TIR1 family protein mutants, Aux / IAA family protein mutants, or DII domain-containing fragment mutants

Abstract

To provide pairs of TIR1 family protein mutants and Aux / IAA family protein mutants or DII domain-containing fragment mutants thereof, which have reduced impact on endogenous plant TIR1 family proteins and Aux / IAA family proteins and are still interactable. [Solution] Introduce a substitution mutation a1 of R489 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding arginine in another amino acid sequence, and / or a substitution mutation a2 of L84 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding leucine in another amino acid sequence, as a mutation into the TIR1 family protein, and introduce a substitution mutation b of G85 in the amino acid sequence shown in SEQ ID NO: 8 or the corresponding glycine in another amino acid sequence, as a mutation into the Aux / IAA family protein or its DII domain-containing fragment.

Description

[Technical Field]

[0001] This invention relates to TIR1 family protein variants, Aux / IAA family protein variants, DII domain-containing fragment variants, and the like. [Background technology]

[0002] Auxin, a plant hormone, controls various phenomena related to plant growth and development. TRANSPORT INHIBITOR RESPONSE 1 (TIR1) is a well-known auxin receptor, a subunit of the SKP1-Cullin-F-box (SCF) ubiquitin ligase complex. When auxin binds to TIR1, its interaction with the DII degron sequence of the AUXIN / INDOLE-3-ACETIC ACID (Aux / IAA) transcription repressor is promoted, leading to ubiquitination. Subsequently, degradation of the Aux / IAA protein by the proteasome releases auxin responsive factors (ARFs), activating the auxin response. Plants possess multiple auxin receptors, including TIR1 and AUXIN-SIGNALING F-BOX (AFB) 1-5 (Arabidopsis thaliana). Interestingly, Aux / IAA forms a larger gene family: the Arabidopsis thaliana genome encodes 29 Aux / IAA genes, 23 of which have well-conserved DII sequences. These redundant protein combinations mediate the complex regulatory mechanisms of auxin recognition in plants.

[0003] To elucidate the complexity of such auxin signaling, artificial TIR1-auxin pairs have been developed using a bump-and-hole approach to design shape-complementary pairs of synthetic ligands and altered receptors (Non-Patent Literature 1). The synthetic convex auxin (indole-3-acetic acid; IAA), 5-(3-methoxyphenyl)indole-3-acetic acid (cvxIAA), is a synthetic convex auxin (TIR1) of Arabidopsis thaliana. F79GIn the presence of ), the cvxIAA-ccvTIR1 pair hijacked the downstream signaling of TIR1 without being affected by the endogenous auxin receptor. The cvxIAA-ccvTIR1 pair deciphered the role of TIR1 in auxin-regulated growth or development. While these studies have provided chemical means to control specific auxin receptors, controlling individual Aux / IAA transcription repressors remains challenging.

[0004] In non-plant cells that do not synthesize auxin, auxin-triggered proteolysis of Aux / IAA has become an innovative research tool for conditionally knocking down specific proteins, known as auxin-inducible degrons (AIDs) (Non-Patent Literature 2). In yeast and mammalian cells accumulating target proteins labeled with plant TIR1 or Arabidopsis thaliana IAA17-derived degrons, exogenous application of auxin induces ubiquitination and proteasomal degradation of the target protein, allowing for the evaluation of the target protein's role under specific conditions. To date, AID technology has been used in various organisms, and its practical application in essential protein research is progressing. The AID method is a powerful tool for conditional proteolysis, and improved AID methods using ccvTIR1 and cvxIAA are unaffected by endogenous auxin. However, these systems affect endogenous Aux / IAA containing DII degrons, making it impossible to apply this method to plant cells. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2018 / 164214 [Non-patent literature]

[0006] [Non-Patent Document 1] Nat Chem Biol. 2018 Mar;14(3):299-305. doi: 10.1038 / nchembio.2555. Epub 2018 Jan 22. [Non-Patent Document 2] Nat Methods. 2009 Dec;6(12):917-22. doi: 10.1038 / nmeth.1401. Epub 2009 pav 15. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide pairs of TIR1 family protein mutants and Aux / IAA family protein mutants or DII domain-containing fragment mutants thereof, which have reduced impact on endogenous plant TIR1 family proteins and Aux / IAA family proteins and are still interactable. [Means for solving the problem]

[0008] In view of the above problems, the inventors diligently conducted research and focused on inserting mutations into both TIR1 family proteins and DII domain-containing fragments of Aux / IAA family proteins. Further research led the inventors to discover that the above problems can be solved by introducing a substitution mutation a1 for R489 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding arginine in another amino acid sequence, and / or a substitution mutation a2 for L84 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding leucine in another amino acid sequence, as mutations in TIR1 family proteins, and by introducing a substitution mutation b for G85 in the amino acid sequence shown in SEQ ID NO: 8 or the corresponding glycine in another amino acid sequence, as mutations in Aux / IAA family proteins or their DII domain-containing fragments. Based on this finding, the inventors furthered their research and completed the present invention. That is, the present invention encompasses the following aspects.

[0009] Item 1. A TIR1 family protein mutant obtained by mutation in a TIR1 family protein, comprising a substitution mutation a1 of R489 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding arginine in another amino acid sequence, and / or a substitution mutation a2 of L84 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding leucine in another amino acid sequence.

[0010] Item 2. The TIR1 family protein variant described in Item 1, wherein the TIR1 family protein is a protein containing an amino acid sequence that has 70% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 6.

[0011] Item 3. The TIR1 family protein variant according to Item 1, wherein the TIR1 family protein comprises the consensus sequence a shown in SEQ ID NO: 7, and the substitution mutation a1 is an arginine substitution mutation within the consensus sequence a.

[0012] Item 4. A TIR1 family protein variant as described in Item 1, wherein the mutated amino acid of the substitution mutation a1 is an amino acid with a molecular weight smaller than arginine.

[0013] Item 5. A TIR1 family protein mutant as described in Item 1, wherein the mutated amino acid of the substitution mutation a2 is an amino acid or acidic amino acid with a molecular weight smaller than leucine.

[0014] Item 6. Aux / IAA family protein variants or DII domain-containing fragment variants obtained by mutation in an Aux / IAA family protein or a DII domain-containing fragment thereof, wherein the mutation includes a G85 substitution mutation b in the amino acid sequence shown in SEQ ID NO: 8 or a corresponding glycine substitution mutation b in another amino acid sequence.

[0015] Item 7. The Aux / IAA family protein is a protein that contains an amino acid sequence having 70% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 8-30, and / or The DII domain-containing fragment is a protein containing an amino acid sequence that has 70% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 31 to 53. Aux / IAA family protein variants or DII domain-containing fragment variants as described in item 6.

[0016] Item 8. The Aux / IAA family protein variant or DII domain-containing fragment variant according to Item 6, wherein the Aux / IAA family protein and / or the DII domain-containing fragment comprises the consensus sequence b shown in SEQ ID NO: 54, and the substitution mutation b is a glycine substitution mutation within the consensus sequence b.

[0017] Item 9. An Aux / IAA family protein mutant or DII domain-containing fragment mutant as described in Item 6, wherein the mutated amino acid of the substitution mutation b is an amino acid with a molecular weight greater than glycine.

[0018] Item 10. A polynucleotide comprising the coding sequence of a TIR1 family protein variant described in any of Items 1-5, or the coding sequence of an Aux / IAA family protein variant or DII domain-containing fragment variant described in any of Items 6-9.

[0019] Item 11. A cell containing at least one selected from the group consisting of a TIR1 family protein variant described in any of Items 1 to 5, an Aux / IAA family protein variant or DII domain-containing fragment variant described in any of Items 6 to 9, and a polynucleotide containing their coding sequences.

[0020] Item 12. A cell or non-human organism containing such cell, comprising a TIR1 family protein variant as described in any of Items 1 to 5, and an Aux / IAA family protein variant or DII domain-containing fragment variant as described in any of Items 6 to 9.

[0021] Item 13. A method comprising contacting a cell described in Item 12 or a non-human organism containing said cell with auxin and / or an auxin derivative.

[0022] Item 14. The method according to item 13, wherein the TIR1 family protein variant comprises another protein domain, and / or the Aux / IAA family protein variant or DII domain-containing fragment variant comprises another protein domain.

[0023] Item 15. The method of Item 14, wherein the Aux / IAA family protein variant or DII domain-containing fragment variant comprises a degradation target protein domain as another protein domain, and the degradation target protein domain is degraded by contacting the auxin and / or auxin derivative with the cell or the non-human organism. [Effects of the Invention]

[0024] According to the present invention, it is possible to provide pairs of TIR1 family protein mutants and Aux / IAA family protein mutants or DII domain-containing fragment mutants thereof, which have reduced effects on endogenous plant TIR1 family proteins and Aux / IAA family proteins and are still interactable. [Brief explanation of the drawing]

[0025] [Figure 1]The crystal structure (PDB; 2P1Q) of the ternary complex of TIR1-auxin-IAA7-DII peptide is shown. (a) Top view and (b) Side view of the ternary complex. TIR1 is shown in cyan, auxin (indole-3-acetic acid) in red, and IAA7-DII peptide (QVVGWPPVRNYRK: SEQ ID NO: 56) in orange. The numbers indicate the LRR order from the N-terminal side of TIR1. [Figure 2] We demonstrate the construction of a modified TIR1 and a modified Aux / IAA that interact orthogonally in the presence of natural auxin (IAA, indole-3-acetic acid). [Figure 3] This shows the interaction between modified IAA7-DII and wild-type TIR1. (a) Schematic diagram of TIR1-FLAG and GST-IAA7-DII used in an in vitro pull-down assay. IAA7-DII is a peptide containing glycine from position 33 to serine 104 of IAA7. It contains a consensus motif (GWPPV: SEQ ID NO: 55) internally. (b) Pull-down assay of modified IAA7-DII, in which G85 of IAA7-DII was modified to 19 other amino acids, and wild-type TIR1-WT. The interaction was examined in the presence of 10 μM IAA, except for "Mock" in the leftmost lane. TIR1-WT-FLAG that interacted with GST-IAA7-DII conjugated to Glutathione Sepharose 4B beads was detected by immunoblotting analysis using an anti-FLAG antibody (anti-FLAG). [Figure 4] The crystal structure (PDB; 2P1Q) of the TIR1-IAA7-DII peptide complex shows the arrangement of TIR1-L84, TIR1-R489, and IAA7-D85. (a) Top view and (b) Side view of the complex crystal structure. The side chain structures of TIR1-L84, TIR1-R489, and IAA7-G85 are shown, in yellow, red, and magenta, respectively. The second loop of TIR1 is shown in green, and the consensus motif of IAA7-DII is shown in violet. [Figure 5]This report describes the investigation of orthogonal interactions focusing on TIR1-L84 and IAA7-DII-G85. (a) In vitro pull-down assays were conducted using wild-type TIR1-FLAG and modified TIR1-FLAG (in which TIR1-L84 was replaced with alanine (A)) and wild-type GST-IAA7-DII and GST-IAA7-DII (in which G85 of IAA7-DII was replaced with arginine (R)). The interactions were examined in the presence of 10 μM IAA. (b) In vitro pull-down assays were conducted using modified TIR1-FLAG (in which TIR1-L84 was replaced with aspartic acid (D)) and GST-IAA7-DII-G85R. The interactions were examined in the presence of 10 μM IAA. [Figure 6] This report describes the investigation of orthogonal interactions between TIR1-R489 and IAA7-DII-G85. (a) In vitro pull-down assay using modified TIR1-FLAG, in which TIR1-R489 was modified with glycine (G), alanine (A), and glutamic acid (E), and GST-IAA7-DII-WT. (b) Pull-down assay between modified IAA7-DII, in which G85 of IAA7-DII was modified with nine other hydrophobic amino acids, and wild-type TIR1-WT and TIR1-R489G. In all cases, the interactions were investigated in the presence of 10 μM IAA. (c) Orthogonal interactions between TIR1-WT, TIR1-R489G and IAA7-DII-WT and IAA7-DII-G85W. [Figure 7] This study demonstrates cvxIAA-dependent interactions between TIR1-F79G R489G and TIR1-F79A R489G and IAA7-DII-G85W. [Figure 8] We demonstrate the construction of a modified TIR1 and a modified Aux / IAA that interact orthogonally in the presence of synthetic auxin (cvxIAA). [Figure 9]This shows that convex IAA7 deglon is resistant to degradation by endogenous auxin but is degraded by the function of concave TIR1 (TIR1R489G). (a) The transcription levels of the IAA7-DII-GFP transgene are shown in wild-type (WT) plants and three transgenic lines: UBQpro:DIIWT-GFP and UBQpro:DIIG85W-GFP. Third rosette leaves from 16-day-old plants were subjected to qRT-PCR analysis. The DII-GFP transcription level was normalized by the ACT2 transcript and is shown as a relative value to the transcription level of DIIG85W-GFP line #2, which is set to 1. The values ​​are mean ± SE (n = 3). (b) The orthogonal projection created from z-stack images (depth 20 μm) of hypocotyl cells of 7-day-old seedlings described in (a) is shown. The graph shows the volume of GFP fluorescence measured from the z-stack images and is shown as a relative value with the fluorescence of DIIG85W-GFP line #2 set to 1. The values ​​are mean ± SE (n = 4). (c) Shows the accumulation of DII-GFP protein in the total protein extract from 6-day-old seedlings described in (a). (d) Shows the accumulation of DII-GFP protein in the total protein extract of seedlings incubated with Mock (solvent) or the proteasome inhibitor 10 μM MG132 or 10 μM bortezomib (BTZ). 7-day-old seedlings of WT, DIIWT-GFP line #2, or line #6 were used for treatment. (e) Shows orthogonal projections created from z-stack images (depth 20 m) of hypocotyl cells in 6-day-old seedlings of DIIG85W-GFP expressing plants with WT(-), FLAG-TIR1WT, or FLAG-TIR1R489G background. The graph shows the volume of GFP fluorescence measured from the Z-stack image, expressed as a relative value with the fluorescence of the WT background set to 1. The values ​​are mean ± SE (n = 6). (f) Accumulation of DII-GFP protein in total protein extract from 6-day-old seedlings as described in (e). (g) Accumulation of DII-GFP and FLAG-TIR1 protein in total protein extract from seedlings possessing DIIG85W-GFP and TIR1R489G. 6-day-old seedlings were incubated with Mock or a proteasome inhibitor.(h) Orthogonal projections created from z-stack images (depth 20 μm) of hypocotyl cells of 6-day-old seedlings of DIIG85W-GFP expressing plants with FLAG-TIR1WT, FLAG-TIR1R489G, and FLAG-TIR1F79A R489G backgrounds are shown. The graph shows the volume of GFP fluorescence measured from the z-stack images, expressed as a relative value with the fluorescence of the TIR1WT background set to 1. The values ​​are mean ± SE (n = 4). (i) Accumulation of DII-GFP and FLAG-TIR1 protein in total protein extracts from 6-day-old seedlings described in (h) is shown. Scale bar, 50 μm; green, GFP; magenta, chlorophyll (Chl) fluorescence. In immunoblot detection, asterisks indicate nonspecific signals, and total protein and ACTIN were detected as loading controls. Dots indicate data points from individual plants. [Figure 10]Demonstration of proteolytic induction in plants: We show that convex modified auxin induces degradation of GFP conjugated with convex IAA7 degron in the presence of double concave TIR1. (a) Orthogonal projection created from z-stack images of hypocotyl cells of seedlings introduced with DIIG85W-GFP and FLAG-TIR1WT. 6-day-old seedlings were incubated for 1 hour with Mock (solvent control), 0.1 μM IAA, cvxIAA, and Ada-IAA. (b) GFP fluorescence levels were measured from the observations in (a) and shown as relative values ​​with the fluorescence level at 0 hours set to 1. Values ​​are mean ± SE (n = 6). (c) Orthogonal projection created from z-stack images of hypocotyl cells of seedlings possessing DIIG85W-GFP and FLAG-TIR1F79A R489G. 6-day-old seedlings were treated as in (a). (d) The volume of GFP fluorescence was measured from the observations in (c) and shown as relative values ​​with the fluorescence level at 0 hours set to 1. The values ​​are mean ± SE (n = 6). (e) Abundance of DII-GFP and FLAG-TIR1 protein in total protein extract from DIIG85W-GFP expressing plants with a FLAG-TIR1WT or FLAG-TIR1F79A R489G background. 6-day-old seedlings were treated as described in (a). (f) Abundance of DII-GFP protein in total protein extract from seedlings possessing DIIG85W-GFP and FLAG-TIR1F79A R489G. 6-day-old seedlings were incubated with cvxIAA or Ada-IAA at the listed concentrations (0, 0.1, 0.01, 0.001, 0.0001 μM) for 1 hour. The graph shows the DII-GFP amount normalized by the ACTIN amount, as a relative value with 0 μM set to 1. The values ​​are mean ± SE (n = 3). (g) This shows the abundance of DII-GFP and FLAG-TIR1 proteins in the total protein extract from plants into which gene constructs expressing both DIIG85W-GFP and TIR1F79G R489G were introduced. 6-day-old seedlings were incubated for 1 hour with Mock, 0.1 μM IAA, 0.1 μM cvxIAA, 0.1 μM Ada-IAA, and 0.01 μM Ada-IAA. In immunoblot detection, asterisks indicate nonspecific signals, and total protein and ACTIN were detected as loading controls.Different lowercase letters indicate statistical significance according to Tukey's test (P < 0.05). Dots represent data points from individual plants (b and d) or plant sample sets (f) in the graph. [Figure 11] The pairing of cvxIAA and double-concave TIR1 does not cause serious defects in plant growth and auxin signaling. (a) Photographs of 16-day-old plants expressing DIIG85W-GFP in a FLAG-TIR1WT or TIR1F79A R489G background, and wild-type (WT) plants grown in soil, showing above-ground fresh weight (shoot FW). Scale bar, 10 mm. Values ​​are mean ± SE (n = 10). (b) Photographs of 7-day-old seedlings of the plant lineage from (a) and root length. Plants were transferred to solid medium and grown, and root length was measured. Arrows indicate root tips; scale bar shows 10 mm. Values ​​are mean ± SE (n = 12). (c) Photographs and root lengths of 7-day-old seedlings carrying DIIG85W-GFP and TIR1F79A R489G, grown in media containing Mock, 0.1 μM IAA, 0.1 μM cvxIAA, 0.1 μM Ada-IAA, or 0.01 μM Ada-IAA. 3-day-old seedlings germinated in media without additional compounds were transplanted into new growth media containing each IAA, grown for 4 days, and root length was measured. Arrows indicate root tips, and the scale bar is 10 mm. Values ​​are mean ± SE (n = 12). Different lowercase letters indicate statistical significance by Tukey's test (P < 0.05). [Figure 12]This paper evaluates available methods for inducing protein degradation in plants. (a) Orthogonal projections are shown from z-stack images (depth 10 μm) of guard cells of the third leaf of a rosette 1 hour after spraying with a mock (solvent control), 0.1 μM cvxIAA, or Ada-IAA solution. As a control (untreated), 16-day-old plants that were not treated were observed. The graph shows the GFP fluorescence levels measured from the observation results, expressed as relative values ​​with the fluorescence level of the untreated leaves set to 1. (b) This shows the abundance of DII-GFP protein in the sprayed plants from (a). The graph shows the DII-GFP levels normalized by the ACTIN level, expressed as relative values ​​with the untreated level set to 1. The values ​​are mean ± SE (n = 4). (c) Shows the abundance of DII-GFP protein in seedlings before transplantation (day 0) or at 1 and 2 days after transplantation into growth media containing Mock (solvent control), 0.1 μM cvxIAA, or 0.1 μM Ada-IAA. The graph shows the relative value of DII-GFP amount normalized by ACTIN amount, with day 0 (0 d) set to 1. The values ​​are mean ± SE (n = 4). (d, e) Shows the changes in orthogonal projection created from z-stack images (depth 20 (g) or 40 (h) μm) of hypocotyl cells (d) or root tip cells (e) of seedlings possessing DIIG85W-GFP and TIR1F79A R489G. Six-day-old seedlings were transplanted into growth media containing Mock, 0.1 μM cvxIAA, or 0.1 μM Ada-IAA, and observed every minute for 30 minutes (g) or 20 minutes (h) on a glass-bottom dish. Images at the time indicated above are shown. The graphs show the time change in GFP fluorescence levels measured from observations, expressed as relative values ​​with the fluorescence level at 0 minutes set to 1. Scale bars, 50 μm; green, GFP; magenta, chlorophyll (Chl) fluorescence. In immunoblot detection, total protein and ACTIN were detected as loading controls. Dots represent data points from individual plants (a, b) or plant sample sets (c) in each graph. Asterisks indicate statistical significance based on Dunnett's test (**, P < 0.01; ***, P < 0.001). [Figure 13]This sequence logo was created using the WebLogo 3 web server (https: / / weblogo.threeplusone.com / ) based on multiple alignment analysis of TIR1 / AFB1-5 (top row) and 23 Aux / IAAs DII deglons (bottom row). The positions of R489 in TIR1 and G85 in IAA7 are indicated by red and magenta arrows, respectively. [Modes for carrying out the invention]

[0026] 1. Definition etc. In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0027] In this specification, "identity" of amino acid sequences refers to the degree of agreement between two or more comparable amino acid sequences. Therefore, the higher the agreement between two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on this BLAST algorithm. The specific methods for these analyses are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). Furthermore, the "identity" of the base sequence is defined in accordance with the above.

[0028] In this specification, "conservative substitution" means that an amino acid residue is substituted for an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Other examples of conservative substitutions include amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0029] In this specification, nucleotides such as DNA and RNA may be subjected to known chemical modifications, as exemplified below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residue of each nucleotide can be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. The hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may also be replaced with -OR (where R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. Furthermore, examples include, but are not limited to, those in which the phosphate or hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group. In addition, BNA (LNA), in which the conformation of the sugar portion of the nucleotide is fixed to the N-type by cross-linking the 2' oxygen and 4' carbon atoms of the sugar portion, can also be preferably used.

[0030] In this specification, a mutation may specifically refer not only to the deletion, substitution, insertion, or addition of amino acids, but also to the addition of other amino acid sequences, such as the addition of other protein domains.

[0031] In this specification, the position of an amino acid in an amino acid sequence may be indicated by a single-letter amino acid designation followed by the amino acid number, counted from the N-terminal amino acid. For example, "R489" indicates arginine, the 489th amino acid from the N-terminus. In this specification, the amino acid number for Aux / IAA family proteins or their DII domain-containing fragments is the amino acid number counted from the N-terminus of the full-length amino acid sequence of the Aux / IAA family protein.

[0032] In this specification, amino acid substitution mutations may be indicated by the single letter notation of the amino acid to be substituted, the amino acid number counted from the N-terminal amino acid, and the single letter notation of the amino acid after the substitution. For example, "R489G" indicates a substitution mutation of arginine, the 489th amino acid from the N-terminus, to glycine.

[0033] In this specification, molecular weight is defined as the sum of the atomic weights of the atoms that make up the molecule.

[0034] In this specification, there are no particular limitations on the types of plants. Examples include angiosperms (dicotyledonous plants, monocotyledonous plants, etc.), gymnosperms, mosses, ferns, and other plants. Specific examples include tomatoes, bell peppers, chili peppers, eggplants, cucumbers, pumpkins, melons, watermelons, and other gourds, cabbage, broccoli, Chinese cabbage, celery, parsley, lettuce, and other raw and spicy vegetables, onions, garlic, and other alliums, soybeans, peanuts, green beans, peas, adzuki beans, and other legumes, strawberries and other fruit vegetables, radishes, turnips, carrots, burdock, and other taproots, taro, cassava, potatoes, sweet potatoes, yams, and other tubers, asparagus, spinach, and other vegetables. Examples include soft vegetables such as sedge and parsley, flowers such as lisianthus, stock, carnation, and chrysanthemum, grains such as rice and corn, grasses such as bentgrass and Korean lawn grass, oil crops such as rapeseed and peanuts, sugar crops such as sugarcane and sugar beet, fiber crops such as cotton and rush, fodder crops such as clover, sorghum, and dent corn, deciduous fruit trees such as apples, pears, grapes, and peaches, citrus fruits such as Satsuma mandarins, lemons, and grapefruits, and woody plants such as azaleas, rhododendrons, and cedars.

[0035] 2. Mutants In one embodiment, the present invention relates to a TIR1 family protein mutant obtained by mutation in a TIR1 family protein, comprising a substitution mutation a1 of R489 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding arginine in another amino acid sequence, and / or a substitution mutation a2 of L84 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding leucine in another amino acid sequence. (This may also be referred to herein as "the TIR1 family protein mutant of the present invention.") Furthermore, in one embodiment, the present invention relates to an Aux / IAA family protein mutant or DII domain-containing fragment mutant obtained by mutation in an Aux / IAA family protein or a DII domain-containing fragment thereof, comprising a substitution mutation b of G85 in the amino acid sequence shown in SEQ ID NO: 8 or the corresponding glycine in another amino acid sequence. (This may also be referred to herein as "the Aux / IAA family protein mutant or DII domain-containing fragment mutant of the present invention.") Furthermore, the TIR1 family protein variants of the present invention and the Aux / IAA family protein variants or DII domain-containing fragment variants of the present invention may be collectively referred to as "variants of the present invention." These will be explained below.

[0036] TIR1 family proteins are F-box proteins that form the E3 ubiquitinating enzyme complex (SCF complex) in the ubiquitin / proteasome system of protein degradation, and are unique to plants. TIR1 family proteins are receptors for the growth hormone auxin, and are known to recognize Aux / IAA family proteins, which are inhibitors of the auxin signaling pathway, and degrade target polypeptides upon receiving auxin.

[0037] The TIR1 family of proteins is not limited to any plant-derived TIR1 family protein. Furthermore, the type of plant from which they originate is also not limited; examples include Arabidopsis thaliana, rice, zinnia, pine, ferns, and Physcomitrella patens. Specific examples of TIR1 family proteins include TIR1 protein, AFB1 protein, AFB2 protein, AFB3 protein, AFB4 protein, and AFB5 protein.

[0038] Examples of TIR1 family proteins include Arabidopsis thaliana TIR1 protein (SEQ ID NO: 1), Arabidopsis thaliana AFB1 protein (SEQ ID NO: 2), Arabidopsis thaliana AFB2 protein (SEQ ID NO: 3), Arabidopsis thaliana AFB3 protein (SEQ ID NO: 4), Arabidopsis thaliana AFB4 protein (SEQ ID NO: 5), and Arabidopsis thaliana AFB5 protein (SEQ ID NO: 6). TIR1 family proteins in other species are either already known or can be easily identified by identity analysis with known TIR1 family proteins.

[0039] TIR1 family proteins are proteins containing an amino acid sequence that has 70% or more identity with the amino acid sequence shown in, for example, any of SEQ ID NOs: 1 to 6. These proteins can be Arabidopsis thaliana TIR1 protein orthologs, Arabidopsis thaliana AFB1 protein orthologs, Arabidopsis thaliana AFB2 protein orthologs, Arabidopsis thaliana AFB3 protein orthologs, Arabidopsis thaliana AFB4 protein orthologs, or Arabidopsis thaliana AFB5 protein orthologs. TIR1 family proteins can be wild-type or endogenous within plant cells. The above identity is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 99% or more, and especially preferably 100%.

[0040] TIR1 family proteins preferably contain consensus sequence a, shown in SEQ ID NO: 7. Consensus sequence a is a sequence containing the target amino acid and surrounding amino acids of substitution mutation a1, described later. Including this sequence makes it easier to exert the effects of substitution mutation a1 (reduction of the impact on endogenous plant Aux / IAA family proteins, and improvement of the ability to interact with the Aux / IAA family protein mutant or DII domain-containing fragment mutant of the present invention). Consensus sequence a is K485-D490 of the amino acid sequence (Arabidopsis thaliana TIR1 protein) shown in SEQ ID NO: 1, and TIR1 family proteins may contain consensus sequence a at a position corresponding to K485-D490 of the amino acid sequence (Arabidopsis thaliana TIR1 protein) shown in SEQ ID NO: 1.

[0041] Note that "corresponding positions" indicate that the two sequences are at the same position on the alignment sequence when compared using BLAST (default settings). For example, the position corresponding to K485-D490 in amino acid sequence A means that, in the alignment sequence obtained by comparing amino acid sequence A and amino acid sequence B, the position in amino acid sequence B is the same as K485-D490 in amino acid sequence A.

[0042] The TIR1 family protein variants of the present invention are formed by mutations in the TIR1 family protein, and the mutations include a substitution mutation a1 of R489 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding arginine in another amino acid sequence, and / or a substitution mutation a2 of L84 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding leucine in another amino acid sequence.

[0043] Other amino acid sequences are those of TIR1 family proteins that consist of amino acid sequences other than the one shown in Sequence ID No. 1.

[0044] The corresponding arginine in other amino acid sequences is shown in the alignment sequence obtained by comparing the amino acid sequence shown in SEQ ID NO: 1 with other amino acid sequences using BLAST (default settings), and in the other amino acid sequences, it represents the arginine at the same position as R489 in the amino acid sequence shown in SEQ ID NO: 1.

[0045] The mutated amino acid of substitution mutation a1 is preferably an amino acid with a smaller molecular weight than arginine, from the viewpoint of making it easier to exert the effects of substitution mutation a1 (reduction of the effect on endogenous plant Aux / IAA family proteins, and improvement of the ability to interact with the Aux / IAA family protein mutant or DII domain-containing fragment mutant of the present invention). Examples of mutated amino acids include glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, and phenylalanine, and preferably glycine, alanine, valine, threonine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, and glutamic acid, and more preferably glycine, alanine, and glutamic acid. In one embodiment, the mutated amino acids are preferably aliphatic amino acids such as glycine, alanine, valine, leucine, and isoleucine; or acidic amino acids such as aspartic acid and glutamic acid. In another embodiment, the mutated amino acids are preferably amino acids other than basic amino acids (arginine, lysine, and histidine).

[0046] From the viewpoint of making it easier to exert the effects of substitution mutation a2 (reduction of the impact on endogenous plant Aux / IAA family proteins, and improvement of the ability to interact with the Aux / IAA family protein mutant or DII domain-containing fragment mutant of the present invention), the mutated amino acid is preferably an amino acid with a smaller molecular weight than arginine or an acidic amino acid. Examples of mutated amino acids include glycine, alanine, serine, proline, valine, threonine, cysteine, isoleucine, aspartic acid, and glutamic acid, with glycine, alanine, aspartic acid, and glutamic acid being preferred, and alanine and aspartic acid being more preferred.

[0047] The TIR1 family protein variants of the present invention may include other mutations besides substitution mutations a1 and a2, insofar as they can interact with the Aux / IAA family protein variants or DII domain-containing fragment variants of the present invention.

[0048] Other preferred mutations include those that reduce auxin receptivity (mutation x). By using an auxin derivative that is relatively highly receptive to the auxin-receptor-reducing TIR1 family protein into which mutation x has been introduced, it is possible to further reduce the influence of endogenous plant auxin while effectively inducing interaction between the TIR1 family protein mutant and the Aux / IAA family protein mutant or its DII domain-containing fragment mutant using the auxin derivative.

[0049] Mutation x can be designed according to known information. For example, a previously published paper (Nature, Vol 446, 5 April 2007, pp640-645) analyzed the interaction region between auxin (indole-3-acetic acid) and its receptor (TIR1), and reported that the phenylalanine residue in loop 2 of TIR1 (for example, in SEQ ID NO: 1, the 79th and 82nd amino acid residues from the N-terminus (phenylalanine residues)) interact with the benzene ring in the auxin indole ring. From this, auxin receptivity can be reduced by substituting the amino acid residue in the auxin receptor that interacts with the benzene ring in the auxin indole ring (preferably the phenylalanine residue in loop 2 of TIR1 (in SEQ ID NO: 1, the 79th and / or 82nd amino acid residues from the N-terminus (phenylalanine residues), or the amino acid residue corresponding to the 79th and / or 82nd amino acid residues from the N-terminus of SEQ ID NO: 1 in the ortholog or paralog of TIRI in SEQ ID NO: 1)) with other amino acid residues.

[0050] Corresponding amino acid residues are those amino acid residues that are aligned at the same position when two amino acid sequences are compared using BLAST's default parameters.

[0051] The "other amino acid residue" that is the substituted amino acid residue in mutation x is preferably an amino acid residue whose side chain molecular weight is smaller than the molecular weight of the side chain of the phenylalanine residue. Examples of such amino acid residues include hydrophobic amino acid residues such as glycine residues, alanine residues, valine residues, isoleucine residues, leucine residues, methionine residues, and cysteine ​​residues; serine residues, etc. Preferably, glycine residues, alanine residues, valine residues, isoleucine residues, serine residues, etc. are preferred, more preferably glycine residues, alanine residues, serine residues, etc., even more preferably alanine residues, serine residues, etc., and even more preferably alanine residues.

[0052] Auxins are plant hormones that regulate plant growth. While indole-3-acetic acid is a typical example of an auxin, other examples of auxins with the above-mentioned effects include 4-chloroindole-3-acetic acid, naphthaleneacetic acid, α-naphthylacetamide, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid.

[0053] The auxin derivative is not particularly limited, as long as it can bind to both the TIR1 family protein variant of the present invention and the Aux / IAA family protein variant or its DII domain-containing fragment variant of the present invention, thereby inducing an interaction between the two.

[0054] Auxin derivatives that exhibit relatively high acceptivity to auxin-receptor-reduced TIR1 family proteins are known, as shown in, for example, Patent Document 1. In one embodiment, such auxin derivatives have the general formula (1):

[0055] [ka]

[0056] [In the formula, n and m are the same or different, and represent 0 or 1. R 1 and R 2 These are the same or different, and each represents a hydrogen atom, an optionally substituted aryl group, or an optionally substituted alkyl group (however, R 1 and R 2 (Except when all are hydrogen atoms at the same time). 3 and R 4 One side represents a carboxyalkyl group, and the other represents a hydrogen atom. X represents -NH- or -CH=CH-. It is a compound represented by [formula].

[0057] n is preferably 0.

[0058] m is preferably 0.

[0059] R 1 or R 2 The aryl group represented by is not particularly limited, but those having 6 to 50 carbon atoms are preferred, those having 6 to 30 carbon atoms are more preferred, those having 6 to 20 carbon atoms are still more preferred, those having 6 to 12 carbon atoms are even more preferred, and those having 6 to 8 carbon atoms are particularly preferred. Specific examples of such aryl groups include a phenyl group, naphthyl group, phenylalkyl group (e.g., benzyl group, phenethyl group, etc.), biphenyl group, pentalenyl group, indenyl group, anthranyl group, tetracenyl group, pentacenyl group, pyrenyl group, perylenyl group, fluorenyl group, phenanthryl group, etc., preferably a phenyl group, naphthyl group, phenylalkyl group, biphenyl group, etc., more preferably a phenyl group, naphthyl group, phenylalkyl group, etc., still more preferably a phenyl group, benzyl group, etc., and even more preferably a phenyl group.

[0060] R 1 or R 2 The substituent that the aryl group represented by may have is not particularly limited, and examples thereof include an optionally substituted alkyl group, an optionally substituted alkoxy group, a halogen atom (F, Br, Cl, etc.), an optionally substituted aryl group, an optionally substituted aryloxy group, a hydroxyl group, a heteroatom-containing group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, -COOR" (R" is a hydrogen atom or a hydrocarbon group), etc., preferably an optionally substituted alkyl group, an optionally substituted alkoxy group, a halogen atom, an optionally substituted aryl group, an optionally substituted aryloxy group, etc., and more preferably an optionally substituted alkyl group, an optionally substituted alkoxy group, a halogen atom, etc.

[0061] R 1 or R 2The alkyl group that the aryl group represented by may have or may be substituted is not particularly limited, and examples include linear, branched, or cyclic alkyl groups having 1 to 20 carbon atoms, preferably 1 to 12, more preferably 1 to 6, even more preferably 1 to 3, and even more preferably 1 carbon atom, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, perfluoromethyl, perfluoroethyl, and cyclohexyl groups.

[0062] R 1 or R 2 The alkoxy group that the aryl group represented by may have or may be substituted is not particularly limited and includes linear or branched alkoxy groups having 1 to 20 carbon atoms, preferably 1 to 12, more preferably 1 to 6, even more preferably 1 to 3, and even more preferably 1 carbon atom, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted alkyl groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, perfluoromethoxy, and perfluoroethoxy groups.

[0063] R 1 or R 2 The halogen atoms that the aryl group represented by the symbol may have are preferably F, Cl, and the like.

[0064] R 1 or R 2The aryl group that the aryl group represented by may have or may be substituted is not particularly limited, and examples include aryl groups having 6 to 12 carbon atoms, preferably 6 to 8 carbon atoms, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted aryl groups include phenyl, naphthyl, benzyl, and phenethyl groups.

[0065] R 1 or R 2 The aryloxy group that the aryl group represented by may have or may be substituted is not particularly limited, and examples include aryloxy groups having 6 to 12 carbon atoms, preferably 6 to 8 carbon atoms, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted aryloxy groups include phenoxy, naphthoxy, benzyloxy, and phenethyloxy groups.

[0066] R 1 or R 2 The heteroatom-containing group that the aryl group represented by may have is preferably a linear, branched, or cyclic group having at least one heteroatom such as a nitrogen atom (N), oxygen atom (O), sulfur atom (S), boron atom (B), phosphorus atom (P), or silicon atom (Si), particularly at least one of a nitrogen atom (N), oxygen atom (O), or sulfur atom (S). Specifically, examples include cyano(-CN) groups, nitro(-NO2) groups, amino groups, and groups obtained by removing one hydrogen atom from heterocycles such as furan rings, thiophene rings, pyrrole rings, silole rings, borol rings, phosphole rings, oxazole rings, thiazole rings, pyridine rings, pyridazine rings, pyrimidine rings, and pyrazine rings. Groups obtained by removing one hydrogen atom from fused rings (thienothiophene rings, quinoline rings, etc.) of the above heterocycles or benzene rings can also be used.

[0067] R 1 or R 2 The alkenyl group that the aryl group represented by may have or may be substituted is not particularly limited and includes linear, branched, or cyclic alkenyl groups having 2 to 20 carbon atoms, preferably 2 to 12, more preferably 2 to 6, and even more preferably 2 to 3 carbon atoms, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted alkenyl groups include vinyl, allyl, 1-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl groups.

[0068] R 1 or R 2 The alkynyl group that the aryl group represented by may have or may be substituted is not particularly limited and includes linear, branched, or cyclic alkynyl groups having 2 to 20 carbon atoms, preferably 2 to 12, more preferably 2 to 6, and even more preferably 2 to 3 carbon atoms, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and phenylacetylyl groups.

[0069] R 1 or R 2 The aryl group represented by -COOR'' may have a hydrogen atom or a hydrocarbon group, and a hydrogen atom or the alkyl group described above is preferred. Specifically, examples of -COOR'' include -COOH, -COOCH3, -COOC2H5, -COOC3H7, -COOC(CH3)2, -COOC4H9, -COOCH(CH3)C2H5, -COOCH2CH(CH3)2, and -COOC(CH3)3.

[0070] R 1 or R 2The number of substituents that the aryl group represented by may have is not particularly limited, for example, 0 to 6, preferably 1 to 3, and more preferably 1 to 2. In a preferred embodiment of the present invention, R 1 or R 2 When the substituents that the aryl group represented by may have are at least one selected from the group consisting of alkyl groups and halogen atoms, the number of substituents is preferably two, and when the substituent is an alkoxy group, the number of substituents is preferably one.

[0071] R 1 or R 2 If the aryl group represented by has two or more substituents, two adjacent substituents may bond to each other to form a ring. Forming a ring means, for example, if the aryl group is a phenyl group, then, for example, in formula (1):

[0072] [ka]

[0073] [In the formula, R' and R'' are R 1 This indicates substituents that the aryl group represented by may have. n is the same as above. The base shown by is, for example, in formula:

[0074] [ka]

[0075] [In the formula, n is the same as above.] This means that it is the group represented by [the symbol].

[0076] R 1 or R 2 The adamantyl group represented by is not particularly limited, and examples include 1-adamantyl groups and 2-adamantyl groups. Among these, the 1-adamantyl group is preferred.

[0077] R 1 or R2 The adamantyl group shown may have substituents such as R 1 or R 2 Examples of substituents that may be present on the aryl group shown can be found.

[0078] R 1 or R 2 The number of substituents that the adamantyl group represented by may have is not particularly limited, for example, 0 to 6, preferably 0 to 3, and more preferably 0.

[0079] R 1 or R 2 The alkyl group represented by is not particularly limited, but examples include linear, branched, or cyclic (preferably cyclic) alkyl groups having 1 to 20 carbon atoms, preferably 3 to 20, more preferably 4 to 15, and even more preferably 6 to 12 carbon atoms. Specific examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and cyclohexyl groups.

[0080] R 1 or R 2 The substituents that the alkyl group shown may have include R 1 or R 2 Examples of substituents that may be present on the aryl group shown can be found.

[0081] R 1 or R 2 The number of substituents that the alkyl group represented by may have is not particularly limited, for example, 0 to 6, preferably 0 to 3, and more preferably 0. 1 or R 2The heterocyclic group represented by is not particularly limited, but examples include groups obtained by removing one hydrogen atom from heterocyclic rings such as furan rings, thiophene rings, pyrrole rings, silole rings, borol rings, phosphole rings, oxazole rings, thiazole rings, pyridine rings, pyridazine rings, pyrimidine rings, and pyrazine rings. In addition, groups obtained by removing one hydrogen atom from fused rings of the above heterocyclic rings or from fused rings of these heterocyclic rings with benzene rings, etc. (such as benzothiephene rings, thienothiophene rings, and quinoline rings) can also be used.

[0082] R 1 or R 2 The substituents that the heterocyclic group shown may have include R 1 or R 2 Examples of substituents that may be present on the aryl group shown can be found.

[0083] R 1 or R 2 The number of substituents that the heterocyclic group represented by may have is not particularly limited, for example, 0 to 6, preferably 0 to 3, and more preferably 0.

[0084] R 1 -(O) n - and R 2 -(O) m - Preferably, one of them is a hydrogen atom. 2 -(O) m The one that is hyphenated is a hydrogen atom.

[0085] R 1 -(O) n -, R 2 -(O) m Specifically, in addition to hydrogen atoms, preferably

[0086] [ka]

[0087] [ka]

[0088] [ka]

[0089] These are some examples, and more

[0090] [ka]

[0091] These are some examples.

[0092] Also, R 1 -(O) n - Preferably, in addition to hydrogen atoms,

[0093] [ka]

[0094] [ka]

[0095] Examples include R 2 -(O) m - Preferably, in addition to hydrogen atoms,

[0096] [ka]

[0097] These are some examples.

[0098] R 3 and R 4 One of them is a carboxyalkyl group, and the other is a hydrogen atom. In a preferred embodiment, R 3 is a hydrogen atom, and R 4This is a carboxyalkyl group. In carboxyalkyl groups, the alkyl group is one of the alkyl groups listed above. That is, carboxymethyl group (-CH2COOH), carboxyethyl group (-C2H4COOH), carboxypropyl group (-C3H6COOH), carboxybutyl group (-C4H8COOH), carboxypentyl group (-C5H 10 COOH), carboxyhexyl group (-C6H 12 Examples include COOH.

[0099] X is preferably -NH-.

[0100] In one embodiment of the present invention, the compound represented by general formula (1) is preferably general formula (1A):

[0101] [ka]

[0102] [where n, m, R 1 , R 2 , R 3 , and R 4 This is the same as above. Examples of compounds represented by the formula (1A1) include, and more preferably, the general formula (1A1):

[0103] [ka]

[0104] [where n, m, R 1 , R 2 , and R 3 The same applies as above. R 4 It is a carboxyalkyl group. Examples of compounds represented by the formula (1A1a) include, and more preferably, compounds represented by the general formula (1A1a):

[0105] [ka]

[0106] [wherein n and R 1 are as defined above. R 4 is a carboxyalkyl group.]] includes compounds represented by

[0107] In another aspect of the present invention, the compound represented by general formula (1) satisfies the following aspect: R 2 -(O) m - is a hydrogen atom, and when n is 0, R 1 is (a) an optionally substituted adamantyl group, (b) an aryl group optionally substituted with at least one substituent selected from the group consisting of aryl groups and aryloxy groups, or optionally substituted with an alkyl group, an alkoxy group, or a halogen atom and at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups, or (c) an alkyl group having 5 to 20 carbon atoms optionally substituted with at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups; or when n is 1, R 1 is (d) an optionally substituted adamantyl group, (e) an optionally substituted phenyl group, an optionally substituted naphthyl group, or a benzyl group substituted with at least one substituent selected from the group consisting of alkyl groups, halogen atoms, aryl groups, and aryloxy groups, or (f) an alkyl group having 6 to 20 carbon atoms optionally substituted with at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups; or R 1 -(O) n - is a hydrogen atom, and when m is 0, R 2 is (g) an optionally substituted adamantyl group, (h) At least one substituent selected from the group consisting of alkoxy groups, aryl groups, and aryloxy groups, or an aryl group substituted with an alkyl group or halogen atom and at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups, (i) an alkyl group having 4 to 20 carbon atoms, which may be substituted; or If m is 1, then R 2 teeth (j) Adamantyl group which may be substituted, (k) A phenyl group or naphthyl group which may be substituted with at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups, or (l) A C4-C20 alkyl group which may be substituted. The configuration is preferable.

[0108] Other mutations besides mutation x can be, for example, amino acid deletions, substitutions, insertions, or additions. The number of amino acids in such mutations can be, for example, 1 to 50, 1 to 20, 1 to 10, or 1 to 5.

[0109] Other mutations besides mutation x may include, for example, the addition of another protein domain. The other protein domain is not particularly limited, as long as it does not affect the domain structure of the TIR1 family protein. Examples of other protein domains include enzyme proteins, transcription factor proteins, structural proteins, muscle proteins, hormone proteins, metal ion and / or nutrient-binding proteins, receptor proteins, antibodies, luminescent proteins, tag proteins, or fragments thereof. The target polypeptide may be the full-length protein or a combination of multiple protein fragments linked together.

[0110] Aux / IAA family proteins are transcription factor proteins that interact with auxin-receiving TIR1 family proteins via their DII domain and are degraded via ubiquitination by the SCF complex containing TIR1 family proteins. They are plants-specific proteins.

[0111] The Aux / IAA family proteins are not limited to any plant-derived Aux / IAA family proteins. Furthermore, the type of plant from which they originate is also not limited; examples include Arabidopsis thaliana, rice, zinnia, pine, ferns, and Physcomitrella patens. Specific examples of Aux / IAA family proteins include IAA1, IAA2, IAA3, IAA4, IAA5, IAA6, IAA7, IAA8, IAA9, IAA10, IAA11, IAA12, IAA13, IAA14, IAA15, IAA16, IAA17, IAA18, IAA19, IAA26, IAA27, IAA28, and IAA29.

[0112] Examples of Aux / IAA family proteins include Arabidopsis thaliana IAA1 protein (SEQ ID NO: 9), Arabidopsis thaliana IAA2 protein (SEQ ID NO: 10), Arabidopsis thaliana IAA3 protein (SEQ ID NO: 11), Arabidopsis thaliana IAA4 protein (SEQ ID NO: 12), Arabidopsis thaliana IAA5 protein (SEQ ID NO: 13), Arabidopsis thaliana IAA6 protein (SEQ ID NO: 14), Arabidopsis thaliana IAA7 protein (SEQ ID NO: 8), Arabidopsis thaliana IAA8 protein (SEQ ID NO: 15), Arabidopsis thaliana IAA9 protein (SEQ ID NO: 16), Arabidopsis thaliana IAA10 protein (SEQ ID NO: 17), Arabidopsis thaliana IAA11 protein (SEQ ID NO: 18), and Arabidopsis thaliana IA Examples include A12 protein (SEQ ID NO: 19), Arabidopsis thaliana IAA13 protein (SEQ ID NO: 20), Arabidopsis thaliana IAA14 protein (SEQ ID NO: 21), Arabidopsis thaliana IAA15 protein (SEQ ID NO: 22), Arabidopsis thaliana IAA16 protein (SEQ ID NO: 23), Arabidopsis thaliana IAA17 protein (SEQ ID NO: 24), Arabidopsis thaliana IAA18 protein (SEQ ID NO: 25), Arabidopsis thaliana IAA19 protein (SEQ ID NO: 26), Arabidopsis thaliana IAA26 protein (SEQ ID NO: 27), Arabidopsis thaliana IAA27 protein (SEQ ID NO: 28), Arabidopsis thaliana IAA28 protein (SEQ ID NO: 29), and Arabidopsis thaliana IAA29 protein (SEQ ID NO: 30). Aux / IAA family proteins in other species are either already known or can be easily identified by identity analysis with known Aux / IAA family proteins.

[0113] Aux / IAA family proteins are proteins containing amino acid sequences that have 70% or more identity with the amino acid sequence shown in, for example, any of Sequence IDs 8-30. These proteins include Arabidopsis thaliana IAA1 protein ortholog, Arabidopsis thaliana IAA2 protein ortholog, Arabidopsis thaliana IAA3 protein ortholog, Arabidopsis thaliana IAA4 protein ortholog, Arabidopsis thaliana IAA5 protein ortholog, Arabidopsis thaliana IAA6 protein ortholog, Arabidopsis thaliana IAA7 protein ortholog, Arabidopsis thaliana IAA8 protein ortholog, Arabidopsis thaliana IAA9 protein ortholog, Arabidopsis thaliana IAA10 protein ortholog, Arabidopsis thaliana IAA11 protein ortholog, and Arabidopsis thaliana IAA12 protein The ortholog may be an ortholog of Arabidopsis thaliana IAA13 protein, Arabidopsis thaliana IAA14 protein, Arabidopsis thaliana IAA15 protein, Arabidopsis thaliana IAA16 protein, Arabidopsis thaliana IAA17 protein, Arabidopsis thaliana IAA18 protein, Arabidopsis thaliana IAA19 protein, Arabidopsis thaliana IAA26 protein, Arabidopsis thaliana IAA27 protein, Arabidopsis thaliana IAA28 protein, or Arabidopsis thaliana IAA29 protein. The Aux / IAA family proteins may be wild-type, intrinsically occurring in plant cells. The above identity is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 99% or more, and especially preferably 100%.

[0114] A DII domain-containing fragment is a fragment of an Aux / IAA family protein that contains a DII domain within an Aux / IAA family protein. The location of the DII domain within an Aux / IAA family protein is known or can be identified based on publicly available information, for example, by identity analysis. DII domain-containing fragments can interact with auxin-receiving TIR1 family proteins.

[0115] Examples of DII domain-containing fragments include the DII domain-containing fragment in Arabidopsis thaliana IAA1 protein (SEQ ID NO: 32), the DII domain-containing fragment in Arabidopsis thaliana IAA2 protein (SEQ ID NO: 33), the DII domain-containing fragment in Arabidopsis thaliana IAA3 protein (SEQ ID NO: 34), the DII domain-containing fragment in Arabidopsis thaliana IAA4 protein (SEQ ID NO: 35), the DII domain-containing fragment in Arabidopsis thaliana IAA5 protein (SEQ ID NO: 36), and the DII domain-containing fragment in Arabidopsis thaliana IAA6 protein. II domain-containing fragment (SEQ ID NO: 37), DII domain-containing fragment within Arabidopsis thaliana IAA7 protein (SEQ ID NO: 31), DII domain-containing fragment within Arabidopsis thaliana IAA8 protein (SEQ ID NO: 38), DII domain-containing fragment within Arabidopsis thaliana IAA9 protein (SEQ ID NO: 39), DII domain-containing fragment within Arabidopsis thaliana IAA10 protein (SEQ ID NO: 40), DII domain-containing fragment within Arabidopsis thaliana IAA11 protein (SEQ ID NO: 41), within Arabidopsis thaliana IAA12 protein DII domain-containing fragment of (SEQ ID NO: 42), DII domain-containing fragment of Arabidopsis thaliana IAA13 protein (SEQ ID NO: 43), DII domain-containing fragment of Arabidopsis thaliana IAA14 protein (SEQ ID NO: 44), DII domain-containing fragment of Arabidopsis thaliana IAA15 protein (SEQ ID NO: 45), DII domain-containing fragment of Arabidopsis thaliana IAA16 protein (SEQ ID NO: 46), DII domain-containing fragment of Arabidopsis thaliana IAA17 protein (SEQ ID NO: 47), Arabidopsis thaliana IAA18 Examples include DII domain-containing fragments within proteins (SEQ ID NO: 48), DII domain-containing fragments within Arabidopsis thaliana IAA19 protein (SEQ ID NO: 49), DII domain-containing fragments within Arabidopsis thaliana IAA26 protein (SEQ ID NO: 50), DII domain-containing fragments within Arabidopsis thaliana IAA27 protein (SEQ ID NO: 51), DII domain-containing fragments within Arabidopsis thaliana IAA28 protein (SEQ ID NO: 52), and DII domain-containing fragments within Arabidopsis thaliana IAA29 protein (SEQ ID NO: 53). DII domain-containing fragments in other species are either already known or can be easily identified through identity analysis with known DII domain-containing fragments.

[0116] The DII domain-containing fragment is a protein containing an amino acid sequence that has 70% or more identity with the amino acid sequence shown in, for example, any of SEQ ID NOs: 31 to 53. The above identity is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 99% or more, and especially preferably 100%.

[0117] The Aux / IAA family protein and / or the DII domain-containing fragment preferably contains consensus sequence b shown in SEQ ID NO: 54. Consensus sequence b is a sequence containing the target amino acid and surrounding amino acids of substitution mutation b described later, and including it makes it easier to exert the effects of substitution mutation b (reduction of the effect on endogenous plant TIR1 family proteins, improvement of the ability to interact with the TIR1 family protein variant of the present invention). Consensus sequence b is G85-P88 of the amino acid sequence (Arabidopsis thaliana IAA7 protein) shown in SEQ ID NO: 8, and the Aux / IAA family protein and / or the DII domain-containing fragment may contain consensus sequence b at the position corresponding to G85-P88 of the amino acid sequence (Arabidopsis thaliana IAA7 protein) shown in SEQ ID NO: 8. The definition of "corresponding position" is as described above.

[0118] The Aux / IAA family protein variant or DII domain-containing fragment variant of the present invention is a mutation of an Aux / IAA family protein or its DII domain-containing fragment, and the mutation includes a substitution mutation b of G85 in the amino acid sequence shown in Sequence ID No. 8 or the corresponding glycine in another amino acid sequence.

[0119] Other amino acid sequences are those of TIR1 family proteins when the Aux / IAA family protein variant or DII domain-containing fragment variant is a protein consisting of an amino acid sequence other than the one shown in Sequence ID No. 8.

[0120] The corresponding glycine in other amino acid sequences is indicated in the alignment sequence obtained by comparing the amino acid sequence shown in SEQ ID NO: 8 with other amino acid sequences using BLAST (default settings), and in the other amino acid sequences, it represents the glycine at the same position as G85 in the amino acid sequence shown in SEQ ID NO: 8.

[0121] The mutated amino acid of substitution mutation b is preferably an amino acid with a molecular weight larger than glycine, from the viewpoint of making it easier to exert the effects of substitution mutation b (reduction of the effect on endogenous plant TIR1 family proteins and improvement of the interaction ability with the TIR1 family protein mutant of the present invention). Examples of mutated amino acids include alanine, serine, proline, valine, threonine, cysteine, isoleucine, leucine, asparagine, aspartic acid, glutamine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan, preferably methionine, histidine, phenylalanine, arginine, tyrosine, and tryptophan, more preferably methionine, phenylalanine, tyrosine, and tryptophan, and particularly preferably tryptophan. Furthermore, examples of mutated amino acids include, in one embodiment, amino acids having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine, and hydrophobic amino acids such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, and tryptophan.

[0122] Furthermore, in one embodiment (particularly when substitution mutation a2 is adopted), the mutated amino acid of substitution mutation b is preferably a basic amino acid, more preferably arginine, lysine, or histidine, and most preferably arginine.

[0123] The Aux / IAA family protein variants or DII domain-containing fragment variants of the present invention may include other mutations besides substitution mutation b, insofar as they are able to interact with the TIR1 family protein variants of the present invention. The definitions of other mutations other than substitution mutation a1 and mutation x described above are applied to such other mutations.

[0124] The variants of the present invention may be drug-linked, insofar as the pairs of variants of the present invention can interact with each other.

[0125] The mutants of the present invention may be chemically modified insofar as they are able to interact with each other.

[0126] The mutants of the present invention have a carboxyl group (-COOH) at the C-terminus, a carboxylate (-COOH) - It may be any of the following: ), amide (-CONH2), or ester (-COOR).

[0127] Here, R in esters can be C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, phenyl, α-naphthyl, etc. 6-12 Aryl group; for example, phenyl-C such as benzyl and phenethyl. 1-2 Alkyl groups; such as α-naphthylmethyl and α-naphthyl-C 1-2 C such as alkyl groups 7-14 Aralkyl groups and pivaloyloxymethyl groups are used.

[0128] In the mutants of the present invention, carboxyl groups (or carboxylates) other than the C-terminus may be amidated or esterified. In this case, the ester may be, for example, the C-terminus ester described above.

[0129] Furthermore, the mutants of the present invention have a protecting group (e.g., a formyl group, an acetyl group, etc.) on the amino group of the N-terminal amino acid residue. 1-6 C such as Alkanoyl 1-6 Protected by an acyl group, etc., the N-terminal glutamine residue which can be cleaved and produced in vivo has been pyroglutamine-oxidized, or the substituent on the side chain of an amino acid within the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) is a suitable protecting group (e.g., formyl group, acetyl group, etc.) 1-6 C such as alkanoyl groups 1-6 This also includes complex proteins such as those protected by acyl groups, or so-called glycoproteins with attached sugar chains.

[0130] The variants of the present invention may be in the form of salts with an acid or a base. The salt is not particularly limited, and either an acidic salt or a basic salt can be used. For example, examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and p-toluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0131] The variants of the present invention may also be in the form of solvates. The solvent is not particularly limited and includes, for example, water, ethanol, glycerol, acetic acid, and the like.

[0132] The variants of the present invention can be readily produced according to known genetic engineering techniques. For example, they can be produced using PCR, restriction enzyme digestion, DNA ligation, in vitro transcription and translation techniques, recombinant protein production techniques, etc.

[0133] 3. Polynucleotides In one embodiment, the present invention relates to a polynucleotide (which may be referred to herein as "the polynucleotide of the present invention") comprising the coding sequence of a variant of the present invention.

[0134] The coding sequence of the mutant of the present invention is not particularly limited, as long as it is a polynucleotide consisting of a base sequence encoding the mutant of the present invention. The coding sequence of the mutant of the present invention may be a single type or a combination of two or more types.

[0135] In one embodiment, the polynucleotide of the present invention preferably has a promoter positioned upstream of the coding sequence of the mutant of the present invention. The promoter is usually positioned so that the mRNA encoding the mutant of the present invention can be expressed. Specific configurations include, for example, a configuration in which the coding sequence of the mutant of the present invention is positioned directly below the 3' end of the promoter (for example, a configuration in which the number of base pairs (bp) between the base at the 3' end of the promoter and the base at the 5' end of the coding sequence of the mutant of the present invention is, for example, 100 bp or less, preferably 50 bp or less). The promoter is not particularly limited and examples include the CaMV35S promoter, the UBQ (ubiquitin) promoter, the NOS promoter, etc.

[0136] In one embodiment, the polynucleotide of the present invention preferably includes a transcription termination signal downstream of the coding sequence of the mutant of the present invention. The termination signal is not particularly limited as long as it is a nucleotide sequence that can terminate transcription from the coding sequence of the mutant of the present invention (and preferably further add a polyA sequence to the mRNA transcribed from the coding sequence of the mutant of the present invention). "Downstream of the coding sequence of the mutant of the present invention" is not particularly limited, but for example, an embodiment may be described in which the number of base pairs (bp) between the 3' end of the coding sequence of the mutant of the present invention and the 5' end of the termination signal is, for example, 500 bp or less, preferably 200 bp or less. Examples of termination signals include the heat shock protein termination signal HspT (Heat shock protein Terminator), NosT (Nopaline synthase Terminator), 35sT (CaMV35S Terminator), etc.

[0137] In one embodiment, the polynucleotide of the present invention preferably has a left border region (LB) of the T-DNA region upstream of the coding sequence of the mutant of the present invention, and a right border region (RB) of the T-DNA region downstream of the coding sequence.

[0138] The polynucleotide of the present invention may include other sequences besides those described above, or further sequences in addition to those described above. The other sequences are not particularly limited, and various known sequences that can be included in subcloning vectors and expression vectors can be used. Examples of such sequences include, for example, origins of replication and drug resistance genes. Examples of drug resistance genes include chloramphenicol resistance genes, tetracycline resistance genes, neomycin resistance genes, erythromycin resistance genes, spectinomycin resistance genes, kanamycin resistance genes, hygromycin resistance genes, puromycin resistance genes, gentamicin resistance genes, phosphinotricin resistance genes, chlorsulfuron resistance genes, methotrexate resistance genes, imidazolinone resistance genes, and glyphosate resistance genes.

[0139] The polynucleotides of the present invention may be in the form of vectors. The type of vector is not particularly limited and includes, for example, binary vectors, plant virus vectors such as tobacco mosaic virus, cucumber mosaic virus, African cassava mosaic virus, apple microglobulin latent virus, barley spotted leaf mosaic virus, bean pod mottle virus, beet curly top virus, brome mosaic virus, cabbage leaf curl virus, cotton leaf crumple virus, cymbidium mosaic virus, grape A virus, pea early browning virus, poplar mosaic virus, potato X virus, rice tungro bacilliform virus, satellite tobacco mosaic virus, tobacco curly shoot virus, tobacco stem necrosis virus, and subcloning vectors.

[0140] The polynucleotides of the present invention can be easily produced according to known genetic engineering techniques. For example, they can be produced using PCR, restriction enzyme digestion, DNA ligation, in vitro transcription, etc.

[0141] 4. Cells, non-human organisms In one embodiment, the present invention relates to a cell or non-human organism (which may be referred to herein as "the cell or non-human organism of the present invention") containing at least one selected from the group consisting of the TIR1 family protein variant of the present invention, the Aux / IAA family protein variant of the present invention, or the DII domain-containing fragment variant, and polynucleotides comprising their coding sequences.

[0142] The cells or non-human organisms of the present invention can be produced by introducing at least one selected from the group consisting of the TIR1 family protein variants of the present invention, the Aux / IAA family protein variants or DII domain-containing fragment variants of the present invention, and polynucleotides containing their coding sequences, into the cells or non-human organisms.

[0143] Cells include plant cells, animal cells (e.g., mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; fish and amphibians such as zebrafish and African clawed frogs; and invertebrates such as C. elegans and fruit flies), fungal cells, and protist cells.

[0144] For non-human organisms, the above description of cell types is applied.

[0145] Since the technology of the present invention has reduced impact on endogenous plant TIR1 family proteins and Aux / IAA family proteins, it can be suitably used in plant cells and plant bodies.

[0146] The method of introduction is not particularly restricted and can be appropriately selected depending on the target of introduction. Examples of introduction methods include the leaf disk method, floral dip method, floral spray method (Agrobacterium methods), particle gun method, virus-mediated nucleic acid delivery, and electroporation. Other examples include calcium phosphate precipitation, DEAE dextran transfection, electroporation, lipofection, and viral vector methods.

[0147] 5. Method In one aspect, the present invention relates to a method (which may be referred to herein as "the method of the present invention") comprising contacting the cells of the present invention or a non-human organism containing said cells with auxin and / or an auxin derivative.

[0148] The method of the present invention can be performed in vitro or in vivo. Furthermore, the method of the present invention may be a method that excludes medical procedures. Furthermore, the method of the present invention may be a method that excludes methods involving humans.

[0149] Auxin and auxin derivatives are defined as described above.

[0150] Auxin and / or auxin derivatives allow the TIR1 family protein variants of the present invention to interact with the Aux / IAA family protein variants or DII domain-containing fragment variants of the present invention. This aspect allows the methods of the present invention to be applied to various methods.

[0151] In one embodiment of the present invention, the method of the present invention may include other protein domains in the TIR1 family protein variant of the present invention, and / or the Aux / IAA family protein variant or DII domain-containing fragment variant of the present invention may include other protein domains.

[0152] In one embodiment of the present invention, if the Aux / IAA family protein variant or DII domain-containing fragment variant of the present invention contains other protein domains (degradation target protein domains), the degradation target protein domains can be degraded by contacting auxin and / or auxin derivatives with the cells or non-human organisms of the present invention.

[0153] In one embodiment of the present invention, by contacting auxin and / or auxin derivatives with the cells or non-human organisms of the present invention, the TIR1 family protein variants (or other protein domains they contain) and the Aux / IAA family protein variants or DII domain-containing fragment variants (or other protein domains they contain) of the present invention can be brought into close proximity or contact. In this more specific embodiment, transcriptional induction is possible. In these uses, if necessary, mutations that suppress target ubiquitination can be introduced into the TIR family proteins. Such mutations can be appropriately designed based on known information.

[0154] Contact with auxin and / or auxin derivatives introduces auxin and / or auxin derivatives into the cell. The concentration of auxin and / or auxin derivatives in the circumcellular fluid (e.g., culture medium) at the time of contact is not particularly limited and can be, for example, 0.01 nM to 1 mM. The lower limit is, for example, 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, or 50 nM, and the upper limit is, for example, 100 μM, 50 μM, 20 μM, 10 μM, 5 μM, 2 μM, 1 μM, or 500 nM. [Examples]

[0155] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0156] Materials and methods Unless otherwise specified in the test examples described later or in the "Brief Description of the Drawings" above, the materials and methods are as follows.

[0157] Pull-down assay The pull-down assay was performed by incubating GST-DII protein and TIR1-FLAG protein bound to glutathione Sepharose beads in assay buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 10% glycerol, 0.1% Tween-20) containing IAA or cvxIAA at the indicated concentration. After washing the beads with the same buffer, the proteins extracted from the beads were subjected to SDS-PAGE (10% acrylamide gel), and immunoblotting analysis was performed using anti-FLAG antibody (Sigma, F3165), SuperSignal WestPico Chemiluminescence reagent (Thermo Scientific), and a Light-Capture cooled CCD camera system (ATTO).

[0158] For the preparation of the GST-DII protein (a protein consisting of GST ligated with a partial-length Arabidopsis thaliana IAA7 (including the DII domain)), IAA7 containing the DII domain (full-length (SEQ ID NO: 8) G33 - S104 (SEQ ID NO: 31)) amplified by PCR was inserted into the BamHI-EcoRI region of the pGEX-2T plasmid, and the GST-fused IAA7-DII protein expressed in E. coli was purified using glutathione Sepharose. The TIR1 protein with a FLAG tag at the C-terminus (Arabidopsis thaliana TIR1: SEQ ID NO: 1) was prepared using a wheat germ extract cell-free system (NUProtein). mRNA was synthesized by reverse transcription using PCR products amplified by first-strand and second-strand PCR according to the product protocol. PCR products were obtained by PCR using specific primers with pGLex313 / TIR1WT and pGLex313 / TIR1F79A as templates. Site-directed mutagenesis of GST-DII and TIR1-FLAG was performed by PCR using mutation primers.

[0159] Plasmids and plant materials for in vivo assays PCR products amplified with PrimeSTAR MAX DNA polymerase (Takara) were ligated to pENTR1A (Invitrogen) or pRED419-amp plasmids using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) or the SLiCE reaction. The cloned fragments were introduced into the plant transformation vector pGWB using the LR clonase II (Invitrogen) reaction. Mutagenesis was performed using the KOD-Plus- Mutagenesis Kit (Toyobo). Arabidopsis thaliana 'Colombia' (Col) was used as the wild type. TIR1 and its mutants, IAA7-DII and its mutant constructs were transformed into Arabidopsis thaliana by floral dipping using Agrobacterium tumefaciens strain GV3101. For the seedling assay, plants were grown in 1 / 2 concentration Murashige Skoog (MS) solid medium containing 2% sucrose and 5 mM MES-KOH pH 5.8. Sterilized seeds were sown in the medium and vernalized by standing in the dark at 4°C for several days, after which they were grown in a 23°C growth chamber under LED light (90-130 μmol m³). -2 s -1 The plants were cultured under a 14-hour light / 10-hour dark photoperiod. Seedlings 5-7 days old were used in the assay. For assays with mature plants, seeds were vernalized by standing them in distilled water in the dark at 4°C for several days, then sown in culture medium, and grown in a growth chamber at 23°C under LED light (90-130 μmol m³). -2 s -1 The plants were grown under a photoperiod of 14 hours of light / 10 hours of darkness. Plants aged 15-16 days were used in the assay.

[0160] Chemical treatment For treatment with IAA (Kanto Chemical), 5-(3-Methoxyphenyl)indole-3-acetic acid (cvxIAA; TCI), or 5-Adamantyl-IAA (Ada-IAA; TCI), seedlings aged 5-7 days were transferred to a 10 mM MES-NaOH pH 5.5 solution containing 1 / 2 concentration MS medium solution with each IAA, and cultured for 1 hour under growth conditions with gentle swirling shaking. For IAA treatment by spraying, a 10 mM MES-NaOH pH 5.5 solution containing each IAA was directly sprayed onto 16-day-old plants grown in soil. The IAA concentrations are shown in the legend of each figure. In the proteasome inhibitor treatment, seedlings were placed in a 10 ml syringe with a 10 mM MES-NaOH pH 5.5 solution containing 10 μM MG132 or 10 μM Bortezomib (BTZ). The solution was then permeated into the seedlings using the negative pressure created by pulling the syringe plunger, and the seedlings were incubated under growth lighting conditions for 4–10 hours. "Mock" refers to a solvent that does not contain the compound, specifically 0.1% (v / v) DMSO.

[0161] Fluorescence detection using a confocal microscope DII WT -GFP (a protein consisting of GFP linked to a partial-length Arabidopsis thaliana IAA7 (including the DII domain)) or DII G85WConfocal images for quantifying the fluorescence signal of -GFP (a protein formed by ligating a partial-length Arabidopsis thaliana IAA7 (including the DII domain) mutant (a mutant of the amino acid corresponding to G85 in the full-length version, with W) to GFP) were acquired using a C2 system (Nikon). GFP fluorescence was detected at 500-550 nm (bandpass filter RPB500-550; Omega Optical) after excitation with a 489.6 nm diode laser. Chlorophyll autofluorescence was detected at 660-720 nm after excitation with a 489.6 nm diode laser. Hypocotyl and mature leaf cells were observed with a water-immersion objective lens (CFI Apochromat LWD Lambda S 40XC, NA = 1.2; Nikon). 3D region z-stack images were acquired (317.44 × 317.44 × 20 μm each for the hypocotyl and 317.44 × 317.44 × 10 μm each for mature leaves). For roots, cells in a region approximately 5 mm from the root tip were observed using an objective lens (Plan Apo Lambda 10x, NA = 0.45; Nikon), and 3D region Z-stack images (512 × 512 × 40 μm each) were acquired. The volume of GFP fluorescence was measured using Imaris software.

[0162] DII G85W -GFP degradation was observed using time-lapse imaging with the LSM900 system (Carl Zeiss). The hypocotyl was observed using a water-immersion objective lens (C-apochromat 40x, NA = 1.2; Carl Zeiss), and a time-series of z-stack images of a 3D region (319.45 × 319.45 × 40 μm) was captured every minute. The root tip was observed using a plan-apochromat objective lens (Plan-apochromat 20x, NA = 0.8; Carl Zeiss), and a time-series of z-stack images of a 3D region (319.45 × 319.45 × 60 μm) was captured every minute. GFP fluorescence was excited with a 488 nm diode laser and then detected at 410-617 nm. Chlorophyll autofluorescence was excited with a 488 nm diode laser and then detected at 650-700 nm.

[0163] Immunoblot detection in total protein extract For protein analysis, approximately 20-40 seedlings were frozen in liquid nitrogen. For protein analysis of sprayed plants, the third rosette leaf from 16-day-old plants was frozen. The frozen plants or leaves were homogenized using a crushing device (Qiagen) and zirconium beads, and then suspended in a homogenization buffer containing 50 mM HEPES-NaOH pH 7.5, 100 mM DTT, 10% (v / v) glycerol, 1% (v / v) Triton X-100, 2% (w / v) SDS, and 1% (v / v) protease inhibitor cocktail (Nacalai). After centrifugation at 20,630 g for 10 minutes at 4°C, the amount of protein in the supernatant was measured using Pierce 660-nm Protein Assay Reagent (Pierce) containing Ionic Detergent Compatibility Reagent (Pierce). The supernatant was incubated at 95°C for 5 minutes to denature it. Equal volumes of protein were subjected to SDS-PAGE using TGX Stain-Free FastCast acrylamide gel (Bio-Rad). Proteins in the gel were detected by UV detection (stain-free gel) and transferred to a nitrocellulose membrane (Trans-blot turbo transfer pack; Bio-Rad). Anti-GFP B-2 antibody (1:1000, sc-9996; SantaCruz), anti-β-actin antibody (1:5000, 60008-1-IG, Proteintech), and anti-FLAG antibody (1:10000, M185-3MS; MBL) were used as primary antibodies. Anti-mouse HRP secondary antibody (1:10000, NA931; Cytiba) was used as the secondary antibody. Chemiluminescent signals, colored using SuperSignal West Dura Extended Duration Substrate (Pierce), were detected using the ChemiDoc MP system (Bio-Rad). Image processing and band intensity quantification were performed using Image Lab Software (Bio-Rad).

[0164] Quantitative reverse transcription (qRT)-PCR analysis To evaluate the transcription of auxin marker genes, RNA samples were prepared from five seedlings, and four RNA samples were subjected to the following analysis under each condition. DII WT -GFP or DII G85W -To detect GFP transcripts, RNA samples were prepared from the third rosette leaf of each strain, and the samples prepared from three individual plants were subjected to the following analyses. Total RNA was extracted using the Maxwell RSC Plant RNA Kit (Promega). Equal volumes of RNA were reverse transcribed using PrimeScrip RT Master Mix (Takara). Quantitative PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and the CFX Connect Real-time system (Bio-Rad).

[0165] Test Example 1. Analysis of orthogonal interactions between modified TIR1 and modified IAA7-DII peptide using an in vitro pull-down assay. The LRR domain of TIR1 has a horseshoe-shaped structure composed of 18 LRRs (leucine-rich repeats), with characteristically long loop structures at the 2nd, 12th, and 14th LRRs (PDB: 2P1Q, Figure 1). Auxin (indole-3-acetic acid; IAA) binds to the central part of the horseshoe-shaped structure of TIR1 by interacting with surrounding amino acid residues. Furthermore, the Aux / IAA protein docks from above onto the auxin-bound TIR1, forming a structure that covers the horseshoe shape of TIR1 like a lid. The consensus motif (GWPPV: SEQ ID NO: 55) located in the DII domain of the Aux / IAA protein is important for interaction with auxin-bound TIR1. The second tryptophan (W) and fourth proline (P) of the GWPPV motif are thought to contribute to interaction with hydrophobic amino acids in the TIR1 binding pocket and packing of the indole ring and side chain of the auxin molecule, while the third proline (P) is thought to be involved in maintaining the arrangement of the above two amino acid residues. With the aim of redesigning the interface between TIR1 and the DII domain of Aux / IAA and constructing an orthogonal interaction system between modified TIR1 and modified DII, we decided to analyze the DII domain (IAA7-DII) of the Arabidopsis thaliana IAA7 protein (one of the Aux / IAA proteins), whose crystal structure has been elucidated, and the Arabidopsis thaliana TIR1 protein as materials.

[0166] To construct an orthogonal system, each modified protein must be designed so that no interaction occurs between modified IAA7-DII and wild-type TIR1, and no interaction occurs between modified TIR1 protein and wild-type IAA7-DII, and furthermore, each modification must not affect the interaction with auxin (Figure 2). As mentioned above, WPP, one of the consensus motifs of IAA7-DII, is considered important not only for interaction with TIR1 but also with auxin, and the fifth V is not conserved in all Aux / IAA, so the first glycine (G) was chosen as a candidate amino acid for modification of IAA7-DII. First, modified IAA7-DII proteins were created by changing this glycine (the 85th amino acid from the N-terminus of IAA7, and therefore referred to as IAA7-G85 in this specification) to other amino acids, and their interaction with wild-type TIR1 was investigated.

[0167] The interaction between the two proteins was investigated using an in vitro pull-down assay with the GST-IAA7-DII protein, which was created using an E. coli protein expression system with the IAA7-DII peptide fused to GST (glutathione S-transferase), and the TIR1-FLAG protein, which was created using a wheat germ in vitro protein synthesis system (Figure 3a). GST was fused to the beads (Glutathione Sepharose 4B) used in the pull-down assay to bind IAA7-DII, and the FLAG peptide was fused to the respective proteins to investigate the presence or absence of interaction using immunoblotting analysis with an anti-FLAG antibody. The results of the interaction analysis revealed that modifications to DII-G85 significantly affected the interaction with wild-type TIR1 (TIR1-WT-FLAG), and in particular, substitutions with hydrophobic amino acids (I, L, M, F, W, Y, V) or R resulted in almost no interaction (Figure 3b). Therefore, modified IAA-DII with DII-G85 substituted with hydrophobic amino acids or R was selected as a candidate.

[0168] According to the crystal structure of the ternary complex of "TIR1"-"auxin"-"IAA7-DII peptide," the amino acids of TIR1 located near IAA7-DII-G85 include L84, which is located in the second loop of TIR1, and R489, which is located in LRR17 of TIR1 (Figure 4), suggesting the possibility of functioning as a counterpart to IAA7-DII-G85. As mentioned above, in order to realize orthogonal interactions, the modified IAA7-DII must not interact with wild-type TIR1. Therefore, we then created multiple amino acid substitution proteins of TIR1-L84 and TIR1-R489 and investigated their interactions with wild-type IAA7-DII (IAA7-DII-WT) to attempt to select a modified TIR1 that does not interact with wild-type IAA7-DII (IAA7-DII-WT).

[0169] When we created and examined TIR1-L84A (Figure 5a) and L84D (Figure 5b), which are amino acid substitutions of TIR1-L84, we found that in both cases, the interaction with wild-type IAA7-DII (IAA7-DII-WT) was significantly reduced, while the interaction with IAA7-DII-G85R was restored. Based on these results, the amino acid substitutions of TIR1-L84A and TIR1-L84D with IAA7-DII-G85R were considered candidate combinations that exhibit orthogonal interactions.

[0170] Next, we investigated amino acid substitutions focusing on TIR1-R489. Since the amino acid substitutions of TIR1-R489, TIR1-R489G, R489A, and R489E, all showed little interaction with wild-type IAA7-DII (IAA7-DII-WT), we selected the R489G mutant protein, which has the shortest side chain due to glycine substitution, as the first candidate to investigate whether it could restore interaction with hydrophobic amino acid substitutions of IAA7-DII-G85. As a result, it was shown that hydrophobic amino acid substitutions of IAA7-DII-G85 did not interact with wild-type TIR1 (TIR1-WT), but did interact with TIR1-R489G (Figure 6). Of these, IAA7-DII-G85W showed the strongest interaction with TIR1-R489G.

[0171] Modified TIR1 (ccvTIR) that orthogonally binds to artificially synthesized auxin (cvxIAA) has been created (Patent Document 1). These modified TIR1s are TIR1-F79G and F79A. We hypothesized that by "transplanting" the newly discovered R489G mutation of TIR1 into the TIR1-F79G and F79A mutant proteins, it might be possible to induce an orthogonal interaction between the dual-modified TIR1 and IAA7-DII-G85W using artificially synthesized auxin (cvxIAA) instead of natural auxin (IAA). Therefore, we created these mutant proteins and subjected them to an in vitro pull-down assay (Figure 7). As previously shown, the artificially synthesized auxin cvxIAA not only induces the interaction between TIR1-F79G and TIR1-F79A and IAA7-DII-WT, but we have now newly revealed that cvxIAA can also induce the interaction between TIR1-F79G R489G and TIR1-F79A R489G and IAA7-DII-G85W (Figure 8).

[0172] Test Example 2. Proteasome degradation via bump-and-hole pair of TIR1 and IAA7 deglon. TIR1 designed R489G and IAA7-DII G85W To investigate whether the pair functions in vivo, we first used a green fluorescent protein (GFP)-labeled IAA7-DII(DII) under a constitutively expressive UBIQUITIN10 (UBQ10) promoter. WT -GFP) or IAA7-DII G85W (DII G85W We created Arabidopsis thaliana expressing -GFP. Three transgenic lines were isolated for each construct, and we confirmed that the transcript of the transgene accumulated in all lines (Figure 9a). However, the GFP signal was only detected in DII G85W -Only the GFP strain was present, and GFP fluorescence was mainly accumulated in the nucleus of the hypocotyl and root cells of the seedlings (Figure 9b). These microscopic observations were confirmed by immunoblotting: GFP is DII G85W - GFP was detected in the total protein fraction of seedlings, but DII WT - Not detected in GFP seedlings (Figure 9c) DII WT -GFP proteins are thought to be ubiquitinated by the function of endogenous auxin receptors including TIR1 and then degraded by the proteasome. Therefore, DII WT When two lines of GFP plants (line #2 and line #6) were incubated with the proteasome inhibitor MG132 or bortezomib (BTZ), nuclear DII was observed in root cells. WT -GFP fluorescence signal appeared. BTZ treatment further revealed DII in hypocotyl cells. WT -Accumulation of GFP signaling was observed. Immunoblot detection of the whole protein extract also showed DII induced by proteasome inhibitors. WT - GFP accumulation was shown (Figure 9d). These results indicate DII WT -GFP is degraded by the endogenous ubiquitin-proteasome system, whereas DII G85W -GFP is shown to be evaded and accumulate. These results indicate the absence of IAA7-DII in vitro in the presence of IAA. G85W Degron and TIR1 WT This is consistent with the decrease in binding affinity (Test Example 1).

[0173] Therefore, in vitro, IAA7-DII via IAA G85W TIR1 has been confirmed to bind with R489G The presence of these interactions and ubiquitination leads to DII G85W - We assumed that GFP would be destabilized. To test this idea, we used a UBQ10 promoter-driven TIR1. WT or TIR1 R489G and DII G85W - Plants containing GFP were prepared. Each TIR1 was tagged with a 3xFLAG tag at its N-terminus to enable band detection by immunoblotting. DII G85W -GFP nuclear fluorescence signal is TIR1 R489G In plants, it disappeared in the cells of the hypocotyl, root, and mature leaves, but TIR1 WT DII did not disappear in plants (Figure 9e). G85W -GFP band is TIR1 R489GIn plants that accumulated TIR1, the decrease was observed, but TIR1 WT In plants that accumulated TIR1, the decrease was not observed (Figure 9f). Treatment with a proteasome inhibitor reduced TIR1 R489G DII in plants G85W - Since GFP accumulation became possible (Figure 9g), DII G85W -GFP protein, after translation, TIR1 R489G It was shown that it is degraded by the proteasome through the action of [TIR1]. These results suggest that the concave modified TIR1 (TIR1) designed in vitro is [TIR1] R489G ) and convex modified IAA7-DII deglon (DII G85W A pair of bump-and-hole structures consisting of ) interacts in vivo, and DII G85W -This supports the idea that GFP leads to proteasomal degradation.

[0174] Test Example 3. A dual bump-and-hole strategy enables the induction of proteolytic degradation through a TIR1-auxin-degron set. The amino acid substitutions F79A or F79G in TIR1 reduce its binding affinity to native auxins, enabling binding to developed convex auxins such as cvxIAA and Ada-IAA. A double-concave TIR1 (IAA7-DII) binds to convex IAA and convex IAA7 degron. G85W As a plant possessing ) TIR1 under UBQ10 promoter control F79A R489G or TIR1 F79G R489G We created a plant that expresses DII. G85W -GFP and TIR1 F79A R489G A plant possessing DII was isolated. In this plant strain, DII G85W -GFP signaling accumulated in the nuclei of hypocotyl, root, and leaf cells (Figure 9h). Immunoblotting revealed DII G85W -GFP is TIR1 R489G TIR1 compared to plants F79A R489G It was confirmed to be stabilized in plants (Figure 9i). Therefore, the F79A mutation in TIR1 is TIR1 R489G By reducing its affinity for endogenous auxin and growing plants under normal conditions without exogenous auxin treatment, DII G85W -It was concluded that this stabilizes GFP.

[0175] Next, double concave TIR1 (TIR1 F79A R489G In plants that have accumulated ), supplying the convex auxin cvxIAA or Ada-IAA allows DII to be produced. G85W - We investigated whether GFP degradation was induced. TIR1 F79A R489G When seedlings accumulating DII are incubated with 0.1 μM cvxIAA or Ada-IAA for 1 hour, DII G85W -GFP nuclear fluorescence signal disappeared (Figure 10c, d). DII in total protein extract G85W -GFP protein levels also decreased in seedlings incubated with 0.1 μM cvxIAA or Ada-IAA (Figure 10e). Such degradation did not occur with the addition of 0.1 μM IAA (Figure 10c, d, e). TIR1 was used as a control. WT In plants accumulating DII, treatment with 0.1 μM I AA, cvxIAA, or Ada-IAA was effective. G85W - No decrease in GFP signaling or protein levels was induced (Figure 10a, b, e).

[0176] In yeast two-hybrid assays, Ada-IAA performed better than cvxIAA in terms of TIR1 F79A Since high affinity for is reported, the effective concentrations of cvxIAA and Ada-IAA were compared. DII G85W - Confocal observation of GFP signaling and DII G85W - GFP immunoblotting detection (Figure 10f) showed that lower concentrations of Ada-IAA (0.1~0.01 μM) were more effective than cvxIAA (0.1 μM) in DII detection. G85W - It was shown to effectively induce GFP degradation. Furthermore, we investigated whether the TIR1 F79G mutation functions similarly to the F79A mutation. For this purpose, a single inserted gene fragment was used to... G85W -GFP and TIR1 F79G R489G or TIR1 F79A R489G A 2-in-1 construct was prepared in which TIR1 is expressed under the control of the UBQ10 promoter. F79G R489G and DII G85WIn plants accumulating -GFP, treatment with 0.1 μM cvxIAA, 0.1 μM Ada-IAA or 0.01 μM Ada-IAA resulted in TIR1 F79A R489G and DII G85W similar to plants accumulating -GFP, degradation of DII G85W -GFP was induced (Figure 10g). From the above, it was confirmed that two types of concave-modified TIR1 (TIR1 F79A R489G , TIR1 F79G R489G ) can interact with the convex IAA7 degron and induce degradation of a protein to which the convex IAA7 degron is added.

[0177] Test Example 4. The TIR1 and cvxIAA pair does not cause serious defects in plant growth and auxin signaling. Next, attention was focused on the effect of expression of the double-modified TIR1 on plant growth. When plants were grown in soil, constitutive expression of TIR1 WT or TIR1 F79A R489G did not exert any obvious effect on the morphology of plants grown in soil or the fresh weight of above-ground parts (shoot FW) (Figure 11a). Root elongation in seedlings was similar among WT, TIR1 WT and TIR1 F79A R489G plants (Figure 11b). It was shown that expression of the double-modified TIR1 does not adversely affect plant growth.

[0178] Next, seedling growth (Figure 11c) and the transcription level of representative auxin marker genes were evaluated when seedlings were exposed to external IAA treatment. The transcription level of auxin marker genes did not change in TIR1 WT or TIR1 F79A R489G plants compared with WT plants under Mock conditions. 0.1 μM IAA similarly induced expression of the tested auxin marker genes in all genotypes. These results indicate that expression of the double-modified TIR1 itself does not affect auxin signal transduction. Importantly, TIR1 F79A R489GRoot growth was strongly inhibited with 0.1 μM IAA, but not with 0.1 μM cvxIAA (Figure 11c). Upregulation of auxin marker genes did not occur with 0.1 μM cvxIAA, regardless of genotype. Therefore, cvxIAA treatment can induce knockdown of the convex IAA7 degron-tagged protein without affecting auxin signaling.

[0179] Test Example 5. Evaluation of in-plant induction method for proteolysis. In the demonstration of plant protein degradation induction by convex auxin and double concave TIR1 (Figure 10), 6-day-old seedlings were immersed in a liquid culture medium containing 1 / 2 concentration Murashige-Skoog (MS) salt and cvxIAA or Ada-IAA, and cultured for 1 hour under growth conditions. As a result, simply immersing in the convex auxin solution induced DII G85W It was revealed that the degradation of deglon was accelerated. Furthermore, we investigated whether similar degradation induction could be induced by other simpler methods. First, TIR1 F79A R489G and DII G85W Mature plants grown in soil for 16 days and accumulating GFP were sprayed with a solution containing 0.1 μM cvxIAA or Ada-IAA (Figure 12a). DII driven by the UBQ10 promoter G85W -GFP fluorescence signal was strong in the nuclei of guard cells in mature leaves (Figure 12a). The fluorescence signal disappeared 1 hour after spraying with 0.1 μM cvxIAA or Ada-IAA (Figure 12a). DII in the total protein fraction of leaves sprayed with 0.1 μM cvxIAA or Ada-IAA G85W -GFP levels decreased to 48.9% or 33.6% of those in untreated leaves, respectively (Figure 12b). Next, by supplying convex IAA to the culture medium, DII in cultured plants G85W We investigated whether the degradation of was induced. Seedlings grown in 1 / 2 concentration MS medium were transplanted into new medium supplemented with 0.1 μM cvxIAA or Ada-IAA (Figure 12c), and after 1 or 2 days, DII G85W -GFP levels decreased. DII 1 day after transplantation into 0.1 μM cvxIAA or 0.1 μM Ada-IAA medium. G85W-The amount of GFP protein was 14.1% or 11.5% compared to before treatment (day 0), respectively. Therefore, the uptake of convex auxin from the culture medium was DII G85W We concluded that it induces the degradation of proteins to which the gene has been grafted.

[0180] Therefore, seedlings embedded in a growth medium containing 0.1 μM cvxIAA or Ada-IAA are placed in a glass-bottom dish, and DII G85W -The degradation of the GFP signal was observed using time-lapse imaging (Figure 12d, e). DII in hypocotyl cells. G85W -GFP fluorescence levels remained stable for more than 30 minutes after transplantation under mock conditions (Figure 12d). When transplanted into 0.1 μM cvxIAA or Ada-IAA medium, most of the GFP signal disappeared within 20 minutes. DII G85W The reduction in GFP signaling was faster in root apical cells compared to hypocotyl cells, with the major GFP signal disappearing within 10 minutes of transplantation into medium containing 0.1 μM cvxIAA or Ada-IAA (Figure 12e). These results indicate that the degradation of proteins tagged by convex IAA7 degron begins within minutes of exposure to convex auxin.

Claims

1. A TIR1 family protein mutant obtained by mutation in a TIR1 family protein, comprising a substitution mutation a1 of R489 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding arginine in another amino acid sequence, and / or a substitution mutation a2 of L84 in the amino acid sequence shown in SEQ ID NO: 1 or the corresponding leucine in another amino acid sequence.

2. The TIR1 family protein variant according to claim 1, wherein the TIR1 family protein is a protein containing an amino acid sequence that has 70% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 6.

3. The TIR1 family protein variant according to claim 1, wherein the TIR1 family protein comprises the consensus sequence a shown in SEQ ID NO: 7, and the substitution mutation a1 is an arginine substitution mutation within the consensus sequence a.

4. The TIR1 family protein mutant according to claim 1, wherein the mutated amino acid of the substitution mutation a1 is an amino acid with a smaller molecular weight than arginine.

5. The TIR1 family protein mutant according to claim 1, wherein the mutated amino acid of the substitution mutation a2 is an amino acid or acidic amino acid with a molecular weight smaller than leucine.

6. Aux / IAA family protein mutant or DII domain-containing fragment mutant obtained by mutation in an Aux / IAA family protein or a DII domain-containing fragment thereof, wherein the mutation includes a G85 substitution mutation in the amino acid sequence shown in Sequence ID No. 8 or a corresponding glycine substitution mutation b in another amino acid sequence.

7. The Aux / IAA family protein is a protein that contains an amino acid sequence having 70% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 8-30, and / or The DII domain-containing fragment is a protein containing an amino acid sequence that has 70% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 31 to 53. The Aux / IAA family protein variant or DII domain-containing fragment variant according to claim 6.

8. The Aux / IAA family protein variant or DII domain-containing fragment variant according to claim 6, wherein the Aux / IAA family protein and / or the DII domain-containing fragment comprises the consensus sequence b shown in SEQ ID NO: 54, and the substitution mutation b is a glycine substitution mutation within the consensus sequence b.

9. The Aux / IAA family protein mutant or DII domain-containing fragment mutant according to claim 6, wherein the mutated amino acid of the substitution mutation b is an amino acid with a molecular weight greater than glycine.

10. A polynucleotide comprising the coding sequence of a TIR1 family protein variant according to any one of claims 1 to 5, or the coding sequence of an Aux / IAA family protein variant or a DII domain-containing fragment variant according to any one of claims 6 to 9.

11. A cell comprising at least one selected from the group consisting of a TIR1 family protein variant according to any one of claims 1 to 5, an Aux / IAA family protein variant or DII domain-containing fragment variant according to any one of claims 6 to 9, and a polynucleotide containing the coding sequence thereof.

12. A cell according to claim 11 or a non-human organism containing the cell, comprising a TIR1 family protein variant according to any one of claims 1 to 5, and an Aux / IAA family protein variant or DII domain-containing fragment variant according to any one of claims 6 to 9.

13. A method comprising contacting the cells described in claim 12 or a non-human organism containing the cells with auxin and / or an auxin derivative.

14. The method according to claim 13, wherein the TIR1 family protein variant comprises another protein domain, and / or the Aux / IAA family protein variant or DII domain-containing fragment variant comprises another protein domain.

15. The method according to claim 14, wherein the Aux / IAA family protein variant or DII domain-containing fragment variant includes a degradation target protein domain as another protein domain, and the degradation target protein domain is degraded by contacting the auxin and / or auxin derivative with the cell or the non-human organism.

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