JP5035769B2 - Tobamovirus resistant plant production method and use thereof - Google Patents

Description

  The present invention relates to a method for producing a tobamovirus-resistant plant and use thereof, and more specifically, a method for producing a tobamovirus-resistant plant using host-side factors involved in RNA replication of tobamovirus and use thereof. It is about.

  Tobamovirus is a positive strand RNA virus belonging to the alphavirus superfamily. Examples of tobamovirus members include tobacco mosaic virus (hereinafter also referred to as “TMV”) and tomato mosaic virus (hereinafter also referred to as “ToMV”).

  The plus-strand RNA virus has a single-stranded RNA (polar RNA) functioning as mRNA in the virus particle as a genome. The majority of plant viruses, including tobamoviruses, and many animal viruses such as hepatitis C virus and poliovirus are positive-strand RNA viruses. When a plus-strand RNA virus enters a host cell, a protein involved in replication (replication protein) is translated from genomic RNA. The replication protein recognizes its own genomic RNA and forms an RNA replication complex on the cytoplasmic surface of the organelle membrane of the host cell. The RNA replication complex replicates genomic plus-strand RNA (offspring genomic RNA) via minus-strand RNA complementary to the plus strand (see Non-Patent Documents 1 and 2).

  It is known that the above-mentioned replication protein of tobamovirus, which is a kind of plus-strand RNA virus, is a protein of about 130 kDa (130K protein) and a protein of 180 kDa (180K protein) synthesized by the lead-through of the stop codon of the 130K protein (See Non-Patent Documents 3 and 4).

  Studies have also been conducted to elucidate the properties of the RNA replication complex and factors on the host side involved in the formation of the RNA replication complex. In general, viral RdRp activity is detected in membrane fractions obtained from cells infected with a plus-strand RNA virus. Therefore, in such studies, various analyzes using viral RdRp isolated and purified from infected leaves are performed. Has been carried out (see Non-Patent Document 5). For example, a study has been reported in which a host protein that is co-purified with RdRp or the above replication protein is identified and the role of the host protein in RNA replication is analyzed (see Non-Patent Document 6).

In addition, for the study of host-side factors involved in the formation of RNA replication complexes, the present inventors have used genetic techniques using Arabidopsis thaliana mutants as host proteins involved in tobamovirus growth, TOM1, TOM2A, TOM2B, and TOM3 have already been identified (see Non-Patent Documents 7 and 8).
Buck, KW (1996) .Comparison of the replication of positive-stranded RNA viruses of plants and animals.Adv.Vrius Res. 47, 159-251. Schwartz, M., Chen, J., Janda, M., Sullivan, M., den Boon, J., and Ahlquist, P. (2002). A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids Mol. Cell 9, 505-514. Ishikawa, M., and Okada, Y. (2004). Replication of tobamovirus RNA. Proc. Jpn. Acad., Ser. B 80, 215-224. Buck, KW (1999). Replication of tobacco mosaic virus RNA. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354, 613-627. Sivakumaran, K., Bao, Y., Roossinck, MJ, and Kao, CC (2000) .Recognition of the core RNA promoter for minus-strand RNA synthesis by the replicases of brome mosaic virus and cucumber mosaic virus.J. Virol. 74, 10323-10331. Takato Watanabe and Tadaaki Hibino. (2000). RNA replication enzyme of plant positive strand RNA virus. Virus 50, 103-108. Yamanaka, T., Ohta, T., Takahashi, M., Meshi, T., Schmidt, R., Dean, C., Naito, S., and Ishikawa, M. (2000). TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein.Proc.Nalt.Acad.Sci.USA 97, 10107-10112. Tsujimoto, Y., Numaga, T., Ohshima, K., Yano, M., Ohsawa, R., Goto, DB, Naito, S., and Ishikawa, M. (2003). Arabidopsis TOBAMOVIRUS MULTIPLICATION (TOM) 2 locus encodes a transmembrane protein that interacts with TOM1. EMBO J. 22, 335-343.

  As disclosed in Non-Patent Documents 1 and 2, an outline of the replication mechanism of a plus-strand RNA virus is known. However, the properties of the RNA replication complex and factors on the host side involved in the formation of the RNA replication complex have not been fully elucidated. For example, regarding factors on the host side involved in the formation of an RNA replication complex, as disclosed in Non-Patent Document 6, a host involved in RNA replication from RdRp or a host protein co-purified with the replication protein. Attempts have been made to find proteins. However, very few examples have been shown that positive-strand RNA virus replication proteins or host proteins co-purified with RdRp have been shown to be involved in the growth of positive-strand RNA viruses in vivo. Since factors on the host side involved in the formation of RNA replication complexes are expected to be used for the production of plants having resistance to plant plus-strand RNA viruses, further elucidation of such factors is desired.

  The present invention has been made in view of the above problems, and its purpose is to impart resistance to tobamoviruses while minimizing the effect on plant growth using host-side factors involved in RNA replication. Another object of the present invention is to provide a method for producing a tobamovirus-resistant plant that can be used, and use thereof.

  As a result of intensive studies in view of the above problems, the present inventors have found that in plants that have decreased expression of an ADP ribosylation factor-like polypeptide (hereinafter also referred to as “ARL polypeptide”), which is a low molecular weight GTP-binding protein, Uniquely found that resistance to tobamovirus is improved with little effect on plant growth, and that the addition of ARL protein to the in vitro ToMV RNA translation / replication system enhances ToMV RNA replication. The present invention has been completed. That is, the present invention includes the following industrially useful inventions.

  (1) A method for producing a tobamovirus-resistant plant, comprising reducing tobamovirus growth-promoting activity by an ADP-ribosylation factor-like polypeptide in a plant body.

(2) The ADP-ribosylation factor-like polypeptide is any one polypeptide selected from the group consisting of the following (a) to (e), and a group consisting of the following (f) to (j): Any one polypeptide selected from the group consisting of the following (k) to (o), and any one selected from the group consisting of the following (p) to (t) The method for producing a tobamovirus-resistant plant according to (1), which is at least two polypeptides selected from the group consisting of any one of these polypeptides.
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or 10 (b) one or several amino acids deleted or inserted in the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 2 or 10; Polypeptide (d) encoded by a polynucleotide consisting of a substituted or added amino acid sequence and having a base sequence shown in SEQ ID NO: 1 or 9 (c) having a tobamovirus growth promoting activity (d) SEQ ID NO: 1 Or a polypeptide encoded by a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in 9 and having a tobamovirus growth-promoting activity (e) Base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 9 A polypeptide (g) consisting of an amino acid sequence shown in SEQ ID NO: 4 or 12 encoded by a polynucleotide that hybridizes with a polynucleotide comprising: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in No. 4 or 12, and having a tobamovirus growth promoting activity (H) a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 or 11 (i) one or several bases deleted or inserted in the base sequence shown in SEQ ID NO: 3 or 11 , Replaced, or added A polypeptide encoded by a polynucleotide comprising a base sequence and having a tobamovirus growth promoting activity (j) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 11 under stringent conditions A polypeptide encoded by soybean polynucleotide and having a tobamovirus growth-promoting activity (k) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or 14 (l) shown in the amino acid sequence shown in SEQ ID NO: 6 or 14 In the amino acid sequence, a polypeptide (m) consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and having a tobamovirus growth promoting activity, the nucleotide sequence shown in SEQ ID NO: 5 or 13 Poly consisting of Polypeptide encoded by nucleotide (n) In the base sequence shown in SEQ ID NO: 5 or 13, encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added. And a polypeptide having a tobamovirus growth-promoting activity (o) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 5 or 13, and Polypeptide (p) having the tobamovirus growth promoting activity Polypeptide (q) consisting of the amino acid sequence shown in SEQ ID NO: 8 or 16 In the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 8 or 16, one or several Deletion of amino acids Polypeptide (s) SEQ ID NO: encoded by a polynucleotide consisting of an inserted, substituted or added amino acid sequence and having the base sequence shown in Polypeptide (r) SEQ ID NO: 7 or 15 having activity to promote tobamovirus growth A polypeptide (t) encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in 7 or 15, and having a tobamovirus growth promoting activity (t ) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 7 or 15, and has a tobamovirus growth-promoting activity.

  (3) The method for producing a tobamovirus-resistant plant according to (1), wherein the expression level of the polynucleotide encoding the ADP-ribosylation factor-like polypeptide is reduced in the plant body.

(4) The polynucleotide is any one selected from the group consisting of the following (A) to (U) and any of the following groups (E) to (C): One polynucleotide, any one polynucleotide selected from the group consisting of the following (ki) to (ke), and any one selected from the group consisting of the following (ko) to (shi) The method for producing a tobamovirus-resistant plant according to (3), which is at least two polynucleotides selected from the group consisting of polynucleotides.
(A) A polynucleotide comprising the base sequence shown in SEQ ID NO: 1 or 9 (ii) In the base sequence shown in SEQ ID NO: 1 or 9, one or several bases are deleted, inserted, substituted, or added And a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1 or 9 that encodes a polypeptide having a tobamovirus growth promoting activity under stringent conditions A polynucleotide that hybridizes and encodes a polypeptide having a tobamovirus growth-promoting activity (e) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 or 11 (O) in the nucleotide sequence represented by SEQ ID NO: 3 or 11, One or several bases deleted, inserted, substituted or appended And a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 11 and having a stringent condition, wherein the polynucleotide encodes a polypeptide having a tobamovirus growth promoting activity In the nucleotide sequence shown in SEQ ID NO: 5 or 13, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 or 13 (SEQ ID NO: 5 or 13) that encodes a polypeptide having a tobamovirus growth promoting activity A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added, and encoding a polypeptide having a tobamovirus growth-promoting activity, as shown in SEQ ID NO: 5 or 13 From a base sequence complementary to the base sequence A polynucleotide encoding the polypeptide having a tobamovirus growth-promoting activity and hybridizing under stringent conditions with a nucleotide sequence shown in SEQ ID NO: 7 or 15 (SEQ ID NO: 5) In the nucleotide sequence shown in 7 or 15, a polynucleotide encoding a polypeptide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added, and having a tobamovirus growth promoting activity. ) A polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 7 or 15 under a stringent condition and encodes a polypeptide having a tobamovirus growth promoting activity.

  (5) The tobamovirus-resistant plant according to (1), wherein a tobamovirus growth-promoting activity by the ADP-ribosylation factor-like polypeptide is reduced by introducing a mutation into the ADP-ribosylation factor-like polypeptide. Production method.

  (6) A kit for producing a tobamovirus-resistant plant comprising a polynucleotide encoding an ADP-ribosylation factor-like polypeptide or a fragment thereof.

  (7) Expression level of ADP ribosylation factor-like polypeptide in plant body, activity level of ADP ribosylation factor-like polypeptide in plant body, and expression level of polynucleotide encoding ADP ribosylation factor-like polypeptide in plant body A screening method for a tobamovirus-resistant plant, characterized in that at least one of them is measured.

(8) The ADP ribosylation factor-like polypeptide is any one polypeptide selected from the group consisting of the following (a) to (e), and a group consisting of the following (f) to (j): Any one polypeptide selected from the group consisting of the following (k) to (o), and any one selected from the group consisting of the following (p) to (t) And at least two polypeptides selected from the group consisting of any one of the polypeptides, wherein the polynucleotide is selected from the group consisting of the following (a) to (u): A nucleotide, any one polynucleotide selected from the group consisting of the following (e) to (ka), and any one polynucleotide selected from the group consisting of the following (ki) to (ke): ,Less than The tobamovirus resistance according to (7), wherein the polynucleotide is any one polynucleotide selected from the group consisting of (ko) to (shi) and at least two polynucleotides selected from the group consisting of Plant screening method.
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or 10 (b) one or several amino acids deleted or inserted in the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 2 or 10; Polypeptide (d) encoded by a polynucleotide consisting of a substituted or added amino acid sequence and having a base sequence shown in SEQ ID NO: 1 or 9 (c) having a tobamovirus growth promoting activity (d) SEQ ID NO: 1 Or a polypeptide encoded by a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in 9 and having a tobamovirus growth-promoting activity (e) Base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 9 A polypeptide (g) consisting of an amino acid sequence shown in SEQ ID NO: 4 or 12 encoded by a polynucleotide that hybridizes with a polynucleotide comprising: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in No. 4 or 12, and having a tobamovirus growth promoting activity (H) a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 or 11 (i) one or several bases deleted or inserted in the base sequence shown in SEQ ID NO: 3 or 11 , Replaced, or added A polypeptide encoded by a polynucleotide comprising a base sequence and having a tobamovirus growth promoting activity (j) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 11 under stringent conditions A polypeptide encoded by soybean polynucleotide and having a tobamovirus growth-promoting activity (k) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or 14 (l) shown in the amino acid sequence shown in SEQ ID NO: 6 or 14 In the amino acid sequence, a polypeptide (m) consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and having a tobamovirus growth promoting activity, the nucleotide sequence shown in SEQ ID NO: 5 or 13 Poly consisting of Polypeptide encoded by nucleotide (n) In the base sequence shown in SEQ ID NO: 5 or 13, encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added. And a polypeptide having a tobamovirus growth-promoting activity (o) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 5 or 13, and Polypeptide (p) having the tobamovirus growth promoting activity Polypeptide (q) consisting of the amino acid sequence shown in SEQ ID NO: 8 or 16 In the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 8 or 16, one or several Of amino acids deleted Polypeptide (s) SEQ ID NO: encoded by a polynucleotide consisting of an inserted, substituted or added amino acid sequence and having the base sequence shown in Polypeptide (r) SEQ ID NO: 7 or 15 having activity to promote tobamovirus growth A polypeptide (t) encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in 7 or 15, and having a tobamovirus growth promoting activity (t ) A polypeptide (A) sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 7 or 15, and has a tobamovirus growth-promoting activity Number 1 or A polynucleotide consisting of the base sequence shown in (9) consisting of a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 9; and Hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 9 encoding a polypeptide having a tobamovirus growth-promoting activity, and proliferating tobamovirus Polynucleotide encoding a polypeptide having enhancing activity (e) Polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 or 11 (O) One or several bases in the base sequence shown in SEQ ID NO: 3 or 11 Consists of nucleotide sequences deleted, inserted, substituted or added And a polynucleotide encoding a polypeptide having a tobamovirus growth-promoting activity (or) hybridizing under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 11, and A polynucleotide encoding a polypeptide having a tobamovirus growth-promoting activity (ki) One or several of the nucleotide sequence shown in SEQ ID NO: 5 or 13 consisting of the base sequence shown in SEQ ID NO: 5 or 13 A polynucleotide encoding a polypeptide having a tobamovirus growth-promoting activity and complementary to the base sequence shown in SEQ ID NO: 5 or 13 Polynucleotides and nucleotides consisting of base sequences A polynucleotide (SEQ ID NO: 7 or 15) comprising the nucleotide sequence shown in SEQ ID NO: 7 or 15 that encodes a polypeptide having a tobamovirus growth-promoting activity that hybridizes under lingent conditions In the nucleotide sequence shown, a polynucleotide (Shi) SEQ ID NO: 7 consisting of a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added, and encoding a polypeptide having a tobamovirus growth promoting activity Alternatively, a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence shown in 15 under a stringent condition and encodes a polypeptide having a tobamovirus growth promoting activity.

  (9) It comprises at least one of an antibody that specifically binds to an ADP ribosylation factor-like polypeptide and a probe that specifically hybridizes to a polynucleotide that encodes the ADP ribosylation factor-like polypeptide. A tobamovirus-resistant plant screening kit.

(10) The polynucleotide according to any one of (a) to (g) below.
(A) A polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 10, 12, 14, and 16 (B) Any one of SEQ ID NOs: 10, 12, 14, and 16 A polynucleotide encoding a polypeptide having a tobamovirus growth-promoting activity, comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in C) A polynucleotide comprising the base sequence shown in any one of SEQ ID NOs: 9, 11, 13, and 15 (d) In the base sequence shown in any one of SEQ ID NOs: 9, 11, 13, and 15 Consists of a base sequence in which one or several bases are deleted, inserted, substituted or added, and Tobamowi A polynucleotide encoding a polypeptide having a growth-promoting activity (e) and a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 9, 11, 13, and 15 and stringent Polynucleotide (f) that encodes a polypeptide that hybridizes under conditions and has a tobamovirus growth-promoting activity (f) positions 27 to 581 of SEQ ID NO: 9, positions 14 to 568 of SEQ ID NO: 11, SEQ ID NO: Polynucleotide (g) comprising the nucleotide sequence shown in any one of positions 16 to 570 of No. 13 and positions 65 to 619 of SEQ ID No. 15 as an open reeling frame No. 27 of SEQ ID No. 9 Positions-581, positions 14-568 of SEQ ID NO: 11, positions 16-570 of SEQ ID NO: 13, and SEQ ID NO: Including a base sequence in which one or several bases are deleted, inserted, substituted, or added as an open reeling frame in the base sequence shown in any one of positions 65 to 619 of No. 5; And a polynucleotide encoding a polypeptide having a tobamovirus growth-promoting activity.

(11) The polypeptide according to any one of the following (h) to (wo).
(H) A polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 10, 12, 14, and 16 (I) The amino acid sequence shown in any one of SEQ ID NOs: 10, 12, 14, and 16 A polypeptide comprising a amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added, and having a tobamovirus growth-promoting activity (SEQ ID NO: 9, 11, 13). And a polypeptide encoded by a polynucleotide consisting of the base sequence shown in any one of 15 (1), one in the base sequence shown in any one of SEQ ID NOs: 9, 11, 13, and 15 Or encoded by a polynucleotide consisting of a base sequence in which several bases have been deleted, inserted, substituted or added, A polypeptide having a tobamovirus growth-promoting activity (V) hybridizing under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in any one of SEQ ID NOs: 9, 11, 13, and 15 A polypeptide encoded by a soybean polynucleotide and having a tobamovirus growth-promoting activity.

  As described above, the method for producing a tobamovirus-resistant plant according to the present invention reduces the tobamovirus growth promoting activity by the ARL polypeptide in the plant body. Therefore, it is possible to suppress the proliferation of tobamovirus in the plant body without affecting the growth of the plant. Therefore, there is an effect that it is possible to produce a tobamovirus resistant plant having a growth ability equivalent to that of a natural plant and having resistance to tobamovirus.

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this.

<I. ARL polypeptide and polynucleotide encoding ARL polypeptide>
ARL polypeptide is one of the low molecular weight GTP binding proteins. The small molecule GTPase exists in a cell bound to GDP or GTP. The three-dimensional structure and interacting proteins differ between the GDP-bound and GTP-bound types. In many of the members of the ADP ribosylation factor (hereinafter, also referred to as “ARF”) family to which the ARL polypeptide belongs, the second amino acid residue is glycine. In these members, the N-terminal methionine residue is removed and the glycine undergoes myristylation. And in the state couple | bonded with GTP, this ARF couple | bonds with a film | membrane via the myristyl group and the hydrophobic (alpha) -helix near N terminal. On the other hand, in the state of binding to GDP, the three-dimensional structure is changed, and the α-helix near the N-terminal is folded inside the protein and is considered to be difficult to bind to the membrane (Pasqualato, S., Renault, L ., and Cherfils, J. (2002). Arf, Arl, Arp and Sar proteins: a family of GTP-binding proteins with a structural device for 'front-back' communication. See EMBO Rep. 3, 1035-1041. ). Among the members of the ARF family, ARF1, which is the most studied, is known to be a protein that is involved in vesicle transport (COPI vesicle transport) between the Golgi and the endoplasmic reticulum and is localized in the cytoplasm and membrane. (See Donaldoson, JG, Honda, A., and Weigert, R. (2005). Multiple activities for Arf1 at the Golgi complex. Biochim Biophys Acta. 1744, 364-373.) GDP-bound ARF1 present in the cytoplasm binds to the membrane via the transmembrane protein p23 / p24 or Membrin present in the Golgi membrane (Gommel et al, 2001, Honda et al., 2004), and then on the Golgi membrane surface. Due to the action of the existing GTP exchange factor, GDP bound to ARF1 is exchanged for GTP. GTP-bound ARF1 induces membrane curvature on the Golgi membrane and recruits coatmers to sprouting transport vesicles. GTP-bound ARF1 that has finished its function is converted to GDP-bound by the action of the GTPase activator and dissociates from the membrane. GTP hydrolytic ability of low molecular weight GTPase itself is low, and GTP-linked fixed type (membrane fixed type) and GDP-linked fixed type (cytoplasmic localized type) allele are produced by substitution of amino acid residues conserved between GTPases. (Okai, T., Araki, M., Tade, M., Tateno, T., Kontani, K., and Katada, T. (2004). Novel small GTPase subfamily capable of associating with tubulin is required for chromosome segregation. See J. Cell Sci. 117, 4705-4715.).

  The ARL polypeptide according to the present invention is a polypeptide belonging to the ARL protein family of plants. In Arabidopsis thaliana (Japanese name: Arabidopsis thaliana, hereinafter also referred to as “At”), it is known that there are at least four polynucleotides encoding polypeptides belonging to the ARL protein family. In the present specification, these four polynucleotides are referred to as arla1a, arla1b, arla1c, and arla1d, respectively, and are collectively referred to as arl polynucleotides. Moreover, when expressing including origin, for example, arla1a derived from Arabidopsis thaliana is referred to as Atarla1a. Furthermore, in this specification, polypeptides encoded by arla1a, arla1b, arla1c, and arla1d are referred to as ARLA1a, ARLA1b, ARLA1c, and ARLA1d, respectively, and are collectively referred to as ARL polynucleotides. Moreover, similarly to polynucleotide, when expressing including origin, for example, ARLA1a derived from Arabidopsis thaliana is referred to as AtARLA1a.

  ARLA1a according to the present invention includes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or 10, a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or 9, and variants thereof Can be mentioned.

  The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is AtARLA1a. The amino acid sequence of AtARLA1a is registered in GenBank as an accession number NP_568553. The polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 is Atalala. The base sequence of Atalla1a is registered in GenBank as an accession number NM_123127. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 is ARLA1a derived from Nicotiana tobacum (hereinafter also referred to as “Nt”). The polynucleotide consisting of the base sequence shown in SEQ ID NO: 9 is Ntarla1a.

  Examples of ARLA1b include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 or 12, a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 or 11, and variants thereof. Can do.

  The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is AtARLA1b. The amino acid sequence of AtARLA1b is registered with GenBank as an accession number NP_190555. In addition, the polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 is Atarla1b. The base sequence of Atala1b is registered in GenBank as an accession number NM_114846. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12 is NtARLA1b. The polynucleotide consisting of the base sequence shown in SEQ ID NO: 11 is Ntarla1b.

  Examples of ARLA1c include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 or 14, a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 or 13, and variants thereof. .

  The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 is AtARLA1c. The amino acid sequence of AtARLA1c is registered with GenBank as an accession number NP_190556. In addition, the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 is Atarla1c. The base sequence of Atala1c is registered in GenBank as an accession number NM_114847. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14 is NtARLA1c. The polynucleotide consisting of the base sequence shown in SEQ ID NO: 13 is Ntarla1c.

  Examples of ARLA1d include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8 or 16, a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7 or 15, and variants thereof. .

  The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8 is AtARLA1d. The amino acid sequence of AtARLA1d is registered in GenBank as an accession number NP_569051. The polynucleotide consisting of the base sequence shown in SEQ ID NO: 7 is Atarla1d. The base sequence of Atalla1d is registered as an accession number NM_126156 in GenBank. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 is NtARLA1d. Further, the polynucleotide consisting of the base sequence shown in SEQ ID NO: 15 is Ntarla1d.

  As used herein with respect to proteins or polypeptides, the term “variant” intends a polypeptide having tobamovirus growth-promoting activity. The “tobamovirus growth-promoting activity” refers to the activity of proliferating tobamovirus, preferably the activity of binding to GTP and proliferating tobamovirus, more preferably the activity of binding to GTP and enhancing the replication of tobamovirus genomic RNA. Intended. Also, as used herein, “ARL polypeptide” is used interchangeably with “ARL peptide” or “ARL protein”.

  The present invention also provides arl polynucleotides. Arl polynucleotides according to the present invention include arla1a-d. The arla1a to d are polynucleotides encoding ARLA1a to d, respectively. Specifically, examples of the above-mentioned arla1a include a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 or 9 and variants thereof. Examples of the arla1b include a polynucleotide having the base sequence shown in SEQ ID NO: 3 or 11 and variants thereof. Examples of the above-mentioned arla1c include a polynucleotide comprising the base sequence shown in SEQ ID NO: 5 or 13 and variants thereof. Examples of the arla1d include a polynucleotide having the base sequence shown in SEQ ID NO: 7 or 15 and variants thereof.

  Among the polynucleotides exemplified above, the polynucleotides consisting of the base sequences shown in SEQ ID NOs: 9, 11, 13, and 15 are Ntarlaa to d, respectively. The open reading frames of Ntarla1a-d (hereinafter also referred to as “ORF”) are the 27th to 581th positions of SEQ ID NO: 9, the 14th to 568th positions of SEQ ID NO: 11, and the SEQ ID NO: 13, respectively. The nucleotide sequence is represented by the 16th to 570th positions and the 65th to 619th positions of SEQ ID NO: 15. Therefore, the polynucleotide according to the present invention includes positions 27 to 581 of SEQ ID NO: 9, positions 14 to 568 of SEQ ID NO: 11, positions 16 to 570 of SEQ ID NO: 13, or SEQ ID NO: A polynucleotide comprising the nucleotide sequence shown in positions 65 to 619 of OR as the ORF, and positions 27 to 581 of SEQ ID NO: 9, positions 14 to 568 of SEQ ID NO: 11, ORF is a base sequence in which one or several bases are deleted, inserted, substituted, or added in the base sequence shown in positions 16 to 570 or positions 65 to 619 of SEQ ID NO: 15. As a polynucleotide. In the present specification, the open reading frame refers to a region that is actually translated into a protein in a polynucleotide, that is, a coding region of the protein.

  As used herein with respect to DNA or polynucleotide, the term “variant” intends a polynucleotide encoding a polypeptide having tobamovirus growth-promoting activity. Also, as used herein, “arl polynucleotide” is used interchangeably with “arl gene”.

  As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein”. In addition, “fragment” of a polypeptide is intended to be a partial fragment of the polypeptide. The polypeptides according to the invention may also be isolated from natural sources or chemically synthesized.

  The term “isolated” polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in a host cell can be isolated in the same manner as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that there is.

  Polypeptides according to the present invention include natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). ) From recombinantly produced products. Depending on the host used in the recombinant production procedure, the polypeptides according to the invention can be myristylated or non-myristylated. Furthermore, the polypeptides according to the invention may also comprise an initiating modified methionine residue in some cases as a result of a host-mediated process.

  The polypeptide according to the present invention may be a polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and may be a complex polypeptide including a structure other than the polypeptide. As used herein, examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited.

  The polypeptide according to the present invention may include an additional polypeptide. Examples of the additional polypeptide include epitope-tagged polypeptides such as His, Myc, and Flag.

  In one embodiment, a polypeptide according to the invention is a variant of an ARL polypeptide. Here, the mutant is a polypeptide having a tobamovirus growth promoting activity. Specifically, the ARLA1a mutant is specifically a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 10. Can be mentioned. Similarly, the ARLA1b variant includes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 or 12. be able to. Examples of the mutant of ARLA1c include a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 6 or 14. . Further, the ARLA1d mutant includes a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8 or 16. Can do.

  Such variants include deletions, insertions, inversions, repeats, and type substitutions (eg, replacement of a hydrophilic residue with another residue, but usually strongly hydrophilic residues are strongly hydrophobic Mutants containing residues that do not substitute) are included. In particular, “neutral” amino acid substitutions in a polypeptide generally have little effect on the activity of the polypeptide.

  It is well known in the art that some amino acids in the amino acid sequence that make up a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants in natural proteins that do not significantly alter the structure or function of the protein, not only artificially modified.

  One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, if the method described in this specification is used, it can be easily determined whether the produced mutant has a desired tobamovirus growth-promoting activity.

  Preferred variants have conservative or non-conservative amino acid substitutions, deletions or additions. These do not change the tobamovirus growth promoting activity of the polypeptides according to the invention.

  Typical conservative substitutions are substitutions of one amino acid with another in the aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

  In another embodiment, the variant of ARLA1a is a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added in the base sequence shown in SEQ ID NO: 1 or 9. And a polypeptide having a tobamovirus growth-promoting activity. Similarly, the variant of ARLA1b is encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 3 or 11. The polypeptide may have a tobamovirus growth-promoting activity. The mutant of ARLA1c is encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 5 or 13, and It may be a polypeptide having enhancing activity. Furthermore, the mutant of ARLA1d is encoded by a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 7 or 15. It may be a polypeptide having a tobamovirus growth promoting activity.

  In yet another embodiment, the ARLA1a mutant is a polymorphism comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added in the base sequence shown in SEQ ID NO: 1 or 9. It may be a polypeptide encoded by a polynucleotide that hybridizes with a nucleotide under stringent conditions and has a tobamovirus growth promoting activity. Similarly, the variant of ARLA1b described above is a stringent gene and a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 3 or 11. It may be a polypeptide encoded by a polynucleotide that hybridizes under various conditions and has a tobamovirus growth promoting activity. The mutant of ARLA1c described above is a stringent condition with a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 5 or 13. It may be a polypeptide that is encoded by a polynucleotide that hybridizes with and has a tobamovirus growth-promoting activity. Furthermore, the mutant of ARLA1d is stringent to a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 7 or 15. It may be a polypeptide encoded by a polynucleotide that hybridizes under conditions and has a tobamovirus growth-promoting activity.

  Hybridization can be performed by well-known methods such as those described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. The appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable.

  As used herein, the term “stringent hybridization conditions” refers to hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate. (Containing pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA) overnight at 42 ° C., then 0.1 × at about 65 ° C. It is intended to wash the filter in SSC. Depending on the polynucleotide that hybridizes to a “portion” of the polynucleotide, at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably about Polynucleotides (either DNA or RNA) that hybridize to polynucleotides longer than 30 nt are contemplated. Polynucleotides (oligonucleotides) that hybridize to “parts” of such polynucleotides are also useful as detection probes as discussed in more detail herein.

  As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to the sequence of deoxyribonucleotides (abbreviated A, G, C, and T). As shown. The “polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or 9 or a fragment thereof” means a polynucleotide comprising the sequence represented by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 1 or 9. Or a fragment portion thereof is contemplated.

  The polynucleotide according to the present invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).

  As used herein, the term “oligonucleotide” is intended to be a combination of several to several tens of nucleotides, and is used interchangeably with “polynucleotide”. Oligonucleotides are called dinucleotides (dimers) and trinucleotides (trimers) as short ones, and are represented by the number of nucleotides polymerized such as 30 mers or 100 mers as long ones. Oligonucleotides can be produced as fragments of longer polynucleotides or chemically synthesized.

  The polynucleotide according to the present invention can also be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'side or 3' side.

  In one embodiment, the polynucleotide according to the present invention is a variant of the ARL polynucleotide. Here, the mutant is a polynucleotide encoding a polypeptide having a tobamovirus growth promoting activity. Specifically, the mutant of arla1a is a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 9. A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of one or several bases deleted, inserted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 1 or 9 Can be mentioned. Similarly, as the mutant of arla1b, a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 3 or 11; Examples of a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted, or added in the nucleotide sequence shown in No. 3 or 11 be able to. As the mutant of arla1c, a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 5 or 13; SEQ ID NO: 5 or Examples of the nucleotide sequence shown in Fig. 13 include polynucleotides that hybridize under stringent conditions with a polynucleotide comprising a nucleotide sequence in which one or several bases have been deleted, inserted, substituted, or added. . Furthermore, the mutant of arla1d is a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 7 or 15; SEQ ID NO: Mention a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in 7 or 15 Can do.

  The polynucleotide according to the present invention may contain a sequence such as an untranslated region (UTR) sequence or a vector sequence (including an expression vector sequence).

  Although it does not specifically limit as a supply source for acquiring the polynucleotide concerning this invention, It is preferable that it is a biological material. As used herein, the term “biological material” intends a biological sample (a tissue sample or cell sample obtained from an organism). In the examples described below, Arabidopsis thaliana and tobacco are used, but not limited thereto.

  The ARL polynucleotide according to the present invention also includes a polynucleotide encoding a fragment of the ARL polypeptide described above.

  The polypeptide or polynucleotide concerning this invention can be used in order to manufacture the plant body to which tobamovirus resistance was provided so that it may mention later. Moreover, according to this invention, it can apply in order to screen the plant body which has tobamovirus resistance. Such a method for producing a tobamovirus-resistant plant and a method for screening a tobamovirus-resistant plant can be used, for example, in the field of plant breeding.

  The uses of the ARL polypeptide and ARL polynucleotide according to the present invention and their variants are not limited to the above-described method for producing a tobamovirus-resistant plant or the method for screening a tobamovirus-resistant plant. Specifically, it should be noted that any application that utilizes the ARL polypeptide involved in tobamovirus growth falls within the scope of the present invention.

  Furthermore, the present invention encodes a variant of ARL polypeptide that does not have tobamovirus growth-promoting activity, and a variant of ARL polynucleotide that does not have tobamovirus growth-promoting activity. Polynucleotides are also included. By expressing such ARL polypeptide mutant and ARL polynucleotide mutant in a plant, a plant having tobamovirus resistance can be produced. Such a plant can be produced, for example, by introducing the ARL polynucleotide mutant into a plant body using a conventionally known transformation technique.

<II. Method for producing ARL polypeptide>
The present invention provides a method of producing an ARL polypeptide. In one embodiment, the method for producing a polypeptide according to the present invention uses a vector containing a polynucleotide encoding an ARL polypeptide.

  In one aspect of this embodiment, in the method for producing a polypeptide according to this embodiment, the above vector is used in a cell-free protein synthesis system. When using a cell-free protein synthesis system, various commercially available kits can be used. Preferably, the method for producing a polypeptide according to this embodiment includes a step of incubating the vector and a cell-free protein synthesis solution.

  The cell-free protein synthesis system is a technique widely used for identification of various proteins encoded by intracellular mRNAs or cloned cDNAs. The cell-free protein synthesis system (cell-free protein synthesis method, cell-free protein translation system) The cell-free protein synthesis solution is also used.

  Cell-free protein synthesis systems include systems using wheat germ extract, systems using rabbit reticulocyte extract, systems using Escherichia coli S30 extract, and cell component extracts obtained from plant devolatilized protoplasts. . Generally, eukaryotic genes are selected for translation of eukaryotic genes, ie, systems using wheat germ extract or systems using rabbit reticulocyte extract. In view of the origin of the gene (prokaryotes / eukaryotes) and the intended use of the protein after synthesis, it may be selected from the above synthesis system.

  Many virus-derived gene products express their activity after translation through complex biochemical reactions involving intracellular membranes such as the endoplasmic reticulum and Golgi apparatus. In order to reproduce the above, it is necessary to add an intracellular membrane component (for example, a microsomal membrane). A cell component extract obtained from a devolatilized protoplast of a plant as described in JP-A-2005-65651 (published on March 17, 2005) is a cell-free protein synthesis solution retaining an intracellular membrane component This is preferable because the addition of a microsomal membrane is not required.

  As used herein, “intracellular membrane component” refers to an organelle composed of a lipid membrane present in the cytoplasm (ie, cells such as the endoplasmic reticulum, Golgi apparatus, mitochondria, chloroplast, vacuole and the like). Inner granules in general) are intended. In particular, the endoplasmic reticulum and the Golgi apparatus play an important role in post-translational modification of proteins, and are essential cellular components for the maturation of membrane proteins and secreted proteins.

  In another aspect of the present embodiment, the polypeptide is preferably used in a recombinant expression system in the method for producing a polypeptide according to the present embodiment. When a recombinant expression system is used, a method in which an arl polynucleotide is incorporated into a recombinant expression vector, introduced into a host capable of expression by a known method, and the ARL polypeptide obtained by translation in the host is purified, etc. Can be adopted. The recombinant expression vector may or may not be a plasmid, as long as the target polynucleotide can be introduced into the host. Preferably, the method for producing a polypeptide according to this embodiment includes a step of introducing the vector into a host.

  Thus, when introducing a foreign polynucleotide into a host, the expression vector preferably incorporates a promoter that functions in the host so as to express the foreign polynucleotide. The method for purifying a recombinantly produced polypeptide differs depending on the host used and the nature of the polypeptide, but the target polypeptide can be purified relatively easily by using a tag or the like.

  It is preferable that the method for producing a polypeptide according to this embodiment further includes a step of purifying the polypeptide from an extract of cells or tissues containing the ARL polypeptide. The step of purifying a polypeptide is to prepare a cell extract from cells or tissues by a well-known method (for example, a method in which a cell or tissue is disrupted and then centrifuged to collect a soluble fraction). Known methods (eg ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography) The step of purifying by a) is preferred, but not limited thereto. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  In another embodiment, the ARL polypeptide production method preferably purifies the polypeptide from cells or tissues that naturally express the ARL polypeptide. The method for producing a polypeptide according to this embodiment preferably includes a step of identifying a cell or tissue that naturally expresses the ARL polypeptide using an antibody or oligonucleotide described below. In addition, the polypeptide production method according to the present embodiment preferably further includes a step of purifying the ARL polypeptide.

  In still another embodiment, the polypeptide production method according to the present invention may chemically synthesize the polypeptide according to the present invention. Those skilled in the art will readily understand that the polypeptide according to the present invention can be chemically synthesized by applying a well-known chemical synthesis technique based on the amino acid sequence of the polypeptide according to the present invention described herein. .

  As described above, the polypeptide obtained by the method for producing a polypeptide according to the present invention may be a naturally occurring mutant polypeptide or an artificially prepared mutant polypeptide.

  The method for producing the mutant polypeptide is not particularly limited. For example, a site-directed mutagenesis method (see, for example, Hashimoto-Gotoh, Gene 152, 271-275 (1995)), a method in which a point mutation is introduced into a base sequence using a PCR method, or a mutant polypeptide is produced, or By using a well-known mutant polypeptide production method such as a mutant strain production method by transposon insertion, a mutant polypeptide can be produced. A commercially available kit may be used for the production of the mutant polypeptide.

  Thus, it can be said that the method for producing a polypeptide according to the present invention may use a known and common technique based on at least the amino acid sequence of the ARL polypeptide or the base sequence of the polynucleotide encoding the ARL polypeptide.

  That is, it is an object of the present invention to provide a method for producing ARL polypeptide, and it should be noted that production methods including steps other than the various steps described above are also within the technical scope of the present invention. There must be.

  A cell-free protein synthesis system is preferred as a method for determining whether or not a variant of ARL polypeptide has a desired tobamovirus growth promoting activity. By adding viral RNA to the cell-free protein synthesis system described below, viral RNA essential for replication can be synthesized by translating viral RNA. This system is also referred to herein as the “in vitro replication system”.

  According to the in vitro replication system, if the prepared ARL polypeptide is added to the in vitro replication system of wild-type ToMV and reacted to detect replication of wild-type ToMV RNA, it is a mutant of ARL polypeptide and tobamovirus A polypeptide having a proliferation promoting activity can be easily selected.

<III. Tobamovirus resistant plant production method and kit>
The method for producing a tobamovirus-resistant plant according to the present invention (hereinafter also referred to simply as “the method for producing a plant according to the present invention”) is also referred to as an ADP ribosylation factor-like polypeptide (hereinafter referred to as “ARL polypeptide”) in the plant body. ) To reduce the tobamovirus growth-promoting activity. According to the said structure, the proliferation of the tobamovirus in a plant body can be suppressed, without affecting the growth of a plant. Therefore, it is possible to produce a tobamovirus-resistant plant having a growth ability equivalent to that of a natural plant and having resistance to tobamovirus. The production method according to the present invention is not particularly limited to a specific configuration other than the above-described steps, for example, a method for reducing the activity of ARL polypeptide in a plant body, a device used, a device used, and the like.

  Hereinafter, the manufacturing method of the plant concerning this invention is demonstrated. As described above, the method for producing a plant according to the present invention only needs to include a step of reducing the tobamovirus growth promoting activity by the ARL polypeptide in the plant body. Specifically, a mutant type in which the tobamovirus growth promoting activity is reduced by reducing the expression level (accumulation amount) of the ARL polypeptide, reducing the expression level of the arl polynucleotide, or introducing a mutation into the arl polynucleotide. Inhibiting the tobamovirus growth promoting activity of the ARL polypeptide in the plant body by expressing the ARL polypeptide or treating the ARL polypeptide with an inhibitor, the tobamovirus growth promoting activity of the ARL polypeptide in the plant body Can be reduced. In the method for producing a plant according to the present invention, any one of the above-mentioned ARLA1a-d may be reduced in any tobamovirus growth-promoting activity, but at least two polypeptides may reduce tobamovirus growth-promoting activity in the plant body. It is preferable that two or three polypeptides of ARLA1a, c, d are more preferable to reduce the tobamovirus growth promoting activity in the plant body. More specifically, ARLA1a and ARLA1c tobamovirus proliferation enhancing activity is preferably reduced, ARLA1c and ARLA1d tobamovirus proliferation enhancing activity is more preferably reduced, and ARLA1a, ARLA1c, and ARLA1d tobamovirus proliferation enhancing activity is reduced. More preferably. By reducing the activity of ARLA1a and ARLA1c to promote tobamovirus growth, it is possible to confer resistance to ToMV to plants. Moreover, resistance to ToMV and TMV can be imparted to plants by reducing the tolvamovirus growth-promoting activity of ARLA1c and ARLA1d. Furthermore, resistance to ToMV and TMV can be imparted to plants by decreasing the tobamovirus growth-promoting activity of ARLA1a, ARLA1c, and ARLA1d. In the present specification, “reducing tobamovirus growth-promoting activity by ARL polypeptide in a plant” is intended to decrease tobamovirus growth-promoting activity by ARL polypeptide in a plant as compared to a wild strain. However, the degree of the decrease is not limited. That is, it also includes completely losing the tobamovirus growth-promoting activity of the ARL polypeptide in the plant body.

  In one embodiment, the plant body to which tobamovirus resistance is imparted according to the plant production method of the present invention may be a plant transformant or a plant variant.

The plant mutant according to this embodiment can be obtained, for example, by selecting a plant having a reduced tobamovirus growth promoting activity of the ARL polypeptide from a naturally occurring mutant. The plant mutant can also be obtained by a method (chemical mutagenesis method) in which seeds are treated with a known compound that is a chemical mutagen. The chemical mutagens include sodium azide (NaN ₃ ), ethyl methanesulfonate (EMS), azidoglycerol (AG, 3-azido-1,2-propanediol), methylnitrosourea (MNU), and Examples thereof include maleic hydrazide (MH). Further, UV irradiation may be used. According to the above method, a mutation of 1 nucleotide or more is introduced into the promoter region of the arl polynucleotide according to the present invention, and transcription of the arl polynucleotide can be down-regulated or knocked out. Thereby, the expression level of the ARL polypeptide concerning this invention falls, As a result, the activity of the ARL polypeptide concerning this invention can be reduced. As another mode, according to the above method, a mutant ARL polypeptide in which a mutation of 1 nucleotide or more is introduced in the coding region of the arl polynucleotide according to the present invention and the tobamovirus growth promoting activity is reduced can be expressed. Thereby, the activity of ARL polypeptide in a plant body can be reduced. In addition, the mutation in the coding region of the gene according to the present invention is not particularly limited, and causes a frame shift or a truncation of a protein as a translation product and / or a decrease in substrate specificity. Includes insertions, deletions and substitutions. The ARL polypeptide is a polypeptide having GTP binding activity as described above. As shown in the examples described later, GTP binding activity is involved in the tobamovirus growth promoting activity of ARL polypeptide. More specifically, small molecule GTPases such as ARL polypeptides exist in a state bound to GDP or GTP in cells. ARL polypeptides are involved in the growth of tobamoviruses in a state bound to GTP. Therefore, the tobamovirus-resistant plant according to the present invention is also produced by introducing a mutation into the ARL polypeptide so that the ARL polypeptide cannot bind to GTP, in other words, so that it can permanently bind to GDP. be able to. The ARL polypeptide bound to GTP is bound to the membrane, and the ARL polypeptide bound to GDP is considered to be localized in the cytoplasm. Therefore, the tobamovirus-resistant plant according to the present invention can also be produced by introducing a mutation into the ARL polypeptide so that the ARL polypeptide does not bind to the membrane, in other words, so that it is constitutively localized in the cytoplasm. can do. Examples of such mutations include substitution of threonine at position 33 with asparagine in a polypeptide (NtARLA1d) consisting of the amino acid sequence shown in SEQ ID NO: 16.

  Moreover, the plant transformant concerning this embodiment can be manufactured by the method demonstrated to the following (i)-(vi), for example.

(I) Mutagenesis Method The polynucleotide according to the present invention is targeted for site-directed mutagenesis using a chimeric RNA / DNA oligonucleotide. The chimeric RNA / DNA oligonucleotide is known to be able to introduce mutations at desired positions in plant cells (Zhu et al., 1999, Proc. Natl. Acad. Sci. 96: 8768-8773; and Beetham et al., 1999, Proc. Natl. Acad. Sci. 96: 8774-8778). A specific method of transformation using the chimeric RNA / DNA oligonucleotide will be described later.

  In addition, whether or not a mutation has been introduced at a desired position can be confirmed and traced by using a conventionally known method such as a PCR-based method.

  Therefore, if the polynucleotide according to the present invention is used as a target for site-directed mutagenesis using a chimeric RNA / DNA oligonucleotide, a mutation can be introduced at a specific site in the polynucleotide according to the present invention.

  According to the above method, a mutation of one or more nucleotides is introduced into the promoter region of the polynucleotide according to the present invention, and transcription of the polynucleotide according to the present invention can be down-regulated or knocked out. Thereby, the expression level of the ARL polypeptide concerning this invention falls, As a result, the activity of the ARL polypeptide concerning this invention can be reduced. As another mode, according to the above method, a mutant ARL polypeptide in which a mutation of 1 nucleotide or more is introduced in the coding region of the arl polynucleotide according to the present invention and the tobamovirus growth promoting activity is reduced can be expressed. Thereby, the activity of ARL polypeptide in a plant body can be reduced.

  The mutation in the coding region of the polynucleotide according to the present invention is not particularly limited, and may cause a frame shift, a truncation of a protein that is a translation product, and / or a decrease in substrate specificity. Insertions, deletions and substitutions.

(Ii) Antisense method The antisense strand of the polynucleotide according to the present invention is expressed in a plant body. A specific method of transformation using an expression vector containing an antisense nucleic acid sequence will be described later.

(Iii) Gene expression having a dominant-negative trait of a target gene According to the present invention inherent in a plant by transforming a polynucleotide having a dominant-negative trait of the polynucleotide according to the present invention (target gene) into the plant Polynucleotide expression can be suppressed. In the present invention, the “polynucleotide encoding a polypeptide having a dominant-negative trait” refers to a polypeptide encoded by an endogenous polynucleotide of the present invention inherent in a plant by expressing the polynucleotide. It refers to a polynucleotide that encodes a polypeptide having a function of eliminating or reducing the activity.

  A specific method for transformation using the polynucleotide will be described later.

(Iv) Co-inhibition Endogenous expression of the polynucleotide of the present invention can be suppressed by transformation of a polynucleotide having the same or similar sequence as the polynucleotide (target gene) sequence of the present invention (co-suppression). Suppression). “Co-suppression” means that when a polynucleotide having the same or similar sequence as a target endogenous polynucleotide is introduced into a plant by transformation, the expression of both the introduced foreign polynucleotide and the target endogenous polynucleotide is suppressed. A phenomenon. Details of the mechanism of co-suppression are not clear but are often observed in plants (see Curr. Biol., 7: R793, 1997; Curr. Biol., 6: 810, 1996). For example, in order to obtain a plant body in which the polynucleotide according to the present invention is co-suppressed, the target plant is obtained by using a vector DNA prepared so that the polynucleotide according to the present invention or a polynucleotide having a similar sequence thereto can be expressed. A plant whose growth is suppressed from the obtained plant may be selected. The polynucleotide used for co-suppression need not be completely identical to the target polynucleotide, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (eg, 95% or more) identical in sequence. Have sex. The sequence identity can be determined by a conventionally known method.

(V) RNA interference (RNAi) method Expression of endogenous polynucleotide can be suppressed by RNA interference caused by transformation of DNA in which sequences identical or similar to target polynucleotide sequences are arranged in inverted repeats ( RNA interference). “RNA interference” refers to the expression of double-stranded RNA derived from foreign DNA when a DNA in which a sequence identical or similar to a target endogenous polynucleotide is placed in inverted repeats is introduced into a plant, resulting in expression of a target poly A phenomenon in which the expression of nucleotides is suppressed. As the mechanism of RNA interference, as a first step, complementary RNA is synthesized using a sequence in which mRNA of a target polynucleotide and a double-stranded RNA derived from an introduced sequence form a complex and associate. As a second step, this complex is fragmented by endogenous RNase. As a third step, it is considered that the double-stranded RNA fragmented to 20 to 30 base pairs functions as a secondary RNA interference signal to degrade the endogenous target polynucleotide mRNA again. A specific method for transformation will be described later.

  For example, in order to obtain a plant body in which the polynucleotide according to the present invention is suppressed by RNA interference, first, a DNA having a polynucleotide according to the present invention or a DNA having a sequence similar thereto is arranged in inverted repeats. A vector DNA prepared so as to be expressed is transformed into a target plant. Next, a plant having a reduced tobamovirus growth promoting activity may be selected from the obtained plant body.

  The polynucleotide used for RNA interference need not be completely the same as the target polynucleotide, but at least 10 bases or more are the same continuously, preferably 20 to 100 bases are the same continuously, Preferably 50 bases are identical in succession. The polynucleotide used for RNA interference may be a polynucleotide having a sequence identity of at least 70% or more, preferably 80% or more, more preferably 90% or more (for example, 95% or more) with the target polynucleotide. Good. Even more preferably, a polynucleotide having a sequence identity of at least 70% or more, preferably 80% or more, more preferably 90% or more (eg, 95% or more) with the target polynucleotide arranged in inverted repeats Is mentioned. In particular, those in which polynucleotides having sequence identity with these target polynucleotides are arranged in inverted repeats with a spacer sequence in between are preferred. The sequence identity can be determined using a conventionally known method.

  The length of the polynucleotide used for RNA interference may be the full length of the target polynucleotide, but it may be at least 25 bases, preferably 50 bases, more preferably 100 bases, and even more preferably 500 bases. Good.

  RNA interference can also be realized by plant virus infection. Plant viruses having single-stranded RNA as a genome take the form of double-stranded RNA during the replication process. Therefore, when the target polynucleotide sequence is inserted into the plant virus genome together with an appropriate promoter and this recombinant virus is infected with a plant, double-stranded RNA of the target polynucleotide sequence is generated along with the replication of the virus. It will be. As a result, the effect of RNA interference can be obtained (see Angell et al., Plant J. 20, 357-362, (1999)).

(Vi) Transposon method Using a transposon gene as a mutagen, a transposon tag line can be produced in a desired plant or the like. Thus, by selecting a plant having a reduced expression level of the polypeptide according to the present invention or a plant having a reduced activity from the produced transposon tag line, a plant having a low tobamovirus growth promoting activity can be produced. it can.

  The transposon gene can be introduced into the plant by the transformation method described above.

  A method for selecting a plant having reduced tobamovirus growth-promoting activity from plants produced by the above method is not particularly limited, and a conventionally known method can be used. For example, the Northern blot method can be exemplified as a method for evaluating the expression level of the polynucleotide. The expression level of the polypeptide may be an immunological method such as an ELISA method or a Western blot method using an antibody that specifically recognizes the polypeptide according to the present invention.

  Here, in the above methods (i) to (vii), a transformation method that can be used will be described. When a recombinant expression vector is used, the recombinant expression vector used for transformation of the plant body is not particularly limited as long as it is a vector capable of expressing the polynucleotide according to the present invention in the plant. Examples of such a vector include a vector having a promoter that constitutively expresses a polynucleotide in a plant cell (eg, cauliflower mosaic virus 35S promoter), or a promoter that is inducibly activated by an external stimulus. The vector which has is mentioned.

  Plants to be transformed in the present invention include whole plants, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundle, It means any of a palisade tissue, a spongy tissue, etc.) or a plant culture cell, or various forms of plant cells (eg, suspension culture cells), protoplasts, leaf sections, callus, and the like. The plant used for transformation is not particularly limited, and may be any plant belonging to the monocotyledonous plant class or the dicotyledonous plant class.

  For introduction of polynucleotides into plants, transformation methods known to those skilled in the art (for example, Agrobacterium method, gene gun, PEG method, electroporation method, etc.) are used. It is divided roughly into the method of introducing into plant cells. When the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium (for example, Agrobacterium tumefaciens), and this strain is introduced into the leaf disk method (Hirofumi Uchimiya). The plant genetic manipulation manual (1990) pages 27-31, Kodansha Scientific, Tokyo) can be used to infect sterile cultured leaf pieces to obtain transformed plants. The method of Nagel et al. (Micribiol. Lett., 67, 325 (1990)) can also be used. In this method, for example, an expression vector is first introduced into Agrobacterium, and then transformed Agrobacterium is plant cell or plant tissue by the method described in Plant Molecular Biology Manual (SBGelvin et al., Academic Press Publishers). It is a method to introduce into. Here, “plant tissue” includes callus obtained by culturing plant cells. When performing transformation using the Agrobacterium method, pBI binary vectors (for example, pBIG, pBIN19, pBI101, pBI121, pBI221, and pPZP202) can be used.

  As a method for directly introducing a polynucleotide into a plant cell or plant tissue, an electroporation method and a gene gun method are known. When using a gene gun, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample prepared in this manner can be processed using a gene transfer apparatus (for example, PDS-1000 (BIO-RAD)). The treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

  A cell or plant tissue into which a polynucleotide has been introduced is first selected for drug resistance such as hygromycin resistance, and then regenerated into a plant by a conventional method. Regeneration of a plant body from a transformed cell can be performed by a method known to those skilled in the art depending on the type of plant cell. The selection marker is not limited to hygromycin resistance, and examples thereof include drug resistance to bleomycin, kanamycin, gentamicin, chloramphenicol and the like.

  When plant cultured cells are used as a host, the recombinant vector can be obtained by, for example, microinjection, electroporation (electroporation), polyethylene glycol, gene gun (particle gun), protoplast fusion, calcium phosphate, etc. It is introduced into a cultured cell and transformed. Callus, shoots, hairy roots, etc. obtained as a result of transformation can be used for cell culture, tissue culture or organ culture as they are, and can be used at a suitable concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).

  Whether or not a polynucleotide has been introduced into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing a DNA-specific primer. PCR can be performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, It can be confirmed that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzymatic reaction.

  Once a transformed plant in which the polynucleotide of the present invention is integrated into the genome is obtained, offspring can be obtained by sexual reproduction or asexual reproduction of the plant. Further, for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, and the like can be obtained from the plant or its progeny, or clones thereof, and the plant can be mass-produced based on them. it can. Therefore, the present invention also includes a plant in which the polynucleotide according to the present invention is introduced so that it can be expressed, or a progeny of the plant having the same properties as the plant, or a tissue derived therefrom.

  In one embodiment, the plant body to which tobamovirus resistance is imparted according to the method for producing a plant of the present invention may be a plant treated with an inhibitor that inhibits the activity of the ARL polypeptide. The inhibitor is preferably one that inhibits the activity of ARLA1 protein. The inhibitor may be a chemically synthesized chemical substance, a naturally occurring chemical substance, or a biomolecule such as an antibody or antibody fragment. Moreover, as one embodiment, a transformant in which an antibody against an ARL polypeptide or a polynucleotide encoding an antibody fragment is introduced into a plant so as to be expressed may be used. The antibody or antibody fragment may be a polyclonal antibody or an antibody fragment thereof, or may be a monoclonal antibody or an antibody fragment thereof. In any case, the antibody or antibody fragment preferably binds to ARLA1 highly specifically and inhibits the activity of the ARLA1 protein.

  The plant body provided with the tobamovirus resistance produced by the method for producing a plant according to the present invention is not particularly limited, and examples thereof include plants that may have the usefulness due to the addition of the tobamovirus resistance. Such plants may be angiosperms or gymnosperms. The angiosperms may be monocotyledons or dicotyledons, but dicotyledons are more preferred.

  The present invention also provides a kit for producing a plant imparted with tobamovirus resistance (hereinafter also simply referred to as “plant production kit”). The plant production kit according to the present invention may be provided with a kit capable of reducing the tobamovirus growth promoting activity in the plant body. For example, a configuration comprising a polynucleotide used in a method for reducing the expression levels of ARL polypeptide and arl polynucleotide in a plant (for example, the above-described methods (i) to (vii)) in a form that can be expressed in the plant. And a configuration comprising an inhibitor that inhibits the activity of the ARL polypeptide in the plant body.

  In addition, it is preferable that the plant production kit according to the present invention includes, in addition to the above-described polynucleotide and inhibitor, reagents necessary for performing the above-described plant production method. That is, a person skilled in the art who has read this specification uses a kit component for producing a plant with tobamovirus resistance used in a method for producing a plant with tobamovirus resistance. Easy to understand.

<IV. Tobamovirus resistant plant screening method and screening kit>
The present invention provides a method for screening a tobamovirus-resistant plant (hereinafter also simply referred to as “plant screening method”). The plant screening method according to the present invention comprises an expression level of an ADP ribosylation factor-like polypeptide in a plant body, an activity level of an ADP ribosylation factor-like polypeptide in the plant body, and an ADP ribosylation factor-like polypeptide in the plant body. It only needs to include a step of measuring at least one of the expression levels of the encoded polynucleotide (hereinafter also referred to as “measurement step”). In one embodiment, the plant screened according to the plant screening method of the present invention may be natural, a transformant, or a mutant.

  As the measurement step, specifically, for example, an ARL protein extracted from a target plant (a crude extract, a partially purified product, or a purified product may be used). A step of measuring the expression level (accumulation amount) of ARL polypeptide in a plant body by reacting with an antibody against ARL protein and detecting an antibody bound to ARL protein can be mentioned. More specifically, a step of measuring the expression level of ARL polypeptide in the plant body using Western blotting or ELISA can be mentioned. According to the above process, a plant with a reduced expression level of ARL polypeptide in the plant can be easily detected (screened) by comparison with the wild type. In many cases, the plant body in which the expression level of the ARL polypeptide is reduced has a reduced tobamovirus growth-promoting activity in the plant. Therefore, the plant having improved resistance to tobamovirus can be easily screened by the above-described steps. . As the antibody used in the above step, an antibody described later can be used.

  In addition, as the measurement step, for example, the active amount of ARL protein extracted from the target plant (may be a crude extract, a partially purified product, or a purified product) The process of measuring can be mentioned. More specifically, a step of measuring the GTP binding activity of the ARL protein can be mentioned. According to the above process, a plant having a reduced amount of ARL polypeptide in the plant can be easily detected (screened) by comparison with the wild type. Plants with reduced activity of ARL polypeptide often have reduced tobamovirus growth-promoting activity in the plant, and therefore, plants with improved resistance to tobamovirus can be easily screened by the above steps. . In the above step, the method for measuring GTP binding activity is not particularly limited, and a conventionally known method may be used.

  Further, in the above measurement step, for example, an oligonucleotide consisting of a fragment of an arl polynucleotide or a complementary sequence thereof is hybridized with genomic DNA, mRNA or cDNA against the target plant and then hybridized with the oligonucleotide. A step of measuring the expression level of the arl polynucleotide by detecting the nucleotide can be mentioned. According to such a process, it is possible to easily detect (screen) the plant body in which the expression level of the arl polynucleotide is reduced by comparison with the wild type strain. In many cases, a plant in which the expression level of arl polynucleotide is reduced has a decreased tobamovirus growth-promoting activity in the plant. Therefore, a plant in which the tobamovirus growth-promoting activity in the plant is reduced can be easily screened by the above steps.

  As the measurement step, the exemplified steps may be used singly or in combination. When used alone, a tobamovirus resistant plant can be screened rapidly. On the other hand, the accuracy of screening can be increased by using a plurality of combinations.

  The oligonucleotides used in the plant screening method according to the present invention can be used as PCR primers or hybridization probes for obtaining arl polynucleotides or fragments thereof. The oligonucleotide can also be used for the following uses. (1) isolation of arl polynucleotides or allelic or splicing variants thereof in a cDNA library; (2) in situ hybridization to metaphase chromosomal spreads to provide the exact chromosomal location of the arl gene ( For example, “FISH”) (Verma et al., Human Chromosomes: described in A Manual of Basic Techniques, Pergamon Press, New York (1988)); and (3) to detect arl mRNA expression in specific tissues. Northern blot analysis.

  It should be noted that those skilled in the art have attributed to the hybridization that occurs between the oligonucleotide used in the plant screening method of the present invention and the target gene (polynucleotide) in any of the applications described above. Is readily understood to be used to hybridize with the gene of interest (polynucleotide).

  A fragment of a polynucleotide according to the present invention comprises at least 7 nt (nucleotide), 10 nt, 12 nt, preferably about 15 nt, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably at least about 40 nt. Although fragments of length are intended, those skilled in the art can appropriately set a preferred length according to the use described above. By “a fragment having a length of at least 20 nt”, for example, a fragment comprising 20 or more consecutive base sequences from the base sequence shown in SEQ ID NO: 1 or a complementary sequence thereof is intended. With reference to this specification, the nucleotide sequence shown in SEQ ID NO: 1 is provided, so that those skilled in the art can easily prepare a DNA fragment based on SEQ ID NO: 1. For example, restriction endonuclease cleavage or ultrasonic shearing can be readily used to create fragments of various sizes. Alternatively, such fragments can be made synthetically. Appropriate fragments (oligonucleotides) are synthesized by Applied Biosystems Incorporated (ABI, 850 Lincoln Center Dr., Foster City, CA 94404) 392 synthesizer and the like.

  Oligonucleotides used in the plant screening method according to the present invention include not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand constituting the same. The oligonucleotide can be used as a tool for gene expression manipulation by an antisense RNA mechanism. With antisense RNA technology, a decrease in the gene product derived from the endogenous gene is observed. By introducing the oligonucleotide, the content of ARL polypeptide can be reduced, and as a result, the tobamovirus growth promoting activity in the plant can be reduced.

  Thus, by using the oligonucleotide used in the plant screening method according to the present invention as a hybridization probe for detecting a polynucleotide encoding an ARL polypeptide or a primer for amplifying the polynucleotide, ARL It is possible to easily detect (screen) a plant or tissue in which the polypeptide expression level is reduced. Furthermore, the above-mentioned oligonucleotide can be used as an antisense oligonucleotide to reduce the expression level of ARL polypeptide in a plant body or its tissue or cell.

  Furthermore, if the plant screening method according to the present invention is used, it is possible to easily detect mutations in ARL polypeptides and arl polynucleotides. Therefore, ARL polypeptides and arl polynucleotides can be obtained from a set of various plant mutants. Plant mutants having mutations can be screened. In the plant screening method according to the present invention, examples of plant mutants that can be screened include the plant mutants described in the above-described plant production method according to the present invention.

  The present invention also provides a kit for screening plants imparted with tobamovirus resistance (hereinafter also simply referred to as “plant screening kit”). The plant screening kit according to the present invention only needs to have a kit capable of reducing the tobamovirus growth promoting activity in the plant body. For example, a kit containing an antibody for measuring the expression level of an ARL polypeptide in a plant body, a kit containing a probe for measuring the expression level of an arl polynucleotide in a plant body, and GTP binding of an ARL polypeptide in the plant body A kit containing a reagent for measuring the activity can be mentioned.

  The plant screening kit according to the present invention includes reagents necessary for carrying out the above-described plant screening method in addition to the antibody, probe, and reagent for measuring GTP binding activity. Preferably it is. That is, a person skilled in the art who has read this specification uses a component of a kit for screening a plant imparted with tobamovirus resistance in a method for screening a plant imparted with tobamovirus resistance. Easy to understand.

<V. Antibody>
The present invention provides an antibody that specifically binds to an ARL polypeptide. The antibody according to the present invention is not limited as long as it can specifically bind to the ARL polypeptide, and may be a polyclonal antibody against the ARL polypeptide, but is preferably a monoclonal antibody against the ARL polypeptide. Monoclonal antibodies have advantages such as uniform properties, easy supply, and semipermanent storage as hybridomas.

As used herein, the term “antibody” refers to immunoglobulins (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ′) ₂ fragments, Fc fragments), eg Examples include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, and anti-idiotype antibodies.

  As described above, ARL polypeptides include ARLA1a-d. In one embodiment, the antibody according to the present invention is preferably an antibody that specifically binds to ARLA1a-d. As used herein, the term “binds specifically to ARLA1a-d” is intended to specifically bind to ARLA1a-d but not to ARL polypeptides other than ARLA1a-d. Is done. Therefore, according to the antibody concerning this invention, the plant body in which the activity of ARLA1a-d fell can be screened easily. The antibody according to the present invention may be an antibody that specifically binds only to a specific ARL polypeptide among ARLA1a to d. According to such an antibody, a plant body in which the activity of only a specific ARL polypeptide is reduced among ARLA1a to d can be screened. That is, a plant screening method comprising a step of incubating an antibody according to the present invention with a tissue extract derived from a target plant, and a plant screening kit comprising the antibody according to the present invention are also resistant to tomovirus. It should be readily understood that it falls within the scope of the present invention for screening plants.

As used herein, the term “an antibody that specifically binds ARLA1a-d” refers to a complete antibody molecule and antibody fragment (eg, Fab and F (ab ') Means containing ₂ fragments). Fab and F (ab ′) ₂ fragments lack the Fc portion of the complete antibody, are removed more rapidly by circulation, and have little nonspecific tissue binding of the complete antibody (Wahl et al. , J. Nucl. Med. 24: 316-325 (1983)). These fragments are therefore preferred.

  “Antibodies” can be prepared by various known methods (eg, HarLow et al., Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, New York (1988); Iwasaki et al., “Monoclonal antibody hybridomas and ELISA, Kodansha (1991). )).

  A monoclonal antibody can be produced using the following method. That is, ARL polypeptide or a fragment thereof, other derivatives or analogs thereof, or cells expressing them are used as an immunogen to immunize mouse spleen lymphocytes, and immunized mouse spleen lymphocytes and mouse myeloma cells A hybridoma is produced by fusing. Subsequently, a monoclonal antibody is produced by this hybridoma. The techniques necessary for obtaining monoclonal antibodies are known in the art. For example, see hybridoma method (see Kohler, G. and Milstein, C., Nature 256, 495-497 (1975)), trioma method, human B-cell hybridoma method (Kozbor, Immunology Today 4, 72 (1983)) And the EBV-hybridoma method (see Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., 77-96 (1985)).

  Peptide antibodies can be produced by methods known in the art. See, for example, Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, FJ et al., J. Gen. Virol. 66: 2347-2354 (1985). I want. In general, animals can be immunized with free peptides. However, anti-peptide antibody titers can be boosted by coupling the peptide to a macromolecular carrier (eg, keyhole limpet hemocyanin (KLH) or tetanus toxoid). For example, peptides containing cysteine can be coupled to a carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides are like glutaraldehyde. Of course, more common linking agents can be used to couple to the carrier. Animals such as rabbits, rats, and mice can be injected either intraperitoneally and / or intradermally with either free or carrier-coupled peptides, eg, emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant. Immunized. In order to provide useful titers of anti-peptide antibodies that can be detected by an ELISA assay using, for example, free peptides adsorbed on a solid surface, for example, at intervals of about 2 weeks. May be needed. The titer of anti-peptide antibodies in serum from immunized animals is increased by selection of anti-peptide antibodies, for example, adsorption to peptides on solid supports and elution of selected antibodies by methods well known in the art. obtain.

  Additional antibodies can be produced in a two-step procedure through the use of anti-idiotype antibodies. Such a method uses the fact that the antibody itself is an antigen, and thus it is possible to obtain an antibody that binds to a secondary antibody. According to this method, an antibody that specifically binds to an ARL polypeptide is used to immunize an animal (preferably a mouse). The splenocytes of such animals are then used to produce hybridoma cells, and the hybridoma cells are capable of binding to an antibody that specifically binds to the ARL polypeptide with an antigen consisting of the ARL polypeptide or fragment thereof. Screened to identify clones producing antibodies that can be blocked. Such antibodies include anti-idiotypic antibodies against antibodies that specifically bind to ARL polypeptides and are used to immunize animals to induce the formation of antibodies that specifically bind to additional ARL polypeptides. Can be done.

It will be apparent to those skilled in the art that Fab and F (ab ′) ₂ and other fragments of the antibodies of the invention can be used in accordance with the methods disclosed herein. Such fragments can typically be produced by proteolytic cleavage using enzymes such as papain (resulting in Fab fragments) or pepsin (resulting in F (ab ′) ₂ fragments). Alternatively, ARL polypeptide binding fragments can be produced by application of recombinant DNA techniques or chemical synthesis.

Thus, the antibody according to the present embodiment only needs to have a fragment (for example, Fab fragment and F (ab ′) ₂ fragment) that specifically binds to the ARL polypeptide, and the Fc fragment of a different antibody molecule. It should be noted that immunoglobulins consisting of are also included in the present invention.

  That is, an object of the present invention is to provide an antibody that specifically binds to an ARL polypeptide and use thereof, and includes the individual immunoglobulin types specifically described herein (IgA, IgD, IgE, IgG or IgM), chimeric antibody production methods, peptide antigen production methods, etc. Therefore, it should be noted that antibodies obtained by methods other than the above methods also belong to the scope of the present invention.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Although this invention is demonstrated more concretely based on an Example and FIGS. 1-4, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

Example 1: Isolation of Ntar polynucleotide
(1) Virus and plant used In this example, TMV-Cg strain (a kind of Tobamovirus cruciferous strain) (Li et al., 1983, Yamanaka et al., 1998) and ToMV-L strain ( A kind of tobamovirus tomato line (Ohno et al., 1984) was used. As plant materials, Arabidopsis thaliana ecotypes Col-0 and No-0 were used. For affinity purification of 180K-FLAG protein, transformed tobacco BY-2 cultured cells (hereinafter also referred to as “ToMV-induced infected BY-2 cells”) capable of inducing replication of ToMV RNA by addition of steroid hormones were used. . For details of the ToMV-induced infected BY-2 cells, see Dohi. K., Nishikiori. M., Tamai. A., Ishikawa. M., Meshi. T. and Mori. M. Inducible virus-mediated expression of Arch. Virol. 151: 1075-1084 (2006), Japanese Patent Application Laid-Open No. 2005-245228 (published on September 15, 2005), Japanese Patent Application Laid-Open No. 2005-110594. See Japanese Patent Publication (published on April 28, 2005), Japanese Patent Application Laid-Open No. 2005-102652 (published on April 21, 2005), and International Publication WO 2005/033306 A1.

(2) Affinity purification of ToMV replication protein (180K-FLAG protein) First, from ToMV-induced infected BY-2 cells, ToMV replication protein and minus-strand RNA were synthesized in the same pattern as in vivo. A cell extract (hereinafter, also referred to as “iBYL”) having an activity to prepare (see JP 2005-237301 A (published September 8, 2005)). Next, an iBYL membrane was prepared from 1.4 ml of iBYL using membrane suspension centrifugation, and 1.2 ml of solubilization buffer (0.7% (wt / vol) LPC (Lysophosphatidylcholine), 30 mM Hepes-KOH , 150 mM NaCl, 1 mM Mg (OAc) ₂ , 0.1 mM DTT, 0.2 mM ATP, 0.2 mM GTP, 0.2 mM UTP) and incubated at 15 ° C. for 20 minutes for solubilization. Next, insoluble components were removed by centrifugation at 15,000 × g, 4 ° C. for 10 minutes. The 600 μl iBYLS15 fraction was subjected to the same solubilization treatment by adding LPC having a final concentration of 0.2%. The membrane fraction and soluble fraction treated with LPC were mixed with 50 μl (packed volume) of anti-FLAG M2 affinity gel (Sigma) previously washed three times with a solubilization buffer, and stirred at 4 ° C. for 1 hour. The resin was washed 5 times with a solubilization buffer containing 0.2% LPC, 250 μl of solubilization buffer containing 0.2% LPC and 100 μg / ml FLAG peptide (Sigma) was added, and 30 minutes at 4 ° C. The ToMV replication protein (180K replication protein) was eluted by stirring. Note that a FLAG tag was added to the C-terminus of the 180K replication protein. It was also confirmed that the replication ability was not impaired by the addition of FLAG.

  Next, in order to measure the ToMV RNA synthesis activity of the eluted fraction, ToMV virion RNA (1 μg / 50-μl reaction solution) was added as an exogenous template to the eluted fraction, and an RdRp reaction was performed. As a result, as shown in FIG. 1, ToMV RNA synthesis activity was detected in the elution fraction.

  Furthermore, the protein contained in the elution fraction was concentrated by acetone precipitation, fractionated by SDS-PAGE, and then analyzed by silver staining (Wako) and Western method. As a result, the elution fraction was co-purified with the translation elongation factor eEF1A, heat shock protein HSP70, and host transmembrane proteins TOM1 and TOM2A genetically shown to be involved in ToMV growth (Nishikiori, M. Dohi, K., Mori, M., Meshi, T., Naito, S. and Ishikawa, M. Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound. J. Virol. 80: 8459-8468 (2006)). This confirmed that the elution fraction contained a protein involved in ToMV growth.

  Furthermore, in the elution fraction obtained by purifying the 180K replication protein using the StrepII tag, it was confirmed that the ToMV RdRp activity dependent on the foreign template and the same host protein as those purified using the FLAG tag were co-purified. Since Strep II purification has the advantage of low contamination with low molecular weight contaminant proteins, it is highly likely that the protein co-purified with the 180K replication protein by the above method is a protein involved in ToMV growth. I can say that. In the purification using the StrepII tag, the recovery rate of the replication protein and RdRp activity is lower than the purification using the FLAG tag. Therefore, in the following experiment, the 180K replication protein obtained by the purification using the FLAG tag was used. The elution fraction was used.

(3) Protein identification by LC-MS / MS analysis
Using Invitrogen's NuPAGE 12% Gel, proteins contained in the elution fraction obtained by purifying the 180K replication protein using the FLAG tag were separated. Proteins in the gel were stained with MS silver staining kit II (Wako). As a result, in addition to the membrane-bound ToMV 180K replication protein, the elution fraction contained many degradation products of the protein, but protein bands thought to be derived from other host proteins were also detected. Therefore, these bands were cut out from an SDS-PAGE gel, treated with trypsin, and the obtained peptide mixture was analyzed by mass spectrometry (LC-MS / MS method). In addition, the Mascot program was used for protein identification by mass spectrometry, and the database including the amino acid sequences of known protein sequence data and NtTOM1, homologue, and NtTOM2A identified in this laboratory was used as the database.

  As a result, as shown in FIG. 1, the purified fraction of the membrane-bound 180K replication protein was derived from a protein derived from an unidentified host of about 20 kDa in addition to peptides derived from NtTOM1 and NtTOM1 homologs and NtTOM2A. Peptide was detected. This unidentified host-derived protein has an ADP ribosylation factor-like protein (ADP ribosylation factor-like protein, belonging to the low molecular weight GTP-binding protein superfamily) because of its homology with the amino acid sequence predicted from the known Solanum tuberosum EST. It was found to be a kind of ARL polypeptide. In addition, genes encoding polypeptides similar to the ARL polypeptide have been widely found in higher eukaryotes including Arabidopsis thaliana, rice, Drosophila and human. In addition, these genes were highly conserved (see FIG. 2). Furthermore, there are four types of genes having high homology with the polynucleotide encoding the ARL polypeptide in Arabidopsis. Here, these four types of homologous genes are referred to as Atalalaa to d.

  Among the ARL polypeptides having high homology with the ARL polypeptide, human HsARL8 and Drosophila ARL polypeptides have been shown to localize in lysosomes (Hoffmann, I and Munro, S. (2006)). An N-terminally acetylated Arf-like GTPase is localized to lysosomes and affects their motility). However, the detailed functions of plant-derived ARL polypeptides are unknown.

(4) Isolation of Ntarl polynucleotide and determination of nucleotide sequence Using the amino acid sequence of the peptide derived from the ARL polypeptide detected by the LC-MS / MS analysis, tobacco N. tabacum (Nt) cDNA (SMART cDNA synthesis kit, clontech), the Ntal cDNA fragment was amplified by the degenerate PCR method. The primers used are shown in Table 1.

  The amplified DNA fragment was cloned using ZeroBlunt TOPO PCR cloning kit (Invitrogen). Ntarl cDNA partial sequence was decoded, and primers were further designed to perform 5'RACE and 3'RACE. Primers of 5 'untranslated region (UTR) and 3' UTR were prepared, and Ntar cDNA was amplified from total RNA obtained from N. tabacum cv. Samsun strain using RT-PCR. The primers used are shown in Table 2.

  By combining these sequence information and EST database (ESTobacco, Advanced Technologies Ltd (Cambridge)), it was considered that tobacco has at least four types of arl homolog genes. Therefore, Ntar cDNA was amplified by RT-PCR. The primers used are shown in Table 3.

  Thus, the base sequences of the four homolog genes of Ntarl were determined. Here, these four homologous genes are referred to as Ntarla1a-d so as to correspond to Atarla1a-d. FIG. 2 shows the deduced amino acid sequence of NtARLA1a-d deduced from the determined base sequence of Ntarla1a-d and the deduced amino acid sequence of AtARLA1a-d deduced from the base sequence of Atarla1a-d. The amino acid sequences of the NtARLA1a-d and AtARLA1a-d had homology with the human (Homo sapiens, Hs) ARL8a, 8b amino acid sequence (see FIG. 2). The Arabidopsis genome encodes 21 genes in the ADP ribosylation factor (ARF) and ARL family (see Vernoud et al., 2003), but has low homology with other ARFs and ARLs. (Identities 34% or less, see TAIR BLASTP, Karlin and Altschul, 1990, 1993).

[Example 2: Examination of tobamovirus growth in Arabidopsis thaliana arla1 mutant strain]
As described above, it was considered that Arabidopsis thaliana encodes four arla1 genes from the Arabidopsis genome sequence. From the EST information and preliminary RT-PCR results, it was found that the transcriptional expression levels of Atala1a-d were in the order of 1d ≧ 1c>1a> 1b. Next, T-DNA or transposon insertion mutant strains were obtained for Atall1a, c, and d, and their effects on tobamovirus growth were examined. As a result, the propagation efficiency of tobamoviruses in the single mutant strains of arla1a, arla1c, and arla1d was wild type. It was about the same as the stock. Since the homology between the Atalla1a to d genes is high (Identities 70% or more, TAIR BLASTP, Karlin and Altschul, 1990, 1993), the possibility of overlapping functions was considered. Thus, an arla1 double mutant was prepared through crossing.

(1) Preparation of arla1 double mutant T-DNA insertion mutants (ecotype Col-0) into Atalla1a and c (locus At5g37680, At3g49870) are obtained from the Salk Institute Genomic Analysis Laboratory website (http: //signal.salk edu) and GABI-kat website (Lietal., inpress, http://www.gabi-kat.de) and obtained from Nottingham Arabidopsis Stock Centre. A transposon insertion mutant strain (Ecotype No-0, Kuromori et al., 2004) into Atalalad (locus At5g67560) was obtained from RIKEN. In order to confirm the T-DNA or transposon insertion site, primers homologous to the site about 500 bp apart (total of about 1000 bp) around the insertion site were prepared based on the genomic sequence information. PCR was performed using these primers and a total of three types of primers, T-DNA or a primer in the transposon region, using Arabidopsis genomic DNA as a template. The primers used are listed in Table 4.

  Arabidopsis genomic DNA was obtained according to the following method. A DNA extraction buffer (200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, 0.5% (wt / vol) SDS) was added to 1-2 rosette leaves about 3 weeks after sowing, and Tissue Lyser (Qiagen) was used to break up the leaves. Total DNA was recovered from the supernatant obtained by centrifugation at 15,000 × g and room temperature for 5 minutes by isopropanol precipitation.

(2) Detection of tobamovirus CP in arla1 double mutant strain Carborundum (600 mesh, Nacalai) was spread on the surface of rosette leaves about 3 weeks after sowing, and the inoculation sources were rubbed together (100 ng virus RNA per leaf) . As the inoculation source, ToMV virion RNA and TMV-Cg virion RNA were used. The method of inoculation and analysis of inoculated leaves were according to the method of Fujisaki et al. (2006).

  Extract buffer twice the wet weight of leaves (50 mM Tris-HCl (pH 7.5), 2.5 mM EDTA, 0.1% (wt / vol) ascorbic acid, complete protease inhibitor (1 tablet / 10 ml)) In addition, the infected leaves were homogenized using a mortar. Furthermore, a gel sample buffer solution (62.5 mM Tris-HCl (pH 6.8), 2.5% (wt / vol) SDS, 5% (vol / vol) β-mercaptoethanol, which is 8 times the wet weight of leaves. 12.5% (vol / vol) glycerol, 0.02% (wt / vol) BPB) was added, and the protein was denatured by heat treatment at 100 ° C. for 3 minutes. The supernatant obtained by centrifugation at 15,000 × g, 4 ° C. for 3 minutes was used as an SDS-PAGE sample. The accumulation of virus CP was analyzed by the Western method (coloring method using BCIP-NBT).

  As a result, as shown in FIG. 3, in the double mutant strain of arla1c and arla1d, the growth of ToMV and TMV-Cg was suppressed at the inoculated leaf level. Moreover, in the double mutant strain of arla1a and arla1c, the growth of ToMV was also suppressed at the inoculated leaf level. In addition, the double mutant strain of arla1c and arla1d and the double mutant strain of arla1a and arla1c showed no difference in growth compared to the wild type strain. From the above, it was considered that the Arabidopsis wild type arla1 gene is a host gene necessary for the proliferation of tobamovirus.

[Example 3: Analysis of wild-type or mutant NtARLA1d using in vitro ToMV RNA translation / replication]
It is possible to reproduce the translation and replication of ToMV RNA using an extract (BYL) prepared from a virus-uninfected tobacco BY-2 cell through a devolatilization treatment (Komoda et al., 2004). . In this system, it is possible to individually analyze the translation process and replication process of ToMV RNA. In order to examine whether NtARLA1 functions in the translation process or replication process of ToMV RNA, wild-type and mutant NtARLA1d proteins were synthesized by in vitro translation and added to the reaction system to translate ToMV RNA. Investigate whether replication is affected.

(1) Expression and purification of NtARLA1d protein in Escherichia coli and preparation of antibody NtARLA1d (17-185aa) having a 6 × His tag and a Thrombin recognition sequence (LVPRGS) at the N-terminus is a pDEST-His vector (Tsunoda et al., 2005) Was expressed in E. coli (BL21 (DE3), Novagen). The primers used for the construction of the expression vector are shown in Table 5.

Soluble His-NtARLA1a was purified using Ni ²⁺ resin (Sigma). Since many purified proteins were contained in the purified fraction, His-NtARLA1d and contaminating proteins were analyzed by SDS-PAGE using 11% (wt / vol) acrylamide and 0.55% (wt / vol) bisacrylamide gel. And separated. From the gel fragment, His-NtARLA1d was eluted using 100 mM phosphate buffer (pH 8.0) containing 0.1% SDS, and about 3 mg was recovered. Using this as an antigen, an anti-NtARLA1d rabbit antibody was prepared.

(2) In vitro ToMV RNA translation and replication ToMV-uninfected BY-2 evacuated protoplast extract (BYL) is Ishibashi, K., Komoda, K., and Ishikawa, M. (2006). In vitro translation and replication of tobamovirus RNA in a cell-free extract of evacuolated tobacco BY-2 protoplasts.In Nagata, T., Matsuoka, K., and Inze, D. (eds.), Tobacco BY-2 cells.Biotechnology and agriculture and forestry, Tobacco BY-2 cells. 53, 183-194. Wild-type and mutant NtARLA1d mRNA were prepared as follows. In order to prepare a template for the in vitro transcription reaction, a region containing the NtARLA1d ORF was amplified by PCR using a synthetic primer having a T7 promoter sequence (Table 1). The DNA fragments encoding the GDP-linked fixed type (T33N) and the GTP-linked fixed type (Q74L) NtARLA1d were amplified by overlapping PCR using synthetic primers (see Table 6) partially modified in nucleotide sequence. .

The 5 ′ end of this DNA fragment was phosphorylated with T4 polynucleotide kinase (Takara) and inserted into the SmaI recognition sequence of pSP64polyA vector (Promega). NtARLA1d mRNA having a 5 ′ cap structure and a 3 ′ polyA ₍₃₀₎ sequence was synthesized in vitro using an AmpliCap T7 High Yield kit (Epicentre) using vector DNA linearized using an EcoRI recognition sequence as a template. Wild type or mutation in 25 μl of BYL translation reaction solution (0.75 mM ATP, 0.1 mM GTP, 25 mM creatine phosphate, 25 μM methionine, 25 μM leucine, 50 μM 18 amino acids, 80 μM spermine, 5 μg creatine kinase, 40 units RNasin) Type NtARLA1d mRNA (500 fmol) was added, and an in vitro translation reaction was performed at 25 ° C. for 30 minutes. Subsequently, ToMV RNA (50 fmol) was added, and an in vitro translation reaction was performed at 25 ° C. for 60 minutes. After translation, 5 μl reaction solution was collected as a protein analysis sample, 5 μl of 5 × RdRp reaction solution was added to the remaining 20 μl, and RNA replication reaction was performed at 25 ° C. for 60 minutes. The RNA after the replication reaction was purified according to the above method, electrophoresed, and detected by autoradiogram.

As a result, when wild-type NtARLA1d was added, the accumulated amount of ToMV replication protein did not change compared to the case where NtARLA1d was not added, but the amount of ³² P-ToMV RNA produced as a result of replication was 2-3. Doubled (see Figure 4). Even when NtARLA1dQ74L was substituted by replacing Glutamine at the 74th amino acid residue of NtARLA1d with leucine, and GTP binding fixed type (membrane bound type) was added, lane ARLA1dQ74L in FIG. 4 was observed. On the other hand, when the threonine of the 33rd amino acid residue was substituted with asparagine and NtARLA1dT33N was added to the GDP binding fixed type (cytoplasmic localized type), there was no effect on the translation and replication of ToMV RNA (Fig. 4 lanes ARLA1dT33N). Therefore, it was suggested that the wild-type NtARLA1d protein is a host protein that supports ToMV RNA replication, and that the membrane-bound NtARLA1d supports tobamovirus RNA replication.

  From the results of the above examples, it was revealed that the ARL polypeptide is essential for tobamovirus RNA replication in plants.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  As described above, the present invention can produce a plant body to which tobamovirus resistance is imparted, and thus can control tobamovirus in various crops. Therefore, the present invention can be applied not only to the breeding field but also to industrial fields such as agriculture and horticulture using plants infected with tobamovirus.

FIG. 1 is a diagram showing the results of SDS-PAGE (silver staining) and ToMV RNA synthesis activity of fractions co-purified with ToMV replication protein from ToMV-induced infected BY-2 cells. FIG. 2 is a diagram showing a comparison of the amino acid sequences of ARL polypeptides derived from each organism. FIG. 3 is a diagram showing the results of examining the tobamovirus growth inhibitory effect in a double mutant of the Arabidopsis thaliana arla1 gene. FIG. 4 is a diagram showing the results of examining the ToMV growth inhibitory effect of wild-type and mutant tobacco ARLA1d proteins using an in vitro ToMV RNA translation / replication system.

Claims (9)

A method for producing a tobamovirus-resistant plant that reduces tobamovirus growth-promoting activity by an ADP-ribosylation factor-like polypeptide in a plant,
The ADP ribosylation factor-like polypeptide is selected from the group consisting of any one of the following (a) to (e) and the following (f) to (j): Any one polypeptide selected from the group consisting of the following (k) to (o) and any one polypeptide selected from the following group (p) to (t) one and polypeptides, at least two features and to belt Bamo method for producing a virus resistant plant that is a polypeptide selected from the group consisting of.
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or 10 (b) one or several amino acids deleted or inserted in the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 2 or 10; Polypeptide (d) encoded by a polynucleotide consisting of a substituted or added amino acid sequence and having a base sequence shown in SEQ ID NO: 1 or 9 (c) having a tobamovirus growth promoting activity (d) SEQ ID NO: 1 Or a polypeptide encoded by a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in 9 and having a tobamovirus growth-promoting activity (e) Base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 9 A polypeptide (g) consisting of an amino acid sequence shown in SEQ ID NO: 4 or 12 encoded by a polynucleotide that hybridizes with a polynucleotide comprising: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in No. 4 or 12, and having a tobamovirus growth promoting activity (H) a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 or 11 (i) one or several bases deleted or inserted in the base sequence shown in SEQ ID NO: 3 or 11 , Replaced, or added A polypeptide encoded by a polynucleotide comprising a base sequence and having a tobamovirus growth promoting activity (j) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 11 under stringent conditions A polypeptide encoded by soybean polynucleotide and having a tobamovirus growth-promoting activity (k) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or 14 (l) shown in the amino acid sequence shown in SEQ ID NO: 6 or 14 In the amino acid sequence, a polypeptide (m) consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and having a tobamovirus growth promoting activity, the nucleotide sequence shown in SEQ ID NO: 5 or 13 Poly consisting of Polypeptide encoded by nucleotide (n) In the base sequence shown in SEQ ID NO: 5 or 13, encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added. And a polypeptide having a tobamovirus growth-promoting activity (o) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 5 or 13, and Polypeptide (p) having the tobamovirus growth promoting activity Polypeptide (q) consisting of the amino acid sequence shown in SEQ ID NO: 8 or 16 In the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 8 or 16, one or several Deletion of amino acids Polypeptide (s) SEQ ID NO: encoded by a polynucleotide consisting of an inserted, substituted or added amino acid sequence and having the base sequence shown in Polypeptide (r) SEQ ID NO: 7 or 15 having activity to promote tobamovirus growth A polypeptide (t) encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in 7 or 15, and having a tobamovirus growth promoting activity (t ) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 7 or 15, and has a tobamovirus growth promoting activity The reduction tobamoviruses hyperproliferation activity, Tobamovirus method for producing a resistant plant according to claim 1, characterized in Rukoto performed by decreased expression level of the polynucleotide encoding the ADP-ribosylation factor-like polypeptide. The polynucleotide is any one polynucleotide selected from the group consisting of the following (a) to (u), and any one polynucleotide selected from the group consisting of the following (e) to (ka): Nucleotides, any one polynucleotide selected from the group consisting of the following (ki) to (ke), and any one polynucleotide selected from the group consisting of the following (ko) to (shi): The method for producing a tobamovirus-resistant plant according to claim 2 , wherein the polynucleotide is at least two polynucleotides selected from the group consisting of:
(A) A polynucleotide comprising the base sequence shown in SEQ ID NO: 1 or 9 (ii) In the base sequence shown in SEQ ID NO: 1 or 9, one or several bases are deleted, inserted, substituted, or added And a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1 or 9 that encodes a polypeptide having a tobamovirus growth promoting activity under stringent conditions A polynucleotide that hybridizes and encodes a polypeptide having a tobamovirus growth-promoting activity (e) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 or 11 (O) in the nucleotide sequence represented by SEQ ID NO: 3 or 11, One or several bases deleted, inserted, substituted or appended And a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 11 and having a stringent condition, wherein the polynucleotide encodes a polypeptide having a tobamovirus growth promoting activity In the nucleotide sequence shown in SEQ ID NO: 5 or 13, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 or 13 (SEQ ID NO: 5 or 13) that encodes a polypeptide having a tobamovirus growth promoting activity A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added, and encoding a polypeptide having a tobamovirus growth-promoting activity, as shown in SEQ ID NO: 5 or 13 From a base sequence complementary to the base sequence A polynucleotide encoding the polypeptide having a tobamovirus growth-promoting activity and hybridizing under stringent conditions with a nucleotide sequence shown in SEQ ID NO: 7 or 15 (SEQ ID NO: 5) In the nucleotide sequence shown in 7 or 15, a polynucleotide encoding a polypeptide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added, and having a tobamovirus growth promoting activity. ) A polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 7 or 15 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity   The method for producing a tobamovirus-resistant plant according to claim 1, wherein the ADP-ribosylation factor-like polypeptide reduces the tobamovirus growth-promoting activity by introducing a mutation into the ADP-ribosylation factor-like polypeptide. A kit for producing a tobamovirus resistant plant comprising a polynucleotide encoding an ADP-ribosylation factor-like polypeptide or a fragment thereof ,
The polynucleotide is any one polynucleotide selected from the group consisting of the following (a) to (u) and any one polynucleotide selected from the group consisting of the following (e) to (ka): Nucleotides, any one polynucleotide selected from the group consisting of the following (ki) to (ke), and any one polynucleotide selected from the group consisting of the following (ko) to (shi): A kit for producing a tobamovirus-resistant plant, wherein the kit is at least two polynucleotides selected from the group consisting of:
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or 9
(Ii) a polypeptide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1 or 9, and having a tobamovirus growth-promoting activity Polynucleotide
(Iii) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or 9 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity
(E) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 or 11
(O) a polypeptide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 3 or 11, and having a tobamovirus growth-promoting activity Polynucleotide
(Or) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 or 11 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity
(G) A polynucleotide comprising the base sequence shown in SEQ ID NO: 5 or 13
(C) A polypeptide consisting of a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 5 or 13, and having a tobamovirus growth promoting activity Polynucleotide
(K) A polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or 13 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity
(Ko) A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7 or 15
(Sa) In the base sequence shown in SEQ ID NO: 7 or 15, a polypeptide comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added, and having a tobamovirus growth-promoting activity Polynucleotide
(Shi) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 7 or 15 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity Of the expression level of ADP-ribosylation factor-like polypeptide in the plant body, the active level of ADP-ribosylation factor-like polypeptide in the plant body, and the expression level of the polynucleotide encoding the ADP-ribosylation factor-like polypeptide in the plant body, A method for screening a tobamovirus resistant plant for measuring at least one of the following:
The ADP ribosylation factor-like polypeptide is selected from the group consisting of any one of the following (a) to (e) and the following (f) to (j): Any one polypeptide selected from the group consisting of the following (k) to (o) and any one polypeptide selected from the following group (p) to (t) One polypeptide and at least two polypeptides selected from the group consisting of:
The polynucleotide is any one polynucleotide selected from the group consisting of the following (a) to (u) and any one polynucleotide selected from the group consisting of the following (e) to (ka): Nucleotides, any one polynucleotide selected from the group consisting of the following (ki) to (ke), and any one polynucleotide selected from the group consisting of the following (ko) to (shi): , at least two features and to belt Bamo virus screening method of resistant plant that is a polynucleotide selected from the group consisting of.
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or 10 (b) one or several amino acids deleted or inserted in the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 2 or 10; Polypeptide (d) encoded by a polynucleotide consisting of a substituted or added amino acid sequence and having a base sequence shown in SEQ ID NO: 1 or 9 (c) having a tobamovirus growth promoting activity (d) SEQ ID NO: 1 Or a polypeptide encoded by a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in 9 and having a tobamovirus growth-promoting activity (e) Base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 9 A polypeptide (g) consisting of an amino acid sequence shown in SEQ ID NO: 4 or 12 encoded by a polynucleotide that hybridizes with a polynucleotide comprising: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in No. 4 or 12, and having a tobamovirus growth promoting activity (H) a polypeptide encoded by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 or 11 (i) one or several bases deleted or inserted in the base sequence shown in SEQ ID NO: 3 or 11 , Replaced, or added A polypeptide encoded by a polynucleotide comprising a base sequence and having a tobamovirus growth promoting activity (j) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 11 under stringent conditions A polypeptide encoded by soybean polynucleotide and having a tobamovirus growth-promoting activity (k) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or 14 (l) shown in the amino acid sequence shown in SEQ ID NO: 6 or 14 In the amino acid sequence, a polypeptide (m) consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and having a tobamovirus growth promoting activity, the nucleotide sequence shown in SEQ ID NO: 5 or 13 Poly consisting of Polypeptide encoded by nucleotide (n) In the base sequence shown in SEQ ID NO: 5 or 13, encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added. And a polypeptide having a tobamovirus growth-promoting activity (o) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 5 or 13, and Polypeptide (p) having the tobamovirus growth promoting activity Polypeptide (q) consisting of the amino acid sequence shown in SEQ ID NO: 8 or 16 In the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 8 or 16, one or several Deletion of amino acids Polypeptide (s) SEQ ID NO: encoded by a polynucleotide consisting of an inserted, substituted or added amino acid sequence and having the base sequence shown in Polypeptide (r) SEQ ID NO: 7 or 15 having activity to promote tobamovirus growth A polypeptide (t) encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in 7 or 15, and having a tobamovirus growth promoting activity (t ) A polypeptide (A) sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 7 or 15, and has a tobamovirus growth-promoting activity Number 1 or A polynucleotide consisting of the base sequence shown in (9) consisting of a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 9; and Hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 9 encoding a polypeptide having a tobamovirus growth-promoting activity, and proliferating tobamovirus Polynucleotide encoding a polypeptide having enhancing activity (e) Polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 or 11 (O) One or several bases in the base sequence shown in SEQ ID NO: 3 or 11 Consists of nucleotide sequences deleted, inserted, substituted or added And a polynucleotide encoding a polypeptide having a tobamovirus growth-promoting activity (or) hybridizing under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 11, and A polynucleotide encoding a polypeptide having a tobamovirus growth-promoting activity (ki) One or several of the nucleotide sequence shown in SEQ ID NO: 5 or 13 consisting of the base sequence shown in SEQ ID NO: 5 or 13 A polynucleotide encoding a polypeptide having a tobamovirus growth-promoting activity and complementary to the base sequence shown in SEQ ID NO: 5 or 13 Polynucleotides and nucleotides consisting of base sequences A polynucleotide (SEQ ID NO: 7 or 15) comprising the nucleotide sequence shown in SEQ ID NO: 7 or 15 that encodes a polypeptide having a tobamovirus growth-promoting activity that hybridizes under lingent conditions In the nucleotide sequence shown, a polynucleotide (Shi) SEQ ID NO: 7 consisting of a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added, and encoding a polypeptide having a tobamovirus growth promoting activity Alternatively, a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence shown in 15 under a stringent condition and encodes a polypeptide having a tobamovirus growth promoting activity A tobamovirus-resistant plant comprising at least one of an antibody that specifically binds to an ADP-ribosylation factor-like polypeptide and a probe that specifically hybridizes to a polynucleotide encoding the ADP-ribosylation factor-like polypeptide A screening kit,
The ADP ribosylation factor-like polypeptide is selected from the group consisting of any one of the following (a) to (e) and the following (f) to (j): Any one polypeptide selected from the group consisting of the following (k) to (o) and any one polypeptide selected from the following group (p) to (t) One polypeptide and at least two polypeptides selected from the group consisting of:
The polynucleotide is any one polynucleotide selected from the group consisting of the following (a) to (u) and any one polynucleotide selected from the group consisting of the following (e) to (ka): Nucleotides, any one polynucleotide selected from the group consisting of the following (ki) to (ke), and any one polynucleotide selected from the group consisting of the following (ko) to (shi): A screening kit for a tobamovirus-resistant plant, which is at least two polynucleotides selected from the group consisting of:
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 10
(B) an amino acid sequence represented by SEQ ID NO: 2 or 10, consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added, and has a tobamovirus growth promoting activity Polypeptide having
(C) a polypeptide encoded by a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 or 9
(D) is encoded by a polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1 or 9, and has a tobamovirus growth promoting activity Polypeptide having
(E) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 9, and has a tobamovirus growth-promoting activity
(F) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or 12
(G) The amino acid sequence shown in SEQ ID NO: 4 or 12, consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added, and has a tobamovirus growth promoting activity Polypeptide having
(H) a polypeptide encoded by a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 or 11
(I) is encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 3 or 11, and has a tobamovirus growth promoting activity Polypeptide having
(J) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 11, and has a tobamovirus growth-promoting activity
(K) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or 14
(L) an amino acid sequence represented by SEQ ID NO: 6 or 14, consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added, and exhibiting tobamovirus growth promoting activity Polypeptide having
(M) a polypeptide encoded by a polynucleotide comprising the base sequence represented by SEQ ID NO: 5 or 13
(N) In the base sequence shown in SEQ ID NO: 5 or 13, encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added, and has a tobamovirus growth promoting activity Polypeptide having
(O) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 5 or 13, and has a tobamovirus growth-promoting activity
(P) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8 or 16
(Q) an amino acid sequence represented by SEQ ID NO: 8 or 16, consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added, and exhibiting tobamovirus growth promoting activity Polypeptide having
(R) a polypeptide encoded by a polynucleotide comprising the base sequence represented by SEQ ID NO: 7 or 15
(S) The base sequence shown in SEQ ID NO: 7 or 15 is encoded by a polynucleotide comprising a base sequence in which one or several bases are deleted, inserted, substituted or added, and has a tobamovirus growth promoting activity. Polypeptide having
(T) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 7 or 15, and has a tobamovirus growth-promoting activity
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or 9
(Ii) a polypeptide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1 or 9, and having a tobamovirus growth-promoting activity Polynucleotide
(Iii) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or 9 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity
(E) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 or 11
(O) a polypeptide comprising a nucleotide sequence in which one or several bases are deleted, inserted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 3 or 11, and having a tobamovirus growth-promoting activity Polynucleotide
(Or) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 or 11 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity
(G) A polynucleotide comprising the base sequence shown in SEQ ID NO: 5 or 13
(C) A polypeptide consisting of a base sequence in which one or several bases are deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 5 or 13, and having a tobamovirus growth promoting activity Polynucleotide
(K) A polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or 13 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity
(Ko) A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7 or 15
(Sa) In the base sequence shown in SEQ ID NO: 7 or 15, a polypeptide comprising a base sequence in which one or several bases are deleted, inserted, substituted, or added, and having a tobamovirus growth-promoting activity Polynucleotide
(Shi) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 7 or 15 under a stringent condition and encodes a polypeptide having a tobamovirus growth-promoting activity The polynucleotide according to any of (a) to (g) below.
Shown in any one of the polynucleotide (b) SEQ ID NO: 10, and 1 2 which encodes a polypeptide comprising an amino acid sequence shown in any one of (i) SEQ ID NO: 10, 12, 14, and 16 A polynucleotide (c) sequence that encodes a polypeptide having a tobamovirus growth-promoting activity, comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in the amino acid sequence Polynucleotide consisting of the base sequence shown in any one of Nos. 9, 11, 13, and 15 (d) One or several in the base sequence shown in any one of SEQ ID Nos. 9 and 11 Consists of a base sequence deleted, inserted, substituted or added, and has a tobamovirus growth-promoting activity Polynucleotides (e) SEQ ID NO: 9 which encodes a polypeptide, and 1 hybridizes 1 at any one polynucleotide under stringent conditions comprising a nucleotide sequence complementary to the nucleotide sequence shown in, and tobamovirus A polynucleotide encoding a polypeptide having a proliferation promoting activity (f) positions 27 to 581 of SEQ ID NO: 9, positions 14 to 568 of SEQ ID NO: 11, positions 16 to 570 of SEQ ID NO: 13 And polynucleotide (g) comprising the nucleotide sequence shown in any one of positions 65 to 619 of SEQ ID NO: 15 as an open reeling frame, positions 27 to 581 of SEQ ID NO: 9, and SEQ ID NO: in # 14 ~ # 568 nucleotide sequence shown in any one of 11, one or several nucleotides are deleted, inserted, location Or include additional base sequence as an open reeling frame, and a polynucleotide encoding a polypeptide having a tobamovirus hyperproliferative activity The polypeptide according to any one of the following (h) to (wo).
Represented by amino acid sequence shown in any one of the polypeptides (i) SEQ ID NO: 10, and 1 2 comprising an amino acid sequence shown in any one of (h) SEQ ID NO: 10, 12, 14, and 16 Polypeptide (nu) SEQ ID NO: 9, 11, 13, and 15 consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence and having a tobamovirus growth promoting activity in the nucleotide sequence shown in any one of the polypeptides (Le) SEQ ID NO: 9, and 1 1, which is encoded by a polynucleotide consisting shown in any one of base sequences in which one or several bases It is encoded by a polynucleotide consisting of a base sequence deleted, inserted, substituted, or added, and tobamovirus is propagated Advancing a polypeptide encoded by a (wo) SEQ ID NO: 9, and 1 1 of any one consisting of a nucleotide sequence complementary to the nucleotide sequence shown in the polynucleotide and a polynucleotide hybridizing under stringent conditions with activity And a polypeptide having a tobamovirus growth-promoting activity