COMPOSITION AND METHOD FOR CONFERRING AND/OR ENHANCING TOLERANCE AGAINST HERBICIDES BY USING VARIANTS OF PPO

Abstract

Provided is a technology for conferring more enhanced tolerance of plants and/or algae against herbicides and/or more greatly enhancing tolerance by using amino acid variants of protoporphyrinogen IX oxidases derived from microorganisms.

Claims

1. A polypeptide selected from the group consisting of: a polypeptide comprising an amino acid sequence of modified SEQ ID NO: 1, wherein one or more amino acid residues selected from the group consisting of R140, F209, V213, A215, G216, V360, S362, F386, L389, L399, I402, and Y422 of the amino acid sequence of SEQ ID NO: 1 are respectively and independently deleted or substituted with an amino acid selected from the group consisting of M(Met), V(Val), I(Ile), T(Thr), L(Leu), C(Cys), A(Ala), S(Ser), F(Phe), P(Pro), W(Trp), N(Asn), Q(Gln), G(Gly), Y(Tyr), D(Asp), E(Glu), R(Arg), H(His), and K(Lys), which is different from the amino acid at the corresponding position of SEQ ID NO: 1; a polypeptide comprising an amino acid sequence of modified SEQ ID NO: 3, wherein one or more amino acid residues selected from the group consisting of R95, V164, I168, A170, G171, I311, V313, F329, L332, L342, I345, and M365 of the amino acid sequence of SEQ ID NO: 3 are respectively and independently deleted or substituted with an amino acid selected from the group consisting of M(Met), V(Val), I(Ile), T(Thr), L(Leu), C(Cys), A(Ala), S(Ser), F(Phe), P(Pro), W(Trp), N(Asn), Q(Gln), G(Gly), Y(Tyr), D(Asp), E(Glu), R(Arg), H(His), and K(Lys), which is different from the amino acid at the corresponding position of SEQ ID NO: 3; and a polypeptide comprising an amino acid sequence with at least 95% identity with the amino acid sequence of the polypeptide.

2. The polypeptide of claim 1, which is selected from the group consisting of: a polypeptide comprising an amino acid sequence of modified SEQ ID NO: 1, wherein one or more amino acid residues selected from the group consisting of R140, F209, V213, A215, G216, V360, S362, F386, L389, L399, I402, and Y422 of the amino acid sequence of SEQ ID NO: 1 are respectively and independently deleted or substituted with an amino acid selected from the group consisting of M(Met), V(Val), I(Ile), T(Thr), L(Leu), C(Cys), S(Ser), and A(Ala), which is different from the amino acid at the corresponding position of SEQ ID NO: 1; a polypeptide comprising an amino acid sequence of modified SEQ ID NO: 3, wherein one or more amino acid residues selected from the group consisting of R95, V164, I168, A170, G171, I311, V313, F329, L332, L342, I345, and M365 of the amino acid sequence of SEQ ID NO: 3 are respectively and independently deleted or substituted with an amino acid selected from the group consisting of M(Met), V(Val), I(Ile), T(Thr), L(Leu), C(Cys), S(Ser), and A(Ala), which is different from the amino acid at the corresponding position of SEQ ID NO: 3; and a polypeptide comprising an amino acid sequence with at least 95% identity with the amino acid sequence of the polypeptide.

3. The polypeptide of claim 1, which is selected from the group consisting of: a polypeptide comprising an amino acid sequence having modification to SEQ ID NO: 1, wherein the modification comprises at least one amino acid mutation selected from the group consisting of Y422M, Y422L, Y422C, Y422V, Y422I, Y422T, A215L, A215C, A215I, V360M, R140A, F209A, V213C, V213S, F386V, L389T, I402T, V360I, V360L, and S362V, in the amino acid sequence of SEQ ID NO: 1; a polypeptide comprising an amino acid sequence having modification to SEQ ID NO: 3, wherein the modification comprises at least one amino acid mutation selected from the group consisting of M365T, M365L, M365C, M365V, M365I, R95A, V164A, I168C, I168S, A170C, A170L, A170I, I311M, F329V, L332T, and I345T, in the amino acid sequence of SEQ ID NO: 3; a polypeptide comprising an amino acid sequence with at least 95% identity with the amino acid sequence of the polypeptide.

4. The polypeptide of claim 3, which is selected from the group consisting of: (1) a polypeptide comprising an amino acid sequence having modification to SEQ ID NO: 1, wherein the modification is selected from the group consisting of amino acid mutations of Y422M, Y422L, Y422C, Y422V, Y422I, Y422T, A215L, A215C, A215I, V360M, R140A, F209A, V213C, V213S, F386V, L389T, I402T, V360I, V360L, S362V, R140A+Y422I, R140A+Y422T, R140A+Y422M, F209A+Y422M, V213C+Y422I, V213C+Y422T, V213C+Y422M, A215C+Y422I, A215C+Y422T, A215C+Y422M, A215L+Y422I, A215L+Y422T, A215L+Y422M, V360M+Y422M, F386V+Y422M, V360M+Y422I, L389T+Y422M, I402T+Y422M, V360I+Y422I, V360I+S362V, S362V+Y422I, R140A+V213C+Y422I, R140A+V213C+Y422M, R140A+A215C+Y422I, R140A+A215L+Y422M, V213C+A215C+Y422I, V213C+A215L+Y422M, V360I+S362V+Y422I, A215C+V360M+Y422M, A215L+V360M+Y422M, A215I+V360M+Y422M, V213C+A215C+Y422M, V213C+A215L+Y422M, R140A+V213C+A215C+Y422I, and R140A+V213C+A215L+Y422M, in the amino acid sequence of SEQ ID NO: 1; a polypeptide comprising an amino acid sequence having modification to SEQ ID NO: 3, wherein the modification is selected from the group consisting of amino acid mutations of M365T, M365L, M365C, M365V, M365I, R95A, V164A, I168C, I168S, A170C, A170L, A170I, I311M, F329V, L332T, I345T, R95A+M365I, R95A+M365V, I168C+M365I, I168C+M365V, A170C+M365I, A170C+M365V, A170L+M365I, A170L+M365V, I311M+M365I, I311M+M365V, L332T+M365I, L332T+M365V, V164A+M365I, F329V+M365I, I345T+M365I, A170C+I311M, A170L+I311M, A170I+I311M, I168C+A170C, I168C+A170L, R95A+I168C+M365I, R95A+I168C+M365V, R95A+A170C+M365I, R95A+I311M+M365I, R95A+I311M+M365V, R95A+L332T+M365I, R95A+L332T+M365V, I168C+A170C+M365V, I168C+I311M+M365I, I168C+I311M+M365V, I168C+L332T+M365I, I168C+L332T+M365V, A170C+I311M+M365I, A170C+L332T+M365V, I311M+L332T+M365I, I311M+L332T+M365V, R95A+I168C+A170C+M365I, R95A+I168C+A170C+M365V, R95A+A170C+I311M+M365V, R95A+A170C+L332T+M365I, R95A+I168C+I311M+M365V, R95A+I168C+L332T+M365I, R95A+I311M+L332T+M365I, R95A+I311M+L332T+M365V, I168C+A170C+I311M+M365I, I168C+A170C+L332T+M365V, A170C+I311M+L332T+M365I, R95A+I168C+A170C+I311M+M365V, R95A+I168C+A170C+L332T+M365I, R95A+I168C+I311M+L332T+M365V, I168C+A170C+I311M+L332T+M365V, and R95A+I168C+A170C+I311M+L332T+M365V, in the amino acid sequence of SEQ ID NO: 3; a polypeptide comprising an amino acid sequence with at least 95% identity with the amino acid sequence of the polypeptide.

5. A polynucleotide encoding the polypeptide of claim 1.

6. A recombinant vector comprising the polynucleotide of claim 5.

7. A recombinant cell comprising the recombinant vector of claim 6.

8. A composition for conferring or enhancing herbicide tolerance of a plant or algae, comprising one or more selected from the group consisting of: the polypeptide of claim 1; a polynucleotide encoding the polypeptide; a recombinant vector comprising the polynucleotide; and a recombinant cell comprising the recombinant vector.

9. The composition of claim 8, wherein the herbicide is an herbicide inhibiting protoporphyrinogen IX oxidase.

10. The composition of claim 8, wherein the herbicide is at least one selected from the group consisting of pyrimidinediones, diphenyl-ethers, phenylpyrazoles, N-phenylphthalimides, phenylesters, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, pyraclonil, flufenpyr-ethyl, and profluazol.

11. The composition of claim 10, wherein the herbicide is at least one selected from the group consisting of butafenacil, saflufenacil, benzfendizone, tiafenacil, fomesafen, oxyfluorfen, aclonifen, acifluorfen, bifenox, ethoxyfen, lactofen, chlomethoxyfen, chlorintrofen, fluoroglycofen-ethyl, halosafen, pyraflufen-ethyl, fluazolate, flumioxazin, cinidon-ethyl, flumiclorac-pentyl, fluthiacet, thidiazimin, oxadiargyl, oxadiazon, carfentrazone, sulfentrazone, azafenidin, pentoxazone, pyraclonil, flufenpyr-ethyl, profluazol, phenopylate, carbamate analogues of phenopylate, and agriculturally acceptable salt thereof.

12. The composition of claim 8, wherein the plant or algae further comprise a second herbicide-tolerant polypeptide or a gene encoding the same, and its tolerance to the second herbicide is conferred or enhanced.

13. The composition of claim 12, wherein the second herbicide is selected from the group consisting of glyphosate, glufosinate, dicamba, 2,4-D(2,4-Dichlorophenoxyacetic acid), isoxaflutole, ALS(acetolactate synthase)-inhibiting herbicide, photosystem II-inhibiting herbicide, phenylurea-based herbicide, bromoxynil-based herbicide, and combinations thereof.

14. The composition of claim 12, wherein the second herbicide-tolerant polypeptide is one or more selected from the group consisting of: glyphosate herbicide-tolerant EPSPS (glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase), GOX (glyphosate oxidase), GAT (glyphosate-N-acetyltransferase) or glyphosate decarboxylase; glufosinate herbicide-tolerant PAT (phosphinothricin-N-acetyltransferase); dicamba herbicide-tolerant DMO (dicamba monooxygenase); 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide-tolerant 2,4-D monooxygenase or AAD (aryloxyalkanoate dioxygenase); ALS (acetolactate synthase)-inhibiting sulfonylurea-based herbicide-tolerant ALS (acetolactate synthase), AHAS (acetohydroxyacid synthase) or AtAHASL (Arabidopsis thaliana acetohydroxyacid synthase large subunit); photosystem II-inhibiting herbicide-tolerant photosystem II protein D1; phenylurea herbicide-tolerant Cytochrome P450; plastid-inhibiting herbicide-tolerant HPPD (hydroxyphenylpyruvate dioxygenase); bromoxynil herbicide-tolerant nitrilase; and combinations thereof.

15. The composition of claim 12, wherein the gene encoding the second herbicide-tolerant polypeptide is one or more selected from the group consisting of: glyphosate herbicide-tolerant cp4 epsps, mepsps, 2mepsps, goxv247, gat4601 or gat4621 gene; glufosinate herbicide-tolerant BAR or PAT gene; dicamba herbicide-tolerant dmo gene; 2,4-D(2,4-dichlorophenoxyacetic acid) herbicide-tolerant AAD-1 or AAD-12 gene; isoxaflutole herbicide-tolerant HPPDPF W336 gene; sulfonylurea herbicide-tolerant ALS, Csr1, Csr1-1, Csr1-2, GM-HRA, S4-HRA, Zm-HRA, SurA or SurB gene; photosystem II-inhibiting herbicide-tolerant psbA gene; phenylurea herbicide-tolerant CYP76B1 gene; bromoxynil herbicide-tolerant bxn gene; and combinations thereof.

16. A transformant of a plant or algae having herbicide tolerance, or a clone or progeny thereof, comprising the polypeptide of claim 1 or a polynucleotide encoding the same.

17. The transformant, clone, or progeny thereof of claim 16, wherein the transformant is an alga, or a cell, protoplast, callus, hypocotyl, seed, cotyledon, shoot, or whole body of a plant.

18. A method of preparing a transgenic plant or algae having herbicide tolerance, the method comprising introducing the the polypeptide of claim 1 or a polynucleotide encoding the same into an alga, or a cell, protoplast, callus, hypocotyl, seed, cotyledon, shoot, or whole body of a plant.

19. A method of conferring or enhancing herbicide tolerance of a plant or algae, the method comprising introducing the the polypeptide of claim 1 or a polynucleotide encoding the same into an alga, or a cell, protoplast, callus, hypocotyl, seed, cotyledon, shoot, or whole body of a plant.

20. A method of controlling weeds in a cropland, the method comprising: providing the cropland with a plant comprising the polypeptide of claim 1 or a polynucleotide encoding the same, and applying an effective dosage of protoporphyrinogen IX oxidase-inhibiting herbicide to the cropland or the plant.

21. The method of claim 20, wherein the step of applying an effective dosage of protoporphyrinogen IX oxidase-inhibiting herbicide to the cropland is performed by applying an effective dosage of two or more kinds of protoporphyrinogen IX oxidase-inhibiting herbicides sequentially or simultaneously.

22. The method of claim 20, wherein the plant further comprises a second herbicide-tolerant polypeptide or a gene encoding the same, and the step of applying an effective dosage of protoporphyrinogen IX oxidase-inhibiting herbicide to the cropland is performed by applying effective dosages of the protoporphyrinogen IX oxidase-inhibiting herbicide and a second herbicide are applied sequentially or simultaneously.

23. A method of removing an undesired aquatic organism from a culture media, the method comprising: providing a culture media with algae comprising the polypeptide of claim 1 or a polynucleotide encoding the same, and applying an effective dosage of protoporphyrinogen IX oxidase-inhibiting herbicide to the culture media.

Description

DESCRIPTION OF DRAWINGS

[0160] FIG. 1 is a map of pET303-CT-His vector.

[0161] FIG. 2 is a photograph showing cell growth level of PPO-deficient BT3 E. coli (BT3(ΔPPO)) transformant transformed with ApPPO1 wild type gene (indicated by ApPPO1WT), or various ApPPO1 mutant genes leading to a mutation of one amino acid, when treated with tiafenacil at a concentration of 0 μM (control), 50 μM, and 100 μM, respectively (upper), and saflufenacil at a concentration of 0 μM (control), 50 μM, and 100 μM, respectively (lower).

[0162] FIG. 3 is a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with ApPPO1WT, or various ApPPO1 mutant genes leading to a mutation of one amino acid, when treated with flumioxazin at a concentration of 0 μM (control), 50 μM, and 200 μM, respectively (upper), and sulfentrazone at a concentration of 0 μM (control), 5 μM, and 25 μM, respectively (lower).

[0163] FIG. 4 is a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with ApPPO1WT, or various ApPPO1 mutant genes leading to a mutation of one amino acid, when treated with fomesafen at a concentration of 0 μM (control), 5 μM, and 25 μM, respectively (upper), and acifluorfen at a concentration of 0 μM (control), 5 μM, and 25 μM, respectively (lower).

[0164] FIG. 5 is a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with ApPPO1WT, or various ApPPO1 mutant genes leading to a mutation of one amino acid, when treated with pyraclonil at a concentration of 0 μM (control), 5 μM, and 25 μM, respectively (upper), and pentoxazone at a concentration of 0 μM (control), 5 μM, and 10 μM, respectively (lower).

[0165] FIG. 6 is a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with ApPPO1WT, or various ApPPO1 mutant genes leading to a mutation of one amino acid, when treated with pyraflufen-ethyl at a concentration of 0 μM (control), 5 μM, and 10 μM, respectively.

[0166] FIGS. 7 to 12 are photographs showing cell growth level of BT3(ΔPPO) transformants transformed with ApPPO1 wild type gene (indicated by ApPPO1WT), or various ApPPO1 mutant genes leading to mutations of two or more amino acids as shown in Table 8, when treated with tiafenacil at a concentration of 0 μM (control), 50 μM, and 200 μM, respectively, flumioxazin at a concentration of 0 μM (control), 50 μM, and 100 μM, respectively, and sulfentrazone at a concentration of 0 μM (control), 200 μM, and 400 μM, respectively.

[0167] FIG. 13 is a photograph showing cell growth level of PPO-deficient BT3 E. coli (BT3(ΔPPO)) transformant transformed with MxPPO wild type gene (indicated by MxPPOWT), or various MxPPO mutant genes leading to a mutation of one amino acid, when treated with tiafenacil at a concentration of 0 μM (control), 200 μM, and 2000 μM, saflufenacil at a concentration of 0 μM (control), 100 μM, and 200 μM, and flumioxazin at a concentration of 0 μM (control), 50 μM, and 100 μM, respectively.

[0168] FIG. 14 is a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with MxPPOWT, or various MxPPO mutant genes leading to mutations of two or more amino acids as shown in Table 10, when treated with tiafenacil at a concentration of 0 μM (control) and 2000 μM, respectively.

[0169] FIGS. 15 to 17 are a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with MxPPOWT, or various MxPPO mutant genes leading to mutations of two or more amino acids as shown in Table 10, when treated with flumioxazin at a concentration of 0 μM (control). 200 μM, and 400 μM, respectively.

[0170] FIGS. 18 to 20 are a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with MxPPOWT, or various MxPPO mutant genes leading to mutations of two or more amino acids as shown in Table 10, when treated with sulfentrazone at a concentration of 0 μM (control), 200 μM, and 1000 μM, respectively.

[0171] FIGS. 21 and 22 are a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with MxPPOWT, or various MxPPO mutant genes made by multiple amino acid changes as shown in Table 10, when treated with flumioxazin at a concentration of 0 μM (control), 400 μM, and 1000 μM, respectively.

[0172] FIGS. 23 and 24 are a photograph showing cell growth level of BT3(ΔPPO) transformant transformed with MxPPOWT, or various MxPPO mutant genes made by multiple amino acid changes as shown in Table 10, when treated with sulfentrazone at a concentration of 0 μM (control), 2000 μM, and 4000 μM, respectively.

[0173] FIG. 25 is a map of pMAL-c2X vector.

[0174] FIG. 26 is a photograph showing seed germination results observed at the 6.sup.th day after sowing the seeds of A. thaliana wild type (Col-0) or transformants of ApPPO1 nutant genes in herbicide-containing medium.

[0175] FIG. 27 is a photograph showing seed germination results of observed at the 6.sup.th day after sowing the seeds of A. thaliana wild type (Col-0) or transformants of an MxPPO and an MxPPO mutant gene in herbicide-containing medium.

MODE FOR INVENTION

[0176] Hereinafter, the present invention will be described in detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

Example 1. Verification of Herbicide Tolerance of ApPPO1 and MxPPO Isolated from Prokaryotes

[0177] PPO gene sequences were obtained from Genebank database of two strains, Auxenochlorella protothecoides and Myxococcus xanthus, respectively. For encoding the PPO protein (ApPPO1; SEQ ID NO: 1) from Auxenochlorella protothecoides, the PPO gene designated as ApPPO1 was isolated from Auxenochlorella protothecoides, and optimized to have the nucleic acid sequence of SEQ ID NO: 7. For encoding the PPO protein (MxPPO; SEQ ID NO: 3) Myxococcus xanthus designated as MxPPO was isolated from Myxococcus xanthus and optimized to have the nucleic acid sequence of SEQ ID NO: 8. In order to obtain the herbicide-binding structure of PPO protein, the herbicides including tiafenacil, saflufenacil, flumioxazin, and sulfentrazone and the PPO proteins including ApPPO1 and MxPPO were used. Homology model of ApPPO1 was constructed from CyPPO10 (the PPO protein originated from Thermosynechococcus elongatus BP-1; SEQ ID NO: 5) structure using SWISS-MODEL protein structure modelling server (https://swissmodel.expasy.org/). The structure information of MxPPO was used from RCSB protein data bank (https://www.rcsb.org/pdb/home/home.do) (PDB ID: 2IVE)Herbicide-interacting structural information of ApPPO1 and MxPPO were superimposed with CyPPO10 bound with herbicides (tiafenacil, saflufenacil, flumioxazin, and sulfentrazone).

[0178] Herbicide-binding information of CyPPO10 was obtained by following procedures: CyPPO10 protein (SEQ ID NO: 5) and tiafenacil, saflufenacil, flumioxazin, and sulfentrazone were examined as the representative protein and herbicides, respectively. The gene encoding the CyPPO10 protein (SEQ ID NO: 6) was cloned to pET29b vector (Catalog Number: 69872-3; EMD Biosciences), and CyPPO10 protein was expressed in E. coli. The expressed CyPPO10 protein was purified through nickel affinity chromatography, to which tiafenacil, saflufenacil, flumioxazin or sulfentrazone was added respectively and herbicide-bound PPO crystals were obtained. Then, the crystals were used for X-ray diffraction by synchrotron radiation accelerator. X-ray diffraction data of the 2.4A resolution of CyPPO10-herbicide complex crystals was obtained, and the three-dimensional structure was determined. Binding information was obtained through analyzing the amino acid residues of CyPPO10 interacting with herbicides.

[0179] Using the information of herbicide-interacting amino acids derived from the structure of CyPPO10-herbicide complexes, information of ApPPO1 and MxPPO amino acid residues which possibly lower the binding affinity of herbicides through mutations were determined.

[0180] As results, amino acid residues including R140, F209, V213, A215, G216, V360, S362, F386, L389, L399, I402 and Y422 of ApPPO1 protein (SEQ ID NO: 1) were involved to interact with herbicides (tiafenacil, saflufenacil, flumioxazin, and sulfentrazone) and those including R95, V164, I168, A170, G171, I311, V313, F329, L332, L342, I345 and M365 of MxPPO protein (SEQ ID NO: 3) were involved to interact with herbicides (tiafenacil, saflufenacil, flumioxazin, and sulfentrazone).

Example 2. Construction of PPO Variants

[0181] In order to enhance PPO-inhibiting herbicide tolerance of ApPPO1 and MxPPO, a mutation(s) at the position interacting with herbicide obtained in the Example 1 was introduced, respectively. Each PPO gene was codon-optimized and synthesized (Cosmogenetech Co., Ltd.) for efficient herbicide tolerance test using BT3, a PPO-deficient E. coli stain.

[0182] Detailed experimental procedure was as follows:

[0183] Using primers listed in Table 2, PCR was carried out to amplify PPO genes under following condition.

[0184] PCR reaction mixture

[0185] Template (synthetic DNA of ApPPO1 and MxPPO) 1 μl

[0186] 10× buffer 5 μl

[0187] dNTP mixture (10 mM each) 1 μl

[0188] Forward primer (10 μM) 1 μl

[0189] Reverse primer (10 μM) 1 μl

[0190] DDW 40 μl

TABLE-US-00002 TABLE 2 Primer list for cloning of ApPPO1 and MxPPO in pET303-CT His SED Se- ID Gene Strain Primer quence No. ApPPO Auxenochlorella ApPPO1_ CCCCTCTA  9 1 protothecoides XbaIF GAATGGCC GAGTACGA CGTTGT TAACGT ApPPO1_ CCCCCTCG 10 XhoIR AGGGTTGC CAGACTTT TAACGT MxPPO Myxococcus MxPPO1_ CCCCTCTA 11 xanthus XbaIF GAATGCAC CATATGCC CCGAAC TAACGT MxPPO1_ CCCCCTCG 12 XhoIR AGAGGCGC GTGTGATG TATTAC

[0191] Pfu-X (Solgent, 2.5 units/μl) 1 μl

[0192] Total 50 μl

TABLE-US-00003 TABLE 1 PCR reaction condition 94° C. 4 min. 1 cycle 94° C. 30 sec. 25 cycles 56° C. 30 sec. 72° C. 1.5 min. 72° C. 5 min. 1 cycle  4° C. 5 min. 1 cycle

[0193] Amplified PCR products above and pET303-CT His vector (VT0163; Novagen; FIG. 1) were digested with XbaI and XhoI restriction enzymes, and ligated to construct pET303-ApPPO1 and pET303-MxPPO plasmids using T4 DNA ligase(RBC, 3 units/μl).

[0194] ApPPO1 and MxPPO genes cloned in pET303-CT His vector were mutated through site-directed mutagenesis using primers listed in Tables 4 and 5, respectively.

[0195] PCR reaction mixture

[0196] Template 1 μl

[0197] 10× buffer 5 μl

[0198] dNTP mixture (10 mM each) 1 μl

[0199] Forward primer (10 μM) 1 μl

[0200] Reverse primer (10 μM) 1 μl

[0201] DDW 40 μl

[0202] Pfu-X (Solgent, 2.5 units/μl) 1 μl

[0203] Total 50 μl

TABLE-US-00004 TABLE 3 PCR reaction condition 94° C. 2 min. 1 cycle 94° C. 30 sec. 17-25 cycles 65° C. 40 sec. 72° C. 3.5 min. 72° C. 5 min. 1 cycle  4° C. 5 min. 1 cycle

TABLE-US-00005 TABLE 4 Primer list for mutagenesis of ApPPO1 gene SEQ ApPPO1 Primer Sequence ID mutation (5′-> 3′) NO Y422M F CTCT 13 TGTC ACTT TATG GGGG GGCT ACCA ACAC R CCCC 14 CATA AAGT GACA AGAG CAGC ACCT TTCC Y422L F CTCT 15 TGTC ACTT TTTG GGGG GGCT AGCA ACAC R CCCC 16 CAAA AAGT GACA AGAG CAGC ACCT TTCC Y422C F TCTT 17 GTCA TGTT TTGG GGGG GCTA CCAA CAC R CCCC 18 CAAA ACAT GACA AGAG CAGC ACCT TTCC Y422V F CTCT 19 TGTC AGTT TTTG GGGG GGCT ACCA ACAC R CCCC 20 CAAA AACT GACA AGAG CAGC ACCT TTC Y422I F CTCT 21 TGTC AATT TTTG GGGG GGCT ACCA ACAC R CCCC 99 AAAA ATTG ACAA GAGC AGCA CCTT TCC Y422T F GTGC 23 TGCT CTTG TCAA CCTT TGGG GGGG CTAC C R GGTA 24 GCCC CCCC AAAG GTTG ACAA GAGC AGC AC A215L F GGGT 25 TTAC CTCG GCGA CCCG GCTA AGTT GAG R GTCG 26 CCGA GGTA AACC CCGC TGCA AAAC GGC A215C F CiGG 27 TTTA CTGC GGCG ACCC GGCT AAGT TGAG R GTCG 28 CCGC AGTA AACC CCGC TGCA AAAC GGC V360M F CCCT 29 CCCA TGGC ATCT GTAG CATT ATCT TACC R TACA 30 GATG CCAT GGGA GGGT AATA GATA GAG C R140A F GATC 31 CGAA GGCG CCCG CGTA CGTT TATT GGGG TG R CACC 32 CCAA TAAA CGTA CGCG GGCG CCTT CGGA TC F209A F CGAC 33 TTAT AGAG CCGG CGTG CAGC GGGG TTTA C R GTAA 34 ACCC CGCT GCAC GCCG GCTC TATA AGTC G V213C F CCGT 35 TTTG CAGC GGGT GCTA CGCC GGCG ACCC G R CGGG 36 TCGC CGGC GTAG CACC CGCT GCAA AACG G F386V F GGTC 37 ACCT AGCG GGCG TGGG CCAG CTAC ACCC TC R GAGG 38 GTGT AGCT GGCC CACG CCCG CTAG GTGA CC L389T F GCGG 39 GCTT TGGC CAGA CCCA CCCT CGTA CTCA G R CTGA 40 GTAC GAGG GTGG GTCT GGCC AAAG CCCG C I402T F CACC 41 ACTC TGGG CACT ACCT ATGC CTCA AGCT TA R TAAG 42 CTTG AGGC ATAG GTAG TGCC CAGA GTGG TG V360I F CTAT 43 TACC CTCC CATC GCAT CTGT AGCA TTAT C R GATA 44 ATGC TACA GATG CGAT GGGA GGGT AATA G V360L F TACC 45 CTCC CCTC GCAT CTGT AGCA TTAT CTTA C R CAGA 46 TGCG AGGG GAGG GTAA TAGA TAGA GC S362V F TACC 47 CTCC CGTC GCAG TTGT AGCA TTAT CTTA C R GTAA 48 GATA ATGC TACA ACTG CGAC GGGA GGGT A V213C + F TTGC 49 A215C AGCG GGTG CTAC TGCG GCGA CCCG GCTA AGT R ACTT 50 AGCC GGGT CGCC GCAG TAGC ACCC GCTG CAA V213C +  F TTGC 51 A215L AGCG GGTG CTAC CTTG GCGA CCCG GCTA AGT R ACTT 52 AGCC GGGT CGCC AAGG TAGC ACCC GCTG CAA

TABLE-US-00006 TABLE 5 Primer list for mutagenesis of MxPPO gene Primer SEQ MxPPO Sequence ID mutation (5′-> 3′) NO M365T F ACTC 53 ATGT ACGG TGGG GGGT GCAA GACA ACC R CCCC 54 ACCG TACA TGAG TATA AGAC ACGC CCAC M365L F TACT 55 CATG TCTG GTGG GGGG TGCA AGAC AACC R CCCA 56 CCAG ACAT GAGT ATAA GACA CGCC CAC M365C F TACT 57 CATG TTGC GTGG GGGG TGCA AGAC AACC R CCCC 58 CACG CAAC ATGA GTAT AAGA CACG CCC M365V F TACT 59 CATG TGTG GTGG GGGG TGCA AGAC AACC R CCCC 60 CCAC CACA CATG AGTA TAAG ACAC GCCC M365I F GTCT 61 TATA CTCA TGTA TCGT GGGG GGTG CAAG AC R GTCT 62 TGCA CCCC CCAC GATA CATG AGTA TAAG AC R95A F GCAA 63 AGAG AGCT TATG TCTA CACG CGAG GACG R GTAG 64 ACAT AAGC TCTC TTTG CAGC CGGA TCGG C VI64A F TAGA 65 TGCA GCGC AGAC AGGG ATAT ATGC CGG R CTGT 66 CTGC GCTG CATC TAAT AGAA CTTG GG I168C F CAGA 67 CAGG GTGC TATG CCGG AGAT GTTG AGC R TCCG 68 GCAT AGCA CCCT GTCT GCAC TGCA TCTA A A170C F GGGA 69 TATA TTGC GGAG ATGT TGAG CAAT TATC R ACAT 70 CTCC GCAA TATA TCCC TGTC TGCA CTGC A170L F GGGA 71 TATA TCTC GGAG ATGT TGAG CAAT TATC R ACAT 72 CTCC GAGA TATA TCCC TGTC TGCA CTGC I311M F TGCC 73 CCCA TGGC TGTA GTTC ATCT CGGA TTC R AACT 74 ACAG CCAT GGGG GCAT AGGC GATA CC F329V F CGAT 75 GGGG TCGG TTTT TTAG TGCC GGCG GAGG R AAAA 76 AACC GACC CCAT CGGG CGCC GGTA AAG L332T F TTCG 77 GTTT TACA GTGC CGGC GGAG GAAC AG R CCGG 78 CACT GTAA AACC GAAC CCAT CGGG CGC I345T F GGGT 79 GCCA CTCA TGCT TCCA CGAC TTTC CCG R GAAG 80 CATG AGTG GCAC CCAA CATC CTTC GCTG I168C + F GTGC 81 A170C AGAC AGGG TGCT ATTG CGGA GATG TTGA G R CTCA 82 ACAT CTCC GCAA TAGC ACCC TGTC TGCA C

[0204] One μl of DpnI (NEB) was treated to each 10 μl of PCR products, and incubated at 37° C. for 30 minutes. DH5alpha competent cell (Biofact Co., Ltd.) was transformed with reaction solution through heat shock method, and was cultured in LB agar media containing carbenicillin (Gold Biotechnology Co., Ltd.). After plasmids were prepared from transformed E. coli, they were sequenced (Cosmogenetech, Co., Ltd.) and confirmed to have correct mutations.

Example 3. Verification of PPO-Inhibiting Herbicide Tolerance of PPO Variants (Test in E. coli)

[0205] The mutated CyPPO gene obtained from the Example 2 was transformed to BT3 (ΔPPO) strain which is deficient of PPO activity and cultured in LB media with PPO-inhibiting herbicide, thereby examining whether growth of transformed BT3 was not inhibited.

[0206] BT3 (ΔPPO) strain was provided by Hokkaido University (Japan) and it is an E. coli strain which is deficient in hemG-type PPO and has kanamycin resistance (refer to “Watanabe N, Che FS, Iwano M, Takayama S, Yoshida S, Isogai A. Dual targeting of spinach protoporphyrinogen IX oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons, J. Biol. Chem. 276(23):20474-20481, 2001; Che FS, Watanabe N, Iwano M, Inokuchi H, Takayama S, Yoshida S, Isogai A. Molecular Characterization and Subcellular Localization of Protoporphyrinogen IX oxidase in Spinach Chloroplasts, Plant Physiol. 124(1):59-70, 2000”).

[0207] Detailed experimental procedure was as follows:

[0208] BT3 competent cells were transformed with the pET303-ApPPO1 and pET303-MxPPO plasmids and those with a mutation(s) constructed in Example 2 respectively, and were cultured in LB agar media containing carbenicillin (Gold Biotechnology, Co., Ltd.).

[0209] Single colony of E. coli transformed with each CyPPO gene was cultured in 3 ml of LB broth containing carbenicillin overnight, and then was subcultured until absorbance (OD.sub.600) reached 0.5 to 1. Then, it was diluted with LB broth to OD.sub.600=0.5. Again, the diluted solution was serially diluted 4 times by a factor of one tenth.

[0210] The LB agar media (LB 25 g/l, Bacto agar 15 g/l) containing carbenicillin (100 μg/ml) and 0 to 4,000 μM of various herbicides dissolved in DMSO was prepared. Next, 10 μl of each diluted solution was dropped on the plate and cultured at 37° C. under light (Tables 7, 9 and 10, FIGS. 2 to 6, 13 to 20) or dark (Tables 8 and 11, FIGS. 7 to 12, 21 to 24) for 16 to 20 hours. Then, the extent of tolerance was evaluated. PPO-inhibiting herbicides used in the experiments were listed in Table 6:

TABLE-US-00007 TABLE 6 PPO-inhibiting herbicides used in the experiments Family Herbicide Pyrimidinedione tiafenacil saflufenacil Diphenyl ether fomesafen acifluorfen N-phenylphthalimides flumioxazin Triazolinones sulfentrazone Oxazolidinediones pentoxazone Phenylpyrazoles pyraflufen-ethyl Others pyraclonil

[0211] The extent of herbicide tolerance of the ApPPO1 or MxPPO mutated genes was evaluated by comparing that of mutated genes with that of ApPPO1 or MxPPO wild type. The relative tolerance was represented with “+” as a factor of 10 times. Evaluation result was listed in Tables 7 to 11 and FIGS. 2 to 24

TABLE-US-00008 TABLE 7 Herbicide tolerance evaluation of mutated ApPPO1 Mutation No. site tiafenacil saflufenacil flumioxazin sulfentrazone Fomesafen 1 A215C + + N.T + + (AC) 2 A215L ++ ++++ ++++ +++ +++ (AL) 3 V360M ++ +++ N.T + + (VM) 4 Y422T ++ ++++ +++ + + (YT) 5 Y422C ++ +++ N.T ++ ++ (YC) 6 Y422M +++ +++++ +++ + + (YM) 7 Y422I (YI) ++ +++++ +++ + + 8 Y422L ++ ++++ +++ + + (YL) WT − − − − − Mutation No. site acifluorfen pyraclonil pentoxazone pyraflufenethyl 1 A215C ++ + + ++ (AC) 2 A215L +++ ++ ++ +++ (AL) 3 V360M ++ + + ++ (VM) 4 Y422T + + ++ ++ (YT) 5 Y422C ++ ++ +++ +++ (YC) 6 Y422M ++ ++ ++ ++ (YM) 7 Y422I (YI) + + ++ ++ 8 Y422L ++ ++ ++ ++ (YL) WT − − − − N.T (Not tested)

TABLE-US-00009 TABLE 8 Herbicide tolerance evaluation of mutated ApPPO1 flumiox- sulfen- No. Mutation site tiafenacil azin trazone 1 R140A + Y422I +++++ +++++ ++++ 2 R140A + Y422T ++++ ++++ +++ 3 R140A + Y422M ++++ ++++ ++++ 4 V213C + Y422I ++++ +++++ +++ 5 V213C + Y422T +++++ +++++ ++++ 6 V213C + Y422M +++++ +++ ++ 7 A215L + Y422I ++++ ++++ ++++ 8 A215L + Y422T + + ++++ 9 A215L + Y422M +++++ +++++ ++++ 10 A215C + Y422I +++++ +++++ ++++ 11 A215C + Y422T +++ +++ +++ 12 A215C + Y422M +++++ +++++ ++++ 13 R140A + V213C + +++++ +++++ ++++ Y422I 14 R140A + V213C + +++++ +++++ ++++ Y422M 15 R140A + A215C + +++++ +++++ ++++ Y422I 16 R140A + A215L + +++++ +++++ ++++ Y422M 17 V213C + A215C + +++++ +++++ ++++ Y422I 18 V213C + A215L + +++++ +++++ ++++ Y422M 19 R140A + V213C + +++++ +++++ ++++ A215C + Y422I 20 R140A + V213C + +++++ +++++ ++++ A215L + Y422M WT − − −

TABLE-US-00010 TABLE 9 Herbicide tolerance evaluation of mutated MxPPO flumiox- No. Mutation site tiafenacil saflufenacil azin 1 A170C + +++ + 2 A170L + ++ ++ 3 I311M ++ ++ ++ 4 M365I + ++ ++ 5 M365L + ++ ++ 6 M365V + +++ ++ WT − − −

TABLE-US-00011 TABLE 10 Herbicide tolerance evaluation of mutated MxPPO flumiox- sulfen- No. Mutation site tiafenacil azin trazone 1 R95A + M365I N.T + + 2 R95A + M365V N.T + + 3 I168C + M365I N.T + + 4 I168C + M365V N.T + + 5 A170C + M365I N.T + + 6 A170C + M365V + ++ + 7 I311M + M365I + ++ + 8 I311M + M365V + ++ + 9 L332T + M365I N.T + + 10 L332T + M365V + + + 11 R95A + I168C + M365I N.T + + 12 R95A + I168C + M365V N.T + + 13 R95A + A170C + M365I N.T + + 14 R95A + I311M + M365I + ++ + 15 R95A + I311M + M365V N.T ++ + 16 R95A + L332T + M365I N.T + + 17 R95A + L332T + M365V N.T + + 18 I168C + A170C + M365V N.T ++ ++ 19 I168C + I311M + M365I + ++ + 20 I168C + I311M + M365V + ++ + 21 I168C + L332T + M365I N.T ++ ++ 22 I168C + L332T + M365V + +++ ++ 23 A170C + I311M + M365I N.T ++ + 24 A170C + L332T + M365V N.T ++ ++ 25 I311M + L332T + M365I + ++ ++ 26 I311M + L332T + M365V + +++ ++ WT − − − N.T (Not tested)

TABLE-US-00012 TABLE 11 Herbicide tolerance evaluation of mutated MxPPO flumiox- sulfen- No. Mutation site azin trazone 1 R95A + I168C + A170C + M365V + ++ 2 R95A + I168C + I311M + M365V + +++ 3 R95A + I168C + L332T + M365I + ++ 4 R95A + A170C + I311M + M365V + + 5 R95A + A170C + L332T + M365I + +++ 6 R95A + I311M + L332T + M365I + +++ 7 I168C + A170C + I311M + M365I + + 8 I168C + A170C + L332T + M365V + ++ 9 A170C + I311M + L332T + M365I + N.T 10 R95A + I168C + A170C + I311M + + +++ M365V 11 R95A + I168C + A170C + L332T + + +++ M365I 12 R95A + I168C + I311M + L332T + + +++ M365V 13 I168C + A170C + I311M + L332T + + ++ M365V 14 R95A + I168C + A170C + I311M + + +++ L332T + M365V WT − − N.T (Not tested)

[0212] In Tables 7 to 11, tolerance level was presented as ‘−’ of tolerance of wild type and of variants equivalent to that of wild type, and was done as ‘+’ per each 10 fold resistance until ‘+++++’ as maximal resistance. (Tolerance level was evaluated by relative growth level of variants to that of wild type in the media containing highest concentration of herbicide; ‘+’=1-9 fold higher tolerance, ‘++’=10-99 fold higher tolerance, ‘+++’=100-999 fold higher tolerance, ‘++++’=1,000-9,999 fold higher tolerance, ‘+++++’=more than 10,000 fold higher tolerance) FIGS. 2 to 12 show the tolerance of ApPPO1 wild type and its variants, and FIGS. 13 to 24 show that of MxPPO wild type and its variants. The concentrations of herbicides were written on the photographs of tolerance test. A dilution series (OD.sub.600=0.5, 0.05, 0.005, 0.0005, 0.00005) was made and spotted on LB agar plates supplemented with herbicides.

[0213] As shown in Tables 7 to 11 and FIGS. 2 to 24, all of BT3 strains transformed with variants of ApPPO1 or MxPPO showed higher tolerance level than that of wild type against various PPO-inhibiting herbicides.

Example 4: Measurement of PPO Enzyme Activity and IC.SUB.50 .Value for Herbicides

[0214] The enzyme activities of variants wherein amino acids of certain position of PPO protein mutated were measured and inhibition assay with the PPO-inhibiting herbicides was conducted.

[0215] Although the solubility of PPO protein is markedly low in aqueous condition, it was greatly increased when maltose binding protein (MBP) was fused to PPO protein. Thus, PPO proteins of wild type and variants were expressed as fused to MBP and were used for experiments.

[0216] In order to express wild type and variant proteins of ApPPO1 and MxPPO, those genes were introduced into pMAL-c2x vector (refer to FIG. 25), respectively.

[0217] Detailed experimental procedure was as follows:

[0218] Using primers listed in Table 13, PCR was carried out to amplify PPO genes under following condition.

[0219] PCR reaction mixture

[0220] Template (synthetic DNA of ApPPO1 or MxPPO) 1 μl

[0221] 10× buffer 5 μl

[0222] dNTP mixture (10 mM each) 1 μl

[0223] Forward primer (10 μM) 1 μl

[0224] Reverse primer (10 μM) 1 μl

[0225] DDW 40 μl

[0226] Pfu-X (Solgent, 2.5 units/μl) 1 μl

[0227] Total 50 μl

TABLE-US-00013 TABLE 12 PCR reaction condition 94° C. 4 min. 1 cycle 94° C. 30 sec. 27 cycles 56° C. 30 sec. 72° C. 5 min. 72° C. 5 min. 1 cycle  4° C. 5 min. 1 cycle

TABLE-US-00014 TABLE 13 Primer list for cloning of ApPPO1 and MxPPO in pMAL-c2x SEQ ID Strain Primer Sequence NO Auxenochlorella ApPPO1_ CCCCGGATC 83 protothecoides BamHIF CATGGCCGA GTACGACGT TGT ApPPO1_ CCCCGTCGA 84 SalIR CTCAGGTTG CCAGACTTT TAACGT Myxococcus MxPPO_ CCCCGGATC 85 xanthus BamHIF CATGCACCA TATGCCCCG AAC MxPPO_ CCCCGTCGA 86 SalIR CTCAAGGCG CGTGTGATG TATTAC

[0228] Amplified PCR products and pMAL-c2x vector (NEB, FIG. 25) were digested with BamHI and SalI restriction enzymes, and ligated to construct pMAL-c2x-ApPPO1 and pMAL-c2x-MxPPO plasmids using T4 DNA ligase (RBC, 3 units/μl).

[0229] ApPPO1 and MxPPO genes cloned in pMAL-c2x vector were mutated through site-directed mutagenesis using primers listed in Tables 4 and 5, respectively.

[0230] PCR reaction mixture

[0231] Template 1 μl

[0232] 10× buffer 5 μl

[0233] dNTP mixture (10 mM each) 1 μl

[0234] Forward primer (10 μM) 1 μl

[0235] Reverse primer (10 μM) 1 μl

[0236] DDW 40 μl

[0237] Pfu-X (Solgent, 2.5 units/μl) 1 μl

[0238] Total 50 μl

[0239] Then, BL21 CodonPlus(DE3) E. coli was transformed with constructs.

[0240] The transformed E. coli were cultured under the following conditions to express PPO proteins:

[0241] Induction: OD.sub.600=0.2, addition of IPTG to 0.3 mM final concentration;

[0242] Culture temperature: 23° C., 200 rpm shaking culture;

[0243] Culture time: 16 hrs;

[0244] Culture volume: 200 ml/1,000 ml flask.

[0245] After harvesting the cells, cell lysis and protein extraction were performed by the following process:

[0246] Extraction buffer: Column buffer (50 mM Tris-Cl, pH 8.0, 200 mM NaCl) 5 ml buffer/g cell;

[0247] Sonication: SONICS&MATERIALS VCX130 (130 watts);

[0248] 15 sec ON, 10 sec OFF for 5 min on ice;

[0249] Centrifugation at 4° C. for 20 minutes (20,000×g); and the supernatant obtained after the centrifugation was diluted at the ratio of 1:6 with column buffer.

[0250] The following process for purification of PPO protein was performed in a 4° C. cold room. Amylose resin (NEB) was packed to 1.5×15 cm column (Bio-Rad, Econo Columns 1.5×15 cm, glass chromatography column, max. vol), and the obtained protein extracts were loaded to the column at a flow rate of 0.2 ml/min. The column was washed with 3 column volumes of buffer and the presence of protein in the washing solution was examined. When the protein was no longer detected, the washing procedure was terminated. Then, the MBP-PPO protein was eluted with approximately 2 column volumes of buffer containing 20 mM maltose. The protein concentration of each eluent was determined and the elution was stopped when the protein was no longer detected. Ten microliter of each fraction was investigated for protein quantification and SDS-PAGE analysis. The highly pure fractions of PPO protein variants were used for the enzyme assay.

[0251] Since protoporphyrinogen IX, a substrate of PPO protein, was not commercially available, it was chemically synthesized in the laboratory. Overall process was performed in dark under nitrogen stream. Nine micrograms of protoporphyrin IX was dissolved in 20 ml of 20% (v/v) EtOH, and stirred under dark condition for 30 minutes. The obtained protoporphyrin IX solution was put into a 15 ml screw tube in an amount of 800 μl, and flushed with nitrogen gas for 5 minutes. To this, 1.5 g of sodium amalgam was added and vigorously shaken for 2 minutes. The lid was opened to exhaust hydrogen gas in the tube. Thereafter, the lid was closed and incubated for 3 minutes. The protoporphyrinogen IX solution was filtered using syringe and cellulose membrane filter. To 600 μl of the obtained protoporphyrinogen IX solution, approximately 300 μl of 2M MOPS [3-(N-morpholino) propanesulfonic acid] was added to adjust pH to 8.0. To determine the enzyme activity of PPO protein, a reaction mixture was prepared with the following composition (based on 10 ml): 50 mM Tris-Cl (pH 8.0); 50 mM NaCl; 0.04% (v/v) Tween 20; 40 mM glucose (0.072 g); 5 units glucose oxidase (16.6 mg); and 10 units catalase (1 μl).

[0252] Hundred and eighty microliters of a reaction mixture containing the purified PPO protein were placed in 96 well plates and 20 μl of purified PPO proteins were added. After 50 μl of the mineral oil was layered, the reaction was initiated by adding the substrate, protoporphyrinogen IX solution, to a final concentration of 50 μM. The reaction proceeded at room temperature for 30 min and the fluorescence of protoporphyrin IX was measured using Microplate reader (Sense, Hidex) (excitation: 405 nm; emission: 633 nm). To calculate the PPO enzyme activity, the protoporphyrinogen IX solution was kept open in the air overnight to oxidize the solution. To this, 2.7 N HCl was added, and the absorbance at 408 nm was measured. A standard curve was generated using standard protoporphyrin IX, and PPO activity was measured by calibration of protoporphyrin IX using the standard curve of protoporphyrin IX.

[0253] The enzyme activities of the obtained PPO wild type and variants were shown in Tables 14 to 15. Activities of variants were presented relatively compared to that of wild type.

[0254] The concentration of the PPO-inhibiting herbicides that inhibits the PPO enzyme activity of each PPO wild type and variants by 50% (IC.sub.50) was measured for each herbicide. The final concentrations of each herbicide were as follows: [0255] tiafenacil, flumioxazin and sulfentrazone: 0, 10, 50, 100, 250, 500, 1000, 2500, 5000, 10000 nM

[0256] The IC.sub.50 value, the concentration of the herbicide inhibiting the PPO enzyme activity to 50%, was calculated by adding the herbicide of the above concentrations.

[0257] The IC.sub.50 value for each herbicide was shown in the following Tables 14 and 15.

TABLE-US-00015 TABLE 14 Determination of IC.sub.50 of ApPPO1 wild type and mutants against various herbicides flumiox- sulfen- Activity tiafenacil azin trazone No. Mutation site (%) (nM) (nM) (nM) 1 WT 100 21 89 348 2 R140A 88 86 202 973 3 F209A 78 69 N.T N.T 4 V213C 85 81 163 526 5 A215C 89 76 N.T N.T 6 A215L 76 3,456 1,552 >10,000 7 V360M 59 75 N.T N.T 8 F386V 86 368 N.T N.T 9 L389T 11 716 N.T N.T 10 I402T 16 488 N.T N.T 11 Y422M 93 457 237 1,084 12 Y422I 91 2,974 911 1,496 13 Y422T 84 3,660 935 3,778 14 R140A + Y422M 29 1,564 332 1,977 15 F209A + Y422M 51 699 N.T N.T 16 V213C + Y422M 29 840 363 1,732 17 A215C + Y422M 58 3,541 N.T N.T 18 A215L + Y422M 34 >5,000 >5,000 >10,000 19 V360M + Y422M 8 1,162 N.T N.T 20 F386V + Y422M 65 756 N.T N.T 21 L389T + Y422M 15 1,956 N.T N.T 22 I402T + Y422M 21 4,187 N.T N.T 23 V360I + Y422I 16 3,282 N.T N.T 24 S362V + Y422I 21 4,836 N.T N.T N.T (Not tested)

TABLE-US-00016 TABLE 15 Determination of IC.sub.50 of MxPPO wild type and mutants against various herbicides flumiox- sulfen- Activity tiafenacil azin trazone No. Mutation site (%) (nM) (nM) (nM) 1 WT 100 242 24 534 2 R95A 43 2,366 154 >10,000 3 V164A 75 367 N.T N.T 4 I168C 47 550 80 1,162 5 A170C 86 1,684 546 4,571 6 A170L 40 >5,000 >5,000 >10,000 7 I311M 87 964 58 1,228 8 F329V 91 239 N.T N.T 9 L332T 87 1,005 78 4,769 10 I345T 33 2,206 N.T N.T 11 M365I 82 1,379 1,327 3,388 12 M365V 77 1,980 1,593 3,590 13 M365T 52 2,772 N.T N.T 14 R95A + M365I 42 >5,000 N.T N.T 15 R95A + M365V 40 >5,000 N.T N.T 16 I168C + M365I 45 1,677 N.T N.T 17 I168C + M365V 42 2,031 N.T N.T 18 A170C + M365I 78 2,449 1,848 >10,000 19 A170C + M365V 71 2,794 N.T N.T 20 A170L + M365I 33 >5,000 N.T N.T 21 A170L + M365V 40 >5,000 N.T N.T 22 I311M + M365I 75 3,327 N.T N.T 23 I311M + M365V 71 3,368 N.T N.T 24 L332T + M365I 80 2,857 N.T N.T 25 L332T + M365V 68 2,591 N.T N.T 26 R95A + I168C + 38 3,982 N.T N.T M365I 27 R95A + A170C + 41 >5,000 N.T N.T M365I 28 R95A + I311M + 37 >5,000 N.T N.T M365V 29 R95A + L332T + 38 >5,000 N.T N.T M365I 30 I168C + A170C + 45 3,577 N.T N.T M365V 31 I168C + I311M + 47 4,671 N.T N.T M365I 32 I168C + L332T + 49 3,196 N.T N.T M365V 33 A170C + I311M + 69 4,572 N.T N.T M365I 34 I311M + L332T + 55 >5,000 N.T N.T M365V 35 R95A + I168C + 33 >5,000 2,477 >10,000 A170C + M365I 36 R95A + A170C + 31 >5,000 N.T N.T I311M + M365V 37 R95A + A170C + 35 >5,000 N.T N.T L332T + M365I 38 R95A + I168C + 37 >5,000 1,891 >10,000 I311M + M365V 39 R95A + I168C + 34 >5,000 2,368 >10,000 L332T + M365I 40 R95A + I311M + 29 >5,000 2,996 >10,000 L332T + M365V 41 I168C + A170C + 44 >5,000 N.T N.T I311M + M365I 42 I168C + A170C + 40 4,537 N.T N.T L332T + M365V 43 A170C + I311M + 52 >5,000 3,627 >10,000 L332T + M365I 44 R95A + I168C + 17 >5,000 N.T N.T A170C + I311M + M365V 45 R95A + I168C + 18 >5,000 N.T N.T A170C + L332T + M365I 46 R95A + I168C + 12 >5,000 3,741 >10,000 I311M + L332T + M365V 47 I168C + A170C + 20 >5,000 N.T N.T I311M + L332T + M365V 48 R95A + I168C + 8 >5,000 >5,000 >10,000 A170C + I311M + L332T + M365V N.T (Not tested)

[0258] As shown in the Tables 14 and 15, it was demonstrated that variants of ApPPO1 and MxPPO proteins showed the significantly increased IC.sub.50 values against each herbicide compared to the wild type. Such results indicate that herbicide tolerance was increased by amino acid substitutions at specified positions of PPO protein. Although the data showed that ApPPO1 and MxPPO protein variants possess reduced enzyme activity compared to the wild type, it might be caused by the difference between the chloroplast environment where PPO functions and in vitro assay condition. Thus, when PPO variants are properly assembled and expressed to chloroplasts in plants, the enzyme activity would not be affected drastically.

Example 5. Generation of Arabidopsis thaliana Transformants Using ApPPO1 or MxPPO Variants and PPO-Inhibiting Herbicide Tolerance Test

[0259] 5-1. Construction of A. thaliana Transformation Vectors and Generation of A. thaliana Transformants

[0260] A. thaliana was transformed with a binary vector having ORF of a selectable marker, Bar gene (glufosinate-tolerant gene), and ORF of each gene of ApPPO1 variants, MxPPO, and MxPPO variants. The transgenic plant was examined for cross-tolerance towards glufosinate and PPO-inhibiting herbicides. The bar gene was also used to examine whether the transgene was stably inherited during generations. NOS promoter and E9 terminator were used for bar gene expression.

[0261] In order to express proteins of ApPPO1 variants, MxPPO, and MxPPO variants in plants, a CaMV35S promoter and a NOS terminator were used. Encoding genes of ApPPO1 variants, MxPPO, and MxPPO variants were introduced into binary vector using XhoI and BamHI restriction enzymes. Furthermore, for confirmation of the protein expression, hemagglutinin (HA) tag was fused to the C-terminal region of PPO protein coding gene using BamHI and SacI restriction enzymes. In addition, in order to transit protein to chloroplast, transit peptide (TP) coding gene (SEQ ID NO: 2) of AtPPO1 gene (SEQ ID NO: 87) was fused to N-terminal region of PPO protein coding gene using XbaI and XhoI restriction enzymes.

[0262] Each constructed vector was transformed to Agrobacterium tumefaciens GV3101 competent cell by freeze-thaw method. Agrobacterium GV3101 competent cells were prepared by following procedures, Agrobacterium GV3101 strain was cultured in 5 ml LB media at 30° C., 200 rpm for 12 hrs. The cells were subcultured in 200 ml of LB media at 30° C., 200 rpm for 3 to 4 hrs, and centrifuged at 3,000×g at 4° C. for 20 minutes. The cell pellet was washed with sterile distilled water, and then resuspended in 20 ml of LB media. Snap frozen 200 μl aliquots with liquid nitrogen were stored in a deep freezer.

[0263] Each transformed Agrobacterium was screened in spectinomycin-containing LB media. The screened colony was cultured in LB broth. After Agrobacterium cell was harvested from the culture media, it was resuspended in the solution containing 5% sucrose (w/v) and 0.05% Silwet L-77 (v/v) (Momentive Performance Materials Co., Ltd.) at an absorbance (OD.sub.600) of 0.8. By floral dipping method, A. thaliana wild type (Col-0 ecotype) was transformed, and then the T.sub.1 seeds were harvested after 1 to 2 months.

[0264] Transgenic plants were screened with glufosinate tolerance which was conferred by Bar gene expression in the binary vector. The obtained T.sub.1 seeds were sown in ½ MS media (2.25 g/l MS salt, 10 g/l sucrose, 7 g/l Agar) supplemented with 50 μM glufosinate, and the surviving plants were selected 7 days after sowing. They were, then, transplanted into soil and grown to obtain T.sub.1 plants.

[0265] In order to examine PPO-inhibiting herbicide tolerance of the transgenic plants, 4-week-old plants were evenly sprayed with herbicide (100 ml of 1 μM tiafenacil and 0.05% Silwet L-77 (v/v)) in 40×60 cm area (0.24 m.sup.2). While wild type A. thaliana (Col-0 ecotype) completely died within 7 days after treatment, each transgenic plant showed no damage to PPO-inhibiting herbicide treatment.

[0266] The T.sub.2 seeds were harvested from T.sub.1 transgenic plants and were sown to ½ MS media (2.25 g/l MS salt, 10 g/l sucrose, 7 g/l Agar) supplemented with 50 μM glufosinate. One week later, surviving plants were transplanted to soil.

[0267] 5-2. Verification of Herbicide Tolerance of Transformed Arabidopsis Plants (T.sub.2) Arabidopsis plants (T.sub.2) transformed with a gene encoding an ApPPO1 variant (Y422I, Y422L, Y422M, Y422V, or A215L+Y422M), MxPPO, or a MxPPO variant (M365I) were tested for their tolerance against herbicides.

[0268] The T.sub.2 seeds of ApPPO1 transgenic plants transformed with a gene encoding each of ApPPO1 variant (Y422I, Y422L, Y422M, Y422V, or A215L+Y422M), MxPPO, or a MxPPO variant (M365I) were sown to ½ MS media containing herbicide. Six days later, the extent of germination of each seeds was evaluated. A wild type A. thaliana (Col-0 ecotype) was used as a control. The obtained results are shown in FIG. 26 (ApPPO1 variant) and FIG. 27 (MxPPO wild type and MxPPO variant).

[0269] The concentrations of herbicide used are as follows:

[0270] FIG. 26: 0.1 μM tiafenacil, 0.3 μM saflufenacil, 0.1 μM flumioxazin, and 1 μM sulfentrazone, respectively; and

[0271] FIG. 27: 10 μM tiafenacil, 0.5 μM flumioxazin, and 5 μM sulfentrazone, respectively.

[0272] The seeds of wild type A. thaliana (Col-0 ecotype) germinated well in herbicide-free media, but did not normally germinate in herbicide-containing media as above. FIG. 26 demonstrates that each seeds of transgenic plants of ApPPO1 variants show excellent germinated rate and survival rate compared to those of the control Col-0. FIG. 27 demonstrates that each seeds of transgenic plants of MxPPO variants show excellent germinated rate and survival rate compared to those of the control Col-0 and MxPPO wild type.