Reaction (PCR) technique (Saiki, R K. et al., Science, 1985, 230, 1350; Saiki, R K. et al., Science, 1988, 239, 487). Thus, DNA with high homology to a DNA that comprises SEQ ID NO: 1 or 3 can generally be isolated from another plant by one skilled in the art. This can be achieved by using, as a probe, a DNA comprising SEQ ID NO: 1 or 3, or one part of this DNA, and using as primers, oligonucleotides that specifically hybridize to a DNA comprising SEQ ID NO: 1 or 3. Thus, a DNA of the present invention also includes DNA that hybridizes to a DNA comprising SEQ ID NO: 1 or 3, isolated using hybridization or PCR technology in this way.
To isolate this kind of DNA, a hybridization reaction is preferably carried out under stringent conditions. Stringent hybridization conditions in the present invention refer to 6 M urea, 0.4% SDS and 0.5×SSC, or to stringent hybridization conditions similar to these. Further stringent conditions, (for example, 6 M urea, 0.4% SDS and 0.1×SSC) are expected to facilitate the isolation of DNA with greater homology. The DNA isolated in this way is thought to have high homology at the amino acid level with the amino acid sequence of SEQ ID NO: 2 or 4. “High homology” with the full length amino acid sequence means sequence identity of at least 70% or more, preferably 80% or more, further preferably 90% or more, and most preferably 95% or more.
Homology in amino acid or nucleotide sequences can be determined using Karlin and Altschul's BLAST algorithm (Proc. Natl. Acad. Sei. USA, 1990, 87, 2264-2268; Karlin, S. & Altschul, SF., Proc. Natl. Acad. Sei. USA, 1993, 90, 5873). Programs called BLASTN and BLASTX have been developed using the BLAST algorithm as a base (Altschul, SF. et al., J. Mol. Biol., 1990, 215, 403). When using BLASTN to analyze nucleotide sequences, the parameters can be set at, for example, score=100 and word length=12. In addition, when using BLASTX to analyze amino acid sequences, the parameters can be set at, for example, score=50 and word length=3. When using BLAST and the Gapped BLAST program, the default parameters for each program are used. Specific techniques for these analysis methods are in the well known (http://www.ncbi.nlm.nih.gov/).
The present invention also provides DNA that codes for proteins with a structural resemblance to the above plant CIGR1 and CIGR2 proteins. This DNA includes DNA coding a protein that comprises the amino acid sequence of the CIGR1 or CIGR2 protein, where one or more amino acid substitutions, deletions, additions and/or insertions have been made.
In preparing the above DNA, one skilled in the art could use well known methods, such as the above-mentioned hybridization technique (Southern, E M., J. Mol. Biol., 1975, 98, 503) or PCR technique (Saiki, R K. et al., Science, 1985, 230, 1350; Saiki, R K. et al., Science, 1988, 239, 487). In addition to these methods, techniques such as, for example, introducing mutations using site-directed mutagenesis (Kramer, W. & Fritz, H J., Methods Enzymol., 1987, 154, 350) are also included. In the natural world, mutations in the nucleotide sequence can cause mutations in the amino acid sequence of the coded protein. In some cases, a nucleotide sequence mutation will not lead to a mutation in the amino acid sequence of the protein (a degenerate mutation). Such degenerately mutated DNAs are also included in the present invention.
The DNA of the present invention includes genomic DNA, cDNA and chemically synthesized DNA. Genomic DNA and cDNA can be produced by practices well used by those skilled in the art. For example, genomic DNA can be prepared as follows: Genomic DNA is extracted from plants comprising genes coding for the above-mentioned plant CIGR1 and CIGR2 proteins, a genomic library is constructed (a plasmid, phage, cosmid, BAC, PAC or the like can be used as the vector) and developed, and colony or plaque hybridization can be carried out using probes prepared by using a DNA coding for an above-mentioned protein as a base. Alternatively, genomic DNA can also be prepared by constructing primers specific to a DNA coding for an above-mentioned plant CIGR1 or CIGR2 protein, and then using these primers to carry out PCR. cDNA can be prepared, for example, as follows: cDNA is synthesized based on mRNA extracted from plants which comprise a gene coding for one of the above-mentioned proteins, this cDNA is inserted into λ ZAP vector or such, a cDNA library is prepared and developed, and cDNA can then be prepared in the same way as above, by carrying out colony or phage hybridization, or PCR.
The DNA of the above invention can be used, for example, in the preparation of recombinant proteins, and in the production of transformant plants whose phenotype has been altered by regulating DNA expression.
A recombinant protein is usually prepared by inserting a DNA of the present invention into an appropriate expression vector, introducing the vector into an appropriate cell, culturing the transformed cells, and purifying the expressed proteins. A recombinant protein can be expressed as a fusion protein with other proteins to make purification easier, for example, as a fusion protein with maltose binding protein in Escherichia coli (New England Biolabs, USA, vector pMAL series), as a fusion protein with glutathione-S-transferase (GST) (Amersham Pharmacia Biotech, vector pGEX series), or tagged with histidine (Novagen, pET series). The host cell is not limited so long as the cell is suitable for expressing the recombinant protein. It is possible to utilize, for example, yeast, plant, insect cells or various other animal cells, besides the above-described E. coli, by altering the expression vector used. A vector can be introduced into a host cell by a variety of methods known to one skilled in the art. For example, a transformation method using calcium ions (Mandel, M. and Higa, A., Journal of Molecular Biology, 1970, 53, 158-162; Hanahan, D., Journal of Molecular Biology, 1983, 166, 557-580) can be used to introduce the vector into E. coli. A recombinant protein expressed in the host cells can be purified and recovered from the host cells or the culture supernatant thereof by known methods in the art. When a recombinant protein is expressed as a fusion protein with maltose binding protein or other partners, the recombinant protein can be easily purified via affinity chromatography. Thus generated proteins encoded by the DNAs of the present invention are also included in the present invention.
The resulting protein can be used to prepare an antibody that binds to the protein. For example, a polyclonal antibody can be prepared from serum by immunizing animals used for immunizations, such as rabbits, with a purified protein of the present invention or a portion thereof, collecting blood after a certain period, and using serum in which clots have been removed. A monoclonal antibody can be prepared by fusing myeloma cells with the antibody-forming cells of animals immunized with the above protein or its portion, isolating a monoclonal cell expressing a desired antibody (hybridoma) and recovering the antibody from the cell. The antibody thus obtained can be utilized to purify or detect a protein of the present invention. The antibodies of the present invention include antisera, polyclonal antibodies, monoclonal antibodies, and fragments thereof.
A plant transformant expressing a DNA of the present invention can be created by inserting the DNA of the present invention into an appropriate vector, introducing this vector into a plant cell, and then, regenerating the resulting transformed plant cell.
Furthermore, a plant transformant can be created in which the expression of a DNA of the present invention is suppressed. This is achieved using a DNA that suppresses the expression of a DNA of the present invention, wherein the DNA is inserted into an appropriate vector, the vector is introduced into a plant cell, and the resulting transformed plant cell is regenerated. The phrase “suppression of expression of a DNA of the present invention” includes suppression of gene transcription and/or suppression of translation to a protein. Furthermore, it also includes a complete inability to express DNA, as well as reduced expression.
Examples of preferable embodiments of a DNA used to inhibit the expression of a DNA of the present invention may include antisense nucleic acids complementary to the transcription product of a DNA of this invention, nucleic acids comprising the ribozyme activity of specifically cleaving a transcription product of a DNA of this invention, nucleic acids which inhibit the expression of a DNA of this invention by the effects of RNAi or co-suppression, and a DNA coding for a protein which comprises a characteristic that is dominant-negative for a transcription product of a DNA of this invention, and such. In the present invention, “nucleic acid” means RNA or DNA.
The expression of a specific endogenous gene in plants can be suppressed by methods utilizing antisense technology conventional to the art. Ecker et al. were the first to demonstrate the antisense effect of an antisense RNA introduced by electroporation into plant cells (J. R. Ecker and R. W. Davis, Proc. Natl. Acad. Sci. USA, 1986, 83, 5372). Thereafter, target gene expression was reportedly reduced in tobacco and petunias by expressing antisense nucleic acids (A. R. van der Krol et al., Nature, 1988, 333, 866). The antisense technique has now been established as a means of suppressing target-gene expression in plants.
Multiple factors cause an antisense nucleic acid to suppress target-gene expression. These include the following: inhibition of transcription initiation by triple strand formation; suppression of transcription by hybrid formation at the site where the RNA polymerase has formed a local open loop structure; transcription inhibition by hybrid formation with the RNA being synthesized; suppression of splicing by hybrid formation at the junction between an intron and an exon; suppression of splicing by hybrid formation at the site of spliceosome formation; suppression of mRNA translocation from the nucleus to the cytoplasm by hybrid formation with mRNA; suppression of splicing by hybrid formation at the capping site or at the poly (A) addition site; suppression of translation initiation by hybrid formation at the binding site for the translation initiation factors; suppression of translation by hybrid formation at the site for ribosome binding near the initiation codon; inhibition of peptide chain elongation by hybrid formation in the translated region or at the polysome binding sites of mRNA; and suppression of gene expression by hybrid formation at the sites of interaction between nucleic acids and proteins. These antisense nucleic acids suppress target gene expression by inhibiting the process of transcription, splicing, translation, or such (Hirashima and Inoue, “Shin Seikagaku Jikken Koza (New Biochemistry Experimentation Lectures) 2, Kakusan (Nucleic Acids) IV, Idenshi No Fukusei To Hatsugen (Replication and Expression of Genes),” Nihon Seikagakukai Hen (The Japanese Biochemical Society) Tokyo Kagaku Dozin, 1993, pp. 319-347).
An antisense nucleic acid of the present invention can suppress target gene expression by any of the above mechanisms. In one embodiment, if an antisense sequence is designed to be complementary to the untranslated region near the 5′ end of a gene's mRNA, it will effectively inhibit translation of the gene. It is also possible to use sequences complementary to the coding regions or to the untranslated region on the 3′ side. Thus, the antisense nucleic acid used in the present invention includes a nucleic acid having antisense sequences against both the untranslated regions and the translated regions of the gene. An antisense nucleic acid to be used is connected downstream of an appropriate promoter, and, preferably, a sequence containing the transcription termination signal is connected on the 3′ side. The nucleic acid thus prepared can be transfected into the desired plant by known methods. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene of the plant to be transformed or a part thereof, but it need not be perfectly complementary so long as it can effectively inhibit gene expression. The transcribed RNA is preferably 90% or more, and most preferably 95% or more complementary to the transcribed products of the target gene. In order to effectively inhibit the expression of the target gene by means of an antisense nucleic acid, the antisense nucleic acid should be at least 15 nucleotides long or more, preferably 100 nucleotides long or more, and still more preferably 500 nucleotides long or more. The antisense nucleic acid to be used is generally shorter than 5 kb, and preferably shorter than 2.5 kb.
Furthermore, DNA encoding ribozymes can also be used to suppress the expression of endogenous genes. A ribozyme means an RNA molecule with catalytic activities. There are many ribozymes having various activities. Research with a focus on ribozymes as RNA-cleaving enzymes has enabled the design of a ribozyme that site-specifically cleaves RNA. While some ribozymes of the group I intron type or the M1RNA contained in RNaseP consist of 400 nucleotides or more, others belonging to the hammerhead type or the hairpin type have an activity domain of about 40 nucleotides (Makoto Koizumi and Eiko Ohtsuka, Tanpakushitsu Kakusan Kohso (Nucleic acid, Protein, and Enzyme), 1990, 35, 2191). 1
The self-cleavage domain of a hammerhead type ribozyme cleaves at the 3′ side of C15 in the sequence G13U14C15. Formation of a nucleotide pair between U14 and A at the ninth position is considered important for the ribozyme activity. It has been shown that the cleavage also occurs when the nucleotide at the 15th position is A or U instead of C (M. Koizumi et al. FEBS Lett., 1988, 228, 228). If the substrate binding site of the ribozyme is designed to be complementary to the RNA sequences adjacent to the target site, one can create a restriction-enzyme-like RNA-cleaving ribozyme which recognizes the sequence UC, UU, or UA within the target RNA (M. Koizumi et al. FEBS Lett., 1988, 239, 285; Makoto Koizumi and Eiko Ohtsuka, Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 1990, 35, 2191; M. Koizumi et al. Nucleic Acids Res., 1989, 17, 7059). For example, in the coding region of the CIGR1 or CIGR2 gene, there are pluralities of sites that can be used as the ribozyme target.
Hairpin-type ribozymes are also useful in the present invention. These ribozymes can be found, for example, in the minus strand of tobacco ringspot virus satellite RNA (J. M. Buzayan, Nature, 1986, 323, 349). The hairpin-type ribozyme has also been shown to create RNA-cleaving ribozymes that specifically target RNA (Y. Kikuchi and N. Sasaki, Nucleic Acids Res., 1991, 19, 6751; Yo Kikuchi, Kagaku To Seibutsu (Chemistry and Biology), 1992, 30, 112).
A ribozyme designed to cleave a target is fused with a promoter, such as the cauliflower mosaic virus 35S promoter, and with a transcription termination sequence, so that it will be transcribed in plant cells. If extra sequences are added to the 5′ end or the 3′ end of the transcribed RNA, the ribozyme activity can be lost. In this case, one can place an additional trimming ribozyme, which functions in cis on the 5′ or the 3′ side of the ribozyme portion, in order to precisely cut the ribozyme portion from the transcribed RNA containing the ribozyme (K. Taira et al., Protein Eng., 1990, 3, 733; A. M. Dzaianott and J. J. Bujarski, Proc. Natl. Acad. Sci. USA, 1989, 86, 4823; C. A. Grosshands and R. T. Cech, Nucleic Acids Res., 1991, 19, 3875; K. Taira et al., Nucleic Acid Res., 1991, 19, 5125). Multiple sites within the target gene can be cleaved by arranging these structural units in tandem to achieve greater effect (N. Yuyama et al., Biochem. Biophys. Res. Commun., 1992, 186, 1271). By using such ribozymes, it is possible to specifically cleave the transcription products of a target gene in the present invention, thereby suppressing expression of that gene.
Inhibition of endogenous gene expression can also be carried out by RNAi (RNA interference), which uses double-stranded RNA (dsRNA) comprising a sequence resembling or identical to a target gene sequence. “RNAi” refers to the phenomenon where the expression of introduced exogenous genes as well as target endogenous genes is inhibited upon introducing cells with dsRNA comprising a sequence which resembles or is identical to the target gene sequence. The details of the RNAi mechanism are not clear, however it is thought that the initially introduced dsRNA is degraded into small pieces, and somehow becomes an indicator of the target gene, resulting in target gene degradation. RNAi is also known to have effect in plants (Chuang, CF. & Meyerowitz, EM., Proc. Natl. Acad. Sci. USA, 2000, 97, 4985). For example, to inhibit the expression of plant gene CIGR1 or CIGR2 using RNAi, dsRNA comprising the CIGR1 or CIGR2 gene sequence, or a sequence resembling one of these genes, can be inserted into a plant. Genes used as RNAi do not need to be completely identical to the target gene, however they comprise at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more identity. Sequence identity can be determined using an above-mentioned method.
Inhibition of endogenous gene expression can also be achieved by co-suppression, caused by a DNA transformant which comprises a sequence identical or similar to a target gene sequence. “Co-suppression” refers to a phenomenon where the expressions of an introduced exogenous gene and a target endogenous gene are inhibited upon using a transformant to introduce plants with a gene which comprises a sequence identical or similar to the target endogenous gene. The details of the co-suppression mechanism are not clear, however at least one part is thought to duplicate the RNAi mechanism. Co-suppression is also observed in plants (Smyth, DR., Curr Biol, 1997, 7, R793; Martienssen, R., Curr Biol, 1996, 6, 810). For example, to produce plants in which the CIGR1 or CIGR2 gene has been co-suppressed, the target plant can be transformed with vector DNA constructed to enable the expression of a DNA which comprises the CIGR1 or CIGR2 gene sequence, or a sequence resembling one of these genes. Genes for use in co-suppression are not required to be completely identical to the target gene, however comprise at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more identity. The degree of sequence identity can be determined using an above-described method.
Expression inhibition of an endogenous gene of the present invention can be achieved by transforming a plant with a gene which encodes a protein comprising characteristics dominant-negative to a protein coding for the target gene. “A gene which codes a protein comprising dominant-negative characteristics” refers to a gene which, when expressed, comprises the function of eliminating or reducing the activity of the plant's original endogenous wild type protein.
In addition, the present invention provides the above-mentioned DNAs, vectors comprising the nucleic acids, transformed plant cells comprising the vectors, transformed plants comprising the transformed plant cells, transformed plants that are offspring or clones of the above transformed plants, and breeding materials from the transformed plants.
Moreover, the present invention provides a method for producing the above-mentioned transformed plants that includes the process of introducing a DNA or nucleic acid of the present invention or a vector of the present invention into plant cells, and regenerating plants from the plant cells.
A DNA or nucleic acid of the present invention can be introduced into plant cells by one skilled in the art using known methods such as the agrobacterium method, electroporation method, and the particle gun method.
The method of Nagel et al., for example, is used for the agrobacterium method (Microbiol. Lett., 1990, 67, 325). According to this method, agrobacterium is transformed by a recombinant vector and introduced to plant cells using a known method such as the leaf disc method. The above vector comprises, for example, a promoter to express a DNA of the present invention in a plant subsequent to introduction into that plant. Generally, a DNA of the present invention is placed downstream of such a promoter and, moreover, a terminator sequence is placed downstream of the DNA. A recombinant vector used for this purpose is suitably determined by one skilled in the art, depending on the transfection method or type of plant. The above-mentioned promoter may be, for example, a cauliflower mosaic virus derived CaMV35S promoter or the ubiquitin promoter from maize (Unexamined Published Japanese Patent Application No. (JP-A) Hei 2-79983).
The above-mentioned terminator may be, for example, a cauliflower mosaic virus-derived terminator or nopalin synthase terminator. However, so long as they function as a promoter or terminator in a plant, they are not limited.
Plants transfected by a DNA or nucleic acid of the present invention may be explants. Alternatively, cultured cells may be prepared from these plants, and such nucleic acids may be introduced into the cultured cells. “Plant cells” in the present invention may be, for example, cells from leaves, roots, stems, flowers, seed scutella, calluses, and cultured cell suspensions.
In addition, to efficiently select transformed plant cells which have been introduced with a DNA or nucleic acid of the present invention, the above recombinant vector preferably harbors an appropriate selective marker gene, or is introduced into plant cells together with a plasmid vector harboring a selective marker gene. Selective marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene, which confers resistance to the antibiotic hygromycin; the neomycin phosphotransferase gene, which confers resistance to kanamycin or gentamycin; and the acetyltransferase gene, which confers resistance to the herbicide, phosphinothricin.
Plant cells transfected with a recombinant vector are plated and cultured on a known selective medium containing an appropriate selective drug, depending on the type of the introduced selective marker gene. In this way, one can obtain transformed plant cultured cells.
A plant is then regenerated from the transformed plant cells into which a DNA or nucleic acid of the present invention has been introduced. Regeneration of a plant can be carried out by methods known to one skilled in the art depending on the plant cell type (Toki et al., Plant Physiol., 1995, 100, 1503-1507). Several techniques have already been established to generate transformed rice plants, and those techniques are widely used in the field of the present invention. For example, rice plants can be regenerated after genes are introduced using (1) polyethylene glycol (suitable for Indica rice varieties) (Datta, S. K. et al., In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.), 1995, 66-74); (2) electric pulse (suitable for Japonica rice varieties) (Toki et al., Plant Physiol., 1992, 100, 1503-1507); (3) particle gun method (Christou et al., Bio/technology, 1991, 9, 957-962); or (4) agrobacteria (Hiei et al., Plant J., 1994, 6, 271-282). In the present invention, these methods can preferably be used.
The plants regenerated from transformed plant cells are subsequently cultured in acclimatization medium. After the acclimatized regenerated plants are grown under normal cultivation conditions, plants can be obtained. Seeds can also be obtained when these plants mature and produce fruit.
The exogenously introduced DNA or nucleic acid in a thus regenerated and grown transformed plant can be confirmed by known methods, such as PCR or Southern hybridization, or by analyzing the nucleotide sequence of the plant's nucleic acid. To extract DNA or nucleic acid from a transformed plant, the known method of J. Sambrook et al. may be used (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989).
To conduct PCR analysis of the exogenous gene comprising a DNA of the present invention, which exists in the regenerated plant body, an amplification reaction is carried out using the template nucleic acid that was extracted from the regenerated plant by the above-mentioned method. When the nucleic acid of the present invention is a DNA, the amplification reaction may be carried out in a reaction mixture containing, as primers, synthesized oligonucleotides comprising nucleotide sequences appropriately selected according to the DNA's nucleotide sequence. An amplified DNA fragment comprising a DNA sequence of the present invention may be obtained by repeating the denaturation, annealing, and extension steps for DNA several dozen cycles of the amplification reaction. The respective amplified DNA fragments can be separated by, for example, electrophoresing the reaction solution containing amplified products on agarose gel. DNA fragments corresponding to a DNA of the present invention can then be confirmed.
Having obtained a transformed plant in which a DNA of the present invention has been inserted into the chromosomes, one can obtain the plant's offspring by sexual or non-sexual reproduction. Also, it is possible to mass-produce such plants by obtaining reproductive materials (such as seeds, fruits, cuttings, stem tubers, root tubers, shoots, calluses, and protoplasts) from the above plant, or its offspring or clones.
Herein below, the present invention will be specifically described with reference to Examples, but it is not to be construed as being limited thereto. The Examples were carried out using the materials and methods described below.
(1) Cultured rice cells: The cultured cells of rice were induced from germinating rice seeds (Oryza sativa cv. Nipponbare) on a N6 agar plate containing 1 ppm 2,4-D (auxin; 2,4-dichlorophenoxyacetic acid). The cells were successively cultured in liquid N6 medium as follows. Once a week, cells of about 1 ml in volume were transferred to 150 ml N6 medium. Every two weeks, sizes of the clusters of cells were reduced by passing through a 20-mesh metallic mesh. Unfiltered cells that were cultured while shaking for four to six days after transferring to a 30-ml medium, were used for Northern blotting's RNA extraction.
(2) RNA extraction: The extraction of total RNA from cultured cells and green leaves of rice was carried out following the phenol-SDS method. A Polytron homogenizer was used to homogenize tissue in the presence of phenol (about 90% saturated with water) of about tenfold or more volume to the tissue volume, and the same volume of extraction buffer solution (50 mM Tris-HCl pH 9.0, 1% SDS, 50 mM NaCl). The aqueous phase obtained by centrifugation was repeatedly extracted with phenol. The total nucleic acid was precipitated by adding 0.6 volume of isopropanol to the extract and then stirring the mixture. After dissolving the precipitate in water, 0.25 volume of 10 M LiCl was added, and the mixture was cooled on ice. High molecular weight RNA was collected by centrifugation, washed with 70% ethanol, and then dissolved in a small volume of water to be used as an RNA sample.
(3) A method for gibberellin treatment of cultured cells and green leaves: The cultured cells were pre-washed with 2,4-D-free N6 medium several times, and then suspended in the same medium and shaken for two hours at 25° C. Gibberellin (GA3 dissolved in ethanol) was added to make the respective final concentrations and the cells were cultured while shaking for a predetermined time and used for RNA extraction. Nipponbare plants, grown three weeks after seeding, were sprayed with gibberellin. The third and fourth leaves were collected at certain intervals and total RNA was extracted. Elicitor treatment was carried out by directly adding aqueous elicitor solutions to cultured cells of rice.
(4) Hybridization: RNA for the Northern hybridization was denatured by the glyoxal method. 2.7 μl of glyoxal (final concentration, 1 M), 1.6 μl of sodium phosphate (pH 7.0, final concentration, 10 mM), and 8 μl of dimethylsulfoxide (final concentration, 50%) were added to 10 μg of total RNA (3.7 μl). The mixture was kept at 50° C. for one hour and then subjected to electrophoresis in 1.4% agarose (10 mM sodium phosphate, pH 7.0). After electrophoresis, RNA was blotted onto a nylon membrane (Biodyne A) and RNA was fixed onto the membrane by treatment at 80° C. for two hours. Hybridization was carried out in a mixture of 50% formamide, 0.1% SDS, 0.1 mg/ml salmon sperm DNA, 5×SSPE (0.9 M NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4), and 5× Denhardt's solution (0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone) at 42° C. for one day. The membrane was then washed with 0.1×SSC (15 mM NaCl, 1.5 mM sodium citrate) three times for five minutes each at room temperature and twice for 30 minutes each at 65° C. and then exposed on X-ray film.
(5) DNA microarray analysis: Microchip (http://cdna01.dna.affrc.go.jp/RMOS/index.html) was prepared using rice ESTs (1265 clones) by the Rice Genome Project. Single-stranded cDNA probes were prepared by the reverse transcription of poly (A)-RNA extracted from the cells with no elicitor treatment and with a 15-minute elicitor treatment, in the presence of cy5-dCTP. The result was analyzed using a Microarray Scanner FLA8000 (Fujifilm).
Of the rice GRAS family members, genes regarded as gibberellin signal repressors (SLR and OsGAI genes) were recently reported (Ogawa, M., Kusano, T., Katsumi, M., and Sano, H. “Rice gibberellin-insensitive gene homolog, OsGAI, encodes a nuclear-localized protein capable of gene activation at transcriptional level.”, Gene, 2000, 245, 21-29; Ikeda, A., Ueguchi-Tanaka, M., Sonoda, Y., Kitano, H., Koshioka, M., Futsuhara, Y., Matsuoka, M., and Yamaguchi, J. “slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8.”, The Plant Cell, 2001, 13, 999-1010). Two MAFF Genebank ESTs (c72495 and AU094860) that showed significantly increased signals on DNA microarrays after a 15-minute elicitor treatment were obtained from the Ministry of Agriculture, Forestry and Fisheries (MAFF) Genebank, and their entire nucleotide sequences were determined. Their deduced amino acid sequences were compared with the known sequences of GRAS family members. The search results indicated that these two sequences had homology with known GRAS family genes (
The products of the genes of the GRAS family are regarded as transcriptional regulators, but it is not known how they are involved in the regulation of gene expression. Arabidopsis SCARECROW does not have the typical nuclear localization signal. However, it is estimated to be a transcription factor because its N-terminal region is rich in serine, threonine, proline, and glutamine. GAI gene, which is one of the Arabidopsis gibberellin signal repressors, and GRS gene very similar to that, have nuclear localization signal-like sequences (Peng, J., Carol, P., Richards, D. E., King, K. E., Cowling, R. J., Murphy, G. P., and Harberd, N. P., Genes and Development (1997) 11, 3194-3205). Furthermore, RGA, which is another repressor, was shown to be localized in the nucleus by experiments using a GFP-fusion protein (Silverstone, A. L., Ciampaglio, C. N., and Sun, T.-P., “The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway”, The Plant Cell (1998) 10, 155-169). To analyze the intracellular localization of the CIGR1 gene and CIGR2 gene products, the chimeric plasmids in which their translation regions were connected in-frame to GFP, were introduced into onion epidermal cells using the particle gun method. Fusion protein behavior was monitored using GFP fluorescence. As a result, images demonstrating the localization of CIGR2-GFP fusion protein in the nucleus were observed (
Both CIGR1 gene and CIGR2 gene were identified as elicitor responsive genes using DNA microarray analysis. The elicitor responsiveness of these two genes was analyzed using Northern blot hybridization method. Significant increases of mRNA amount were observed in both genes five minutes after treatment with chitin heptamer. Expression continued to increase for up to 90 minutes (
Ashikari et al. (Ashikari, M., Wu, J., Yano, M., Sasaki, T., and Yoshimura, A., “Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the alpha-subunit of GTP-binding protein”, Proc. Natl. Acad. Sci. U.S.A. (1999) 96, 10284-10289) and Fujisawa et al. (Fujisawa, Y., Kato, T., Ishikawa, A., Kitano, H., Sasaki, T., Asahi, T., and Iwasaki, Y., “Suppression of the heterodimeric G protein causes abnormal morphology, including dwarfism, in rice”, Proc. Natl. Acad. Sci. U.S.A. (1999) 96, 7575-7580) independently found that gene D1, which causes dwarf mutant d1 in rice, encodes the α-subunit of the trimeric G protein. In animal cells, trimeric G proteins have been known to be coupled with the seven-transmembrane receptors and play important roles in the transduction of extracellular information (Neer, E. J., “Heterotrimeric G proteins: organizers of transmembrane signals”, Cell (1995) 50, 1011-1019).
On the other hand, analysis using their inhibitors and activators suggested that the G proteins were involved in the transduction of elicitor signals (Legendre, L., Heinsyein, P. F., and Low, P. S., “Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells”, J. Biol. Chem. (1992) 267, 20140-20147). However, some problems were pointed out; e.g., the specificity of these inhibitors was not necessarily clear (Ephritikhine, G., Pradier, J.-M., and Guern, J., “Complexity of GTP γS binding to tobacco plasma membranes”, Plant Physiol. Biochem. (1993) 31, 573-584). Thus, no clear conclusion has been obtained concerning the involvement of the D1 gene product.
Tsukada et al. compared in detail the various elicitor responses of calluses derived from d1 strain seeds with those of the wild type, and demonstrated that there was no significant difference between them (Tsukada, K., Ishizaka, M., Fujisawa, Y., Iwasaki, Y., Yamaguchi, T., Minami, E., and Shibuya, N., “Rice receptor for chitin oligosaccharide elicitor does not couple to heterotrimeric G-protein: Elicitor responses of suspension cultured rice cells from Daikoku dwarf (d1) mutants lacking a functional G-protein α-subunit”, Physiol. Plantrum (2002) 116, 373-382).
Preliminary experiments demonstrated that the time-courses of elicitor induction of CIGR1 gene and CIGR2 gene in the d1 strain were similar to those in the wild type.
On the other hand, Ueguchi-Tanaka et al. showed that D1 gene is involved in the transduction of gibberellin signals, using as an indicator the induction of α-amylase in aleurone in germinating seeds (Ueguchi-Tanaka, M., Fujisawa, Y., Kobayashi, M., Ashikari, M., Iwasaki, Y., Kitano, H., and Matsuoka, M., “Rice dwarf mutant d1, which is defective in the α subunit of the heterotrimeric G protein, affects gibberellin signal transduction”, Proc. Natl. Acad. Sci. U.S. A. (2000) 97, 11638-11643). Prior to this study, Schumacher et al. (Schumacher, K., Schmitt, T., Rossberg, M., Schmitz, G., and Theres, K., “The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family”, Proc. Natl. Acad. Sci. U.S. A. (1999) 96, 290-295) showed that the tomato lateral bud suppressor gene product belonged to the same gene family as SCARECROW. They pointed out the possibility of an interaction between this gene product and gibberellin in the discussion on its role in morphogenesis.
Therefore, the present inventors examined the gibberellin responsiveness of CIGR1 and CIGR2 genes. Suspension-cultured cells were treated with GA3, which is one of the active-type gibberellins, and changes in the expression of these two genes were analyzed over time. In both genes, there was no significant change in the amount of expression up to three hours after treatment. To maintain cell division activity, the medium used for culturing plant cells contained 2,4-D, which is one of auxins that are not easily metabolized by plants. It was thought that 2,4-D might suppress the action of gibberellin. Therefore, the cells were pre-washed with 2,4-D-free medium, and after two hours of pre-culture in the same medium, the cells were treated with GA3. Transient expression with a maximum at ten minutes after treatment was observed (
In the prior art, studies on the transduction of gibberellin signals were carried out mostly by analyzing the induction of α-amylase in aleurone tissues in cereal seeds. The above-mentioned result is the first example that uses cultured cells. Responsiveness to gibberellin was then analyzed in more detail using the expressions of CIGR1 gene and CIGR2 gene in cultured cells as indicators.
Unlike auxins and cytokinins, gibberellins are defined, not by their physiological activities, but as compounds that have the ent-gibberellane skeleton. Therefore, there are large variations in their activities. In order to judge which of the physiological activity or the ent-gibberellane skeleton itself induces CIGR1 gene and CIGR2 gene expression, the induction of expression of these two genes by active type (GA1, GA3, and GA4) and inactive type (GA13 and GA17) (Crozier, A., Kuo, C. C., Durley, R. C., and Pharis, R. P., “The biological activities of 26 gibberellins in nine plant bioassays”, Canadian J. Botany (1970) 48, 867-877.) gibberellins was analyzed. The results showed that only active type gibberellins induced the expression of both genes (
Cultured plant cells are thought to be in an undifferentiated state in the presence of auxins. They grow under heterotrophic conditions as they are devoid of chloroplasts. Due to such factors, cultured cells are quite different from whole plants in their histological and physiological characteristics. Therefore, the possibility cannot be denied that the induction of CIGR1 and CIGR2 gene expression by gibberellin is a special phenomenon in cultured cells. Thus, the responses of these two genes to gibberellin were analyzed in green leaves of rice. The third and fourth leaves of rice, three weeks after seeding, were sprayed with GA3 (50 μM) and total RNA was extracted at certain intervals over time to analyze changes in the expression level of these genes. Both genes showed a very rapid transient expression with a maximum at 30 minutes after spraying (
Kuo et al. (Kuo, A., Cappelluti, S., Cervantes-Cervantes, M., Rodriguez, M., and Bush, D. S., “Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression and cell death induced by gibberellin in wheat aleurone cells”, The Plant Cell (1996) 8, 259-269.) found that the induction of α-amylase in the aleurone layer of wheat by gibberellin was specifically inhibited by okadaic acid, one of the protein phosphatase inhibitors. Okadaic acid is known to inhibit PP1 and PP2B among the protein phosphatases of animals.
On the other hand, protein kinase inhibitors, staurosporine and K252a, hardly inhibited induction, so it was presumed that phosphorylation of proteins, especially by protein phosphatases, was significantly involved in the signal transduction from gibberellin to the α-amylase gene.
In contrast, the data to date of the present inventors shows that the induction of expression of elicitor-responsive genes is strongly inhibited by pretreatment with K-252a (He, D.-Y., Yazaki, Y., Nishizawa, Y., Takai, R., Yamada, K., Sakano, K., Shibuya, N., and Minami, E., “Gene activation by cytoplasm acidification in suspension-cultured rice cells in response to the potent elicitor, N-acetylchitoheptaose”, Mol. Plant-Microbe Int. (1998) 12, 1167-1174; Nishizawa, Y., Kawakami, A., Hibi, T., He, D.-Y., Shibuya, N., and Minami, E., “Regulation of the chitinase gene expression in suspension-cultured rice cells by N-acetylchitooligosaccharides: differences in the signal transduction pathways leading to the activation of elicitor-responsive genes”, Plant Mol. Biol. (1999) 39, 907-914).
In addition, the effects of various inhibitors on the inductions by gibberellin and elicitors of CIGR1 gene and CIGR2 gene in the cultured rice cells were examined. Referring to the results of Kuo et al., cells pretreated with okadaic acid were tested. In these cells, while the induction of both genes by chitin heptamer was hardly inhibited, induction by GA3 was inhibited almost completely (
The induction of α-amylase by gibberellin in the aleurone layer of wheat is reported to be inhibited by okadaic acid but not by K-252-A (Kuo, A., Cappelluti, S., Cervantes-Cervantes, M., Rodriguez, M., and Bush, D. S., “Okadaic acid, a protein phosphatase inhibitor, blocks calcium change, gene expression and cell death induced by gibberellin in wheat aleurone cells”, The Plant Cell (1996) 8, 259-269).
The results shown in
Richards et al. (Richards, D. E., Peng, J., and Harberd, N. P., BioEssays (2000) 22, 573-577) have proposed a hypothesis that the GRAS family corresponds to STATs, which are transcriptional factors found widely inmetazoan organisms. In the C-terminal regions of GRAS, structures similar to the SH2 region are found; the SH2 region is a structure common in the STAT family, transcription factors in animals. Near the C-terminal of the SH2 region, a tyrosine residue that is highly conserved in animal STATs and susceptible to phosphorylation, and an arginine residue that is supposed to interact electrostatically with the phosphorylated tyrosine, are conserved. The homology in the N-terminal regions of amino acid sequences is lower than that in the C-terminal regions. This is generally found in the GRAS family. The homology of CIGR1 gene and CIGR2 gene at the nucleotide level was 57%. From the result of genomic Southern hybridization, it was estimated that one copy of each of these two genes was present (
From these results, each of the full length strand cDNAs was thought to hybridize specifically with its corresponding gene product. Both genes do not comprise the DELLA sequence, a common structure in the GAI/RGA subfamily which is a negative regulator of gibberellin signals.
To elucidate the function of the CIGR2 gene, the cDNA was inserted into a binary vector (pTA7001), which comprises a promoter responsive to dexamethasone, a synthetic steroid hormone that does not exist in plants. Using Agrobacterium methods, the plasmid was introduced into rice (Nipponbare). The derived transformed rice was inbred, and second generation seeds were obtained. Each independent line was spray-treated with dexamethasone (DEX; 30 μM, ethanol solution). Over a time course, total RNAs were extracted by phenol methods from the above ground part of the plant. Northern hybridization was carried out using the cDNA of 32P-labeled CIGR2 as a probe. A line in which CIGR2 gene expression was strongly induced twelve hours after dexamethasone treatment was discovered (
The present invention provides plant elicitor- and gibberellin-responsive genes. In plants, elicitors are known to induce a variety of defense-related enzyme genes, bringing about a defense reaction. Thus, it is highly expected that CIGR1 and GIGR2 genes, which can be induced by elicitors, will be valuable in disease-resistant recombinant crops.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2001-387862 | Dec 2001 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP02/13375 | Dec 2002 | US |
| Child | 10871083 | Jun 2004 | US |
