EA045758B1 - GFAP OPTIONS AND METHODS OF APPLICATION - Google Patents

Description

Field to which the invention relates

The present invention relates to plant molecular biology, in particular to novel GPPD polypeptides that confer improved resistance to GPPD-inhibiting herbicides.

Prerequisites for creating the invention

4-Hydroxyphenylpyruvate dioxygenases (HPPDs) are enzymes that catalyze the conversion of para-hydroxyphenylpyruvate (herein abbreviated as HPP), a breakdown product of tyrosine, into homogentisate (herein abbreviated as GG), a precursor of tocopherol and plastoquinone in plants (Crouch N.P. et al (1997), Tetrahedron, 53, 20, 6993-7010, Fritze et al (2004), Plant Physiology 134:1388-1400). Tocopherol acts as a membrane-associated antioxidant. Plastoquinone, firstly, acts as an electron carrier between PSII and the cytochrome b6/f complex, and secondly, it is a redox cofactor for phytoene desaturase involved in the biosynthesis of carotenoids.

To date, more than 1000 nucleic acid sequences from various organisms that are present in the NCBI database have been annotated as encoding putative GPPD domain proteins. However, for most of them, it has not been demonstrated that the protein will have HFPD enzymatic activity, either in an in vitro or in planta approach, nor that such a HFPD protein can confer herbicide resistance to herbicides that inhibit HFPD at expression in the plant. Some GPPD proteins and their primary sequences have been described in the prior art, in particular GPPD proteins from bacteria such as Pseudomonas (Ruetschi et al., Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567), Kordia (WO 2011/076889) Synechococcus (WO 2011/076877), Acidobacterium and Mucilaginibacter (WO 2015/022634), Rhodococcus (WO 2011/076892), protists such as Blepharisma (WO 2011/076882), euryarchaeota such as Picrophilus (WO 2011/076885), algae such as Chlamydomonas reinhardtii (ES 2275365; WO 2011145015), Scenedesmus (WO 2015/022634), plants such as Arabidopsis (WO 96/38567, GENBANK® AF047834), carrots (WO 96/ 38567, GENBANK® 87257), Avena sativa (WO 2002/046387, WO 2011/068567), wheat (WO 2002/046387), Brachiaria platyphylla (WO 2002/046387), Cenchrus echinatus (WO 2002/046387), Lolium rigid um( WO 2002/046387), Festuca arundinacea (WO 2002/046387), Setaria faberi (WO 2002/046387), Eleusine indica (WO 2002/046387), Sorghum (WO 2002046387, WO 2012021785), corn (WO 2012/021785), Coptis japonica (WO 2006/132270), Lemna (WO 2015/022634), or mammals such as mouse or pig, or fungi such as Coccicoides (GENBANK® COITRP).

Inhibition of HPPD results in uncoupling of photosynthesis, deficiency of accessory light-harvesting pigments and, most importantly, destruction of chlorophyll by UV radiation and reactive oxygen species (bleaching) due to lack of photoprotection normally provided by carotenoids (Norris et al. (1995), Plant Cell 7 : 2139-2149). Discoloration of photosynthetically active tissues leads to inhibition of plant growth and death.

Several GPPD-inhibiting molecules (hereinafter referred to as GPPD-inhibiting herbicides), which inhibit the conversion of GPPD to GG, despite specific binding to the enzyme, have proven to be very effective herbicides.

Currently, most commercially available GPPD-inhibiting herbicides belong to one of the following chemical families:

1) triketones, for example, benzobicyclone [i.e. 3-[2-chloro-4-(methylsulfonyl)benzoyl]-4(phenylsulfanyl)bicyclo[3.2.1]oct-3-en-2-one]; sulcotrione [i.e. 2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione], mesotrione [i.e. 2-[4-(methylsulfonyl)-2nitrobenzoyl]-1,3-cyclohexanedione] (abbreviated herein as MST); tembotrione [i.e. 2-[2chloro-4-(methylsulfonyl)-3-[(2,2,2,-trifluoroethoxy)methyl]benzoyl]-1,3-cyclohexanedione]; tefuryltrione [i.e. 2-[2-chloro-4-(methylsulfonyl)-3-[[(tetrahydro-2-furanyl)methoxy]methyl]benzoyl]-1,3cyclohexanedione]]; bicyclopyrone [i.e. 4-hydroxy-3-[[2-[(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3pyridinyl]carbonyl]bicyclo[3.2.1]oct-3-en-2-one]; Phenquinotrione [i.e. 2-[[8-chloro-3,4-dihydro-4-(4methoxyphenyl)-3-oxo-2-quinoxalinyl]carbonyl]-1,3-cyclohexanedione], and as described in WO 2007088876, WO 2009016841, WO 2010089993, WO 2010116122, WO 2012002096, WO 201131658, WO 2012136703, JP 2013040141, WO 2013080484, WO 2014014904, WO 2014031971, US 20140106968;

2) diketonitriles, for example, 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1-[4-(methylsulfonyl)-2-trifluoromethylphenyl] - 3-(1 methylcyclopropyl)propane-1,3-dione;

3) isoxazoles, for example, isoxaflutole [i.e. (5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4(trifluoromethyl)phenyl]methanone]. In plants, isoxaflutole (abbreviated herein as IFT) is rapidly metabolized to DKN, a diketonitrile compound that exhibits GPPD inhibitory properties;

4) hydroxypyrazoles, for example, pyrazoxifene [i.e. 2-[[4-(2,4-dichlorobenzoyl)-1,3-dimethyl-1Hpyrazol-5-yl]oxy]-1-phenylethanone]; benzophenap [i.e. 2-[[4-(2,4-dichloro-3-methylbenzoyl)-1,3-dimethyl-1Hpyrazol-5-yl]oxy]-1-(4-methylphenyl)ethanone]; pyrazolinate [i.e. (2,4-dichlorophenyl)[1,3-dimethyl-5-[[(4-

- 1 045758 methylphenyl)sulfonyl]oxy]-1H-pyrazol-4-yl]methanone]; pyrasulfotol [i.e. (5-hydroxy-1,3-dimethyl1H-pyrazol-4-yl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone]; topramesone [i.e. [3-(4,5dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl-1H-pyrazol-4yl)methanone]; tolpyralate [i.e. 1-[[1-ethyl-4-[3-(2-methoxyethoxy)-2-methyl-4-(methylsulfonyl)benzoyl]1 H-pyrazol-5-yl]oxy]ethyl methyl carbonate];

5) No. (1,2,5-oxadiazol-3-yl)benzamides, as described in WO 2011035874 and WO 2012123416, WO 2012123409, EP 2562174, WO 2013064459, WO 2013087577, WO 2013124238, WO 2013124228, WO 2013164333, WO 2013037342 , WO 2014053473, WO 2014086737, WO 2015007662, WO 2015007632, WO 2015007633 and as described in WO 2013072300, WO 2013072402, WO 2013072450, WO 201 4184014, WO 2014184019, WO 2014184058, WO 2014192936, WO 2015052152, WO 2015052178 and Ш1,3 ,4-oxadiazol-2yl)benzamides as described in WO 2012126932 and EP 2562174, WO 2013064459, WO 2013087577, WO 2013124238, WO 2013124228, WO 2013124245, WO 20131 64333, WO 2013037342, WO 20141053473, WO 2014086737, WO 2015007662, WO 2015007632 , WO 2015007633; for example, 2-methyl-N5-methyl-1,3,4oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2); 2-chloro-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide; 2chloro-3-(ethylsulfonyl)-K-(5-methyl-1,3,4-oxadiazol-2-yl)-4-(trifluoromethyl)benzamide;

6) N-(tetrazol-5-yl)- or N(triazol-5-yl)arylcarboxamides, as described in WO 2012028579 and WO 2012123409, WO 2013017559, EP 2562174, WO 2013064459, WO 2013064457, WO 201 3087577, WO 2013104705, WO 2013124238, WO 2013124228, WO 2013124245, WO 2013164331, WO 2013164333, WO 2013174843, WO 2013037342, WO 2014053473, WO 201408673 7, WO 2015007662, WO 2015007632, WO2015007633; for example, 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide; 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide; 2-chloro-3(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4(trifluoromethyl)benzamide and as described in WO 2013072528, WO 2013076315, WO 2013076316, WO 2013083859, WO 2013092834, WO 2013139760, WO 2013144231, WO 2014126070, WO 2014135654, WO 2014184015, WO 2014184016, WO 2014184017, WO 201418407 3, WO 2014184074, WO 2014192936, WO 2015022284, WO 2015052153, WO 2015052173;

7) pyridazinone derivatives, as described in WO 2013050421 and WO 2013083774, WO 2014154828, WO 2014154882;

8) oxoprazine derivatives, as described in WO 2013054495;

9) N(triazol-2-yl)arylcarboxamides, as described in WO 2013144234, WO 2015007564;

10) triazinones, as described in WO 2014154829; And

11) pyrazolones, as described in EP 2881387 and EP 2881388.

These GPPD inhibitory herbicides can be used against grasses and/or broadleaf weeds in fields of crops that exhibit metabolic resistance, such as maize (Zea mays), rice (Oryza sativa) and wheat (Triticum aestivum), in which they degrade quickly (Schulz et al. (1993), FEBS letters, 318, 162-166; Mitchell et al. (2001), Pest Management Science, Vol 57, 120-128; Garcia et al. (2000), Biochem. , 39, 7501-7507; Pallett et al. (2001), Pest Management Science, Vol 57, 133-142). In order to broaden the range of applications of these GPPD-inhibiting herbicides, several efforts have been made to impart a level of resistance acceptable under field agronomic conditions to plants, particularly plants with no or insufficient metabolic tolerance.

With the exception of an attempt to bypass HFPD-mediated homogentisate production (US 6,812,010), overexpression of the sensitive enzyme was carried out in order to produce sufficient quantities of the target enzyme in the plant against the herbicide (WO96/38567). Overexpression of HPPD polypeptides resulted in better pre-emergence resistance to the diketonitrile derivative (herein abbreviated as DKN) - IFT, but the level of resistance was insufficient to cause resistance in post-emergence treatments (Matringe et al. (2005), Pest Management Science 61: 269276).

The third strategy was to mutate GPPD in such a way as to obtain a target enzyme that, while retaining its ability to catalyze the conversion of GPPD to GG, is less sensitive to GPPD inhibitors than the natural GPPD polypeptide before mutation.

This strategy has been successfully used to produce plants resistant to 2-cyano-3cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1-[4(methylsulfonyl)-2 -trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione (EP496630), two GPPD inhibitory herbicides belonging to the diketonitrile family (WO99/24585). Pro215Leu, Gly336Glu, Gly336Ile, and in particular Gly336Trp (mutated amino acid positions are indicated by reference to the wild type Pseudomonas fluorescens GPPD polypeptide corresponding to SEQ ID NO: 1 in accordance with the present invention) have been identified as mutations responsible for increased resistance to treatment with these diketonitriles herbicides.

More recently, the introduction of the Pseudomonas fluorescens GPPD gene into the plastid genome of tobacco and soybeans

- 2,045,758 beans were shown to be more effective than nuclear transformation in conferring resistance to post-emergence treatment through IFT (Dufourmantel et al. (2007), Plant Biotechnol J.5(1):118-33).

In WO 2004/024928, the inventors sought to increase the biosynthesis of prenylquinone (eg, the synthesis of plastoquinones, tocopherols) in plant cells by increasing the flux of the GFP precursor into the plant cells. This was achieved by connecting the synthesis of the mentioned precursor to the shikimate pathway through overexpression of the enzyme prephenate dehydrogenase (PDH). They also noted that transforming plants with a gene encoding the PDH enzyme and a gene encoding the GPPD enzyme makes it possible to increase the resistance of these plants to herbicides that inhibit GPPD.

Application WO 2009/144079 discloses nucleic acid sequences encoding hydroxyphenylpyruvate dioxygenase (HPPD) with specific mutations at position 336 of the GPPD protein of Pseudomonas fluorescens and their use for producing plants resistant to herbicides that inhibit HFPD.

Application WO 2002/046387 identified several plant-derived GPPD polypeptide domains that may be essential for conferring resistance to various GPPD-inhibiting herbicides, but provided no in planta or biochemical data to support the effects of the described domain functions.

Application WO 2008/150473 exemplified a combination of two different resistance mechanisms - a modified Avena sativa gene encoding a mutant GPPD enzyme and a maize monooxygenase CYP450 (nsf1 gene) - to obtain improved resistance to GPPD-inhibiting herbicides, but was not disclosed data demonstrating synergistic effects based on the combination of both proteins.

In addition, a method for creating plants resistant to herbicides that inhibit GPPD by overexpressing not only the gene encoding resistant GPPD, such as from Avena sativa (US 2011/0173718) or Arabidopsis (WO 2013/064964, WO 014/177999) is disclosed. , as well as in combination with some plant genes encoding the HST protein (homogentisate solanesyl transferase). However, the level of resistance to some selected GPPD-inhibiting herbicides was quite limited.

WO 2011/094199 and US2011/0185444 assessed the resistance of several hundred wild-type soybean lines to the GPPD inhibitor IFT.

Very few lines showed the required level of herbicide resistance. A putative QTL (quantitative trait locus) responsible for resistance was identified. In this region of the genome, the gene encoding the ABC transporter was identified as the main trait responsible for improved resistance to the herbicide inhibiting GPPD. However, transgenic plants expressing the identified genes did not show any improvement in resistance to the tested GPPD-inhibiting herbicides.

Applications WO 2010/085705 and US 2014/0053295 disclose certain mutants of the Avena sativa GPPD polypeptide. WO 2010/085705 showed that some of the variants exhibited improved in vitro resistance to the triketone Mesotrione (herein abbreviated as MST), however, only a small number of mutants were expressed in tobacco plants. In addition, none of the tobacco plants expressing these mutants exhibited increased resistance to MST or IFT compared with tobacco plants expressing the wild-type Avena sativa GPPD gene. Application US 2014/0053295 describes that few Avena sativa GPPD mutants were expressed in soybean plants and had good levels of MST resistance as known from plants expressing the wild type Avena sativa GPPD gene. However, other herbicides such as tembotrione or IFT induced significantly higher leaf damage in these soybean plants.

US 2012/0042413 describes mutant maize GPPD polypeptides that have GPPD activity, and also demonstrate a certain insensitivity to at least one herbicide that inhibits GPPD and in addition proposes a certain set of mutations at different positions of the GPPD polypeptides and ultimately discloses biochemical data, and the resistance levels of plants containing some of these mutated HPPD polypeptides. EP 2453012 describes some mutants of GPPD polypeptides; however, the increased resistance of the described mutants in planta to some herbicides that inhibit GPPD is not shown.

WO 2014/043435 describes recombinant nucleic acid molecules encoding Pseudomonas spp. GPPD polypeptides consisting of an amino acid sequence comprising a proline at an amino acid position corresponding to amino acid position 335 SEQ ID NO: 1; or proline at an amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1 and tryptophan at an amino acid position corresponding to amino acid position 336 of SEQ ID NO: 1; or serine at an amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, serine at an amino acid position corresponding to amino acid position 336 of SEQ ID NO: 1, threonine at an amino acid position corresponding to

- 3045758 amino acid position 339 of SEQ ID NO: 1, and glutamine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO: 1; or tryptophan at an amino acid position corresponding to amino acid position 188 of SEQ ID NO: 1 and tryptophan at an amino acid position corresponding to amino acid position 336 of SEQ ID NO: 1; or proline at an amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, serine at an amino acid position corresponding to amino acid position 336 of SEQ ID NO: 1, and glutamic acid at an amino acid position corresponding to amino acid position 340 of SEQ ID NO: 1; or proline at an amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, tryptophan at an amino acid position corresponding to amino acid position 336 of SEQ ID NO: 1, alanine at an amino acid position corresponding to amino acid position 339 of SEQ ID NO: 1, and glutamine at amino acid position position corresponding to amino acid position 340 SEQ ID NO: 1.

Currently described and partially commercialized GPPD inhibitory herbicides act as slow-binding or slow-binding inhibitors (see Morrison (1982) Trends Biochem. Sci. 7, 102-105). These inhibitors bind slowly (i.e., with a low rate of association, kon) but not covalently to the GPPD polypeptide (i.e., they produce time-dependent inhibition), and are released very slowly (i.e., at an extremely low rate dissociation, koff) due to their extremely strong interaction with the enzyme.

These inhibitors bind so strongly that stoichiometric titrations with the enzyme are possible.

It is becoming increasingly clear that slow-binding or slow-binding inhibitors are not only exceptionally potent GPPD inhibitors, but also have attributes that make them attractive agrochemicals for weed control. The slow rate of dissociation enhances the effectiveness of the inhibitor to such an extent that ideally only one molecule of inhibitor per active site of the GPPD polypeptide is sufficient to completely inhibit its activity and maintain this level of inhibition for a long period of time even in the absence of free inhibitor molecules in the plant cell. This determines the low application rates of these inhibitors necessary to combat unwanted weeds in areas where agricultural crops are cultivated.

The properties of slow-binding or slow-binding inhibitors are advantageous when the goal is to achieve GPPD inhibition and herbicidal activity. However, these same properties are a big drawback when it is necessary to create GPPD polypeptides that are resistant to these inhibitors.

Mutations in the GPPD polypeptide that only reduce the affinity of the inhibitor for the enzyme (ki) cannot completely overcome GPPD inhibition, since binding of the inhibitor and inhibition of the GPPD polypeptide may still occur, therefore, the achieved level of inhibition will be maintained over a long period of time, even with absence of free inhibitor in the plant cell. In addition, the partially commercially available GPPD inhibitory herbicides belong to structurally distinct chemical classes, such as triketones, diketonitriles, isoxazoles, hydroxypyrazoles, N-(1,2,5-oxαdiαzol-3-yl)benzamides, N-(1, 3,4-oxadiazol-2yl)benzamides, N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides, pyridazinone derivatives, oxoprazine derivatives, N-(triazol-2-yl), triazinones and pyrazolones. Current GPPD polypeptides described in the art exhibit a fairly narrow range of resistance to structurally different GPPD inhibitory herbicides.

Due to the above kinetic properties of all currently described and partially commercially available GPPD-inhibiting herbicides, no GPPD-inhibiting herbicide-resistant plants with complete resistance to GPPD-inhibiting herbicides have been reported to date, despite numerous efforts to develop them. .

The essence of the invention

The present invention describes GPPD polypeptides and plants containing them that exhibit complete resistance to one or more GPPD-inhibiting herbicides belonging to different chemical classes. It turned out that in order to create such GPPD polypeptides with increased maximum and broad resistance to certain classes of GPPD-inhibiting herbicides, it is important to reduce the affinity for the GPPD polypeptide (ki) relative to the corresponding GPPD-inhibiting herbicides and at the same time provide a higher dissociation rate ( koff) slow-binding or slow-binding inhibitor, as known from wild-type GPPD polypeptides and some mutant GPPD polypeptides, to achieve a high level of inhibitor stability.

In the present invention, this problem was solved by developing a set of GPPD polypeptides that either have no or only significantly reduced affinity for GPPD-inhibiting herbicides and, at the same time, the rate of dissociation of GPPD-inhibiting herbicides is increased to such an extent that the herbicides HFPD inhibitors no longer act as slowly

- 4 045758 binding or slow, tightly binding inhibitors, and instead become completely reversible inhibitors.

The present invention provides compositions and methods for producing a new set of GPPD polypeptides having the above characteristics (i.e., having no or only significantly reduced affinity for GPPD-inhibiting herbicides, an increased dissociation rate of GPPD enzyme-inhibiting herbicides; herbicides that inhibit GPPD enzyme more do not act as slow-binding or slow-binding inhibitors, but instead become fully reversible inhibitors). The compositions include GPPD polypeptides and isolated, recombinant or chimeric nucleic acid molecules encoding such GPPD polypeptides, vectors and host cells containing these nucleic acid molecules. The compositions also include antibodies to these polypeptides. The nucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Nucleotide sequences can be synthetic sequences designed for expression in an organism, including, but not limited to, a microorganism or a plant.

The compositions contain nucleic acid molecules encoding herbicide-resistant GPPD polypeptides, including nucleic acid molecules encoding a GPPD polypeptide containing (a) proline at an amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (b) histidine or aspartic acid at a position corresponding to amino acid position 336 of SEQ ID NO: 1, and (c) a serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1 and, optionally, one or more additional amino acid substitutions at positions corresponding to amino acid positions 204, 213, 264, 268 , 270, 310, 315, 330, 331, 338, 339, 340, 344, 345 SEQ ID NO: 1, including the GPPD polypeptides set forth in any of SEQ ID NO: 3-108, as well as fragments thereof.

The compositions also contain transformed plants, plant cells, tissues and seeds that are resistant to GPPD inhibitor herbicides due to the introduction of a nucleic acid sequence in accordance with the invention into the genome of the plants, plant cells, tissues and seeds. The introduction of the sequence allows herbicides that inhibit GPPD to be applied to plants to selectively kill weeds or other non-transformed plants that are sensitive to the GPPD inhibitor, rather than the transformed organism. The sequences can also be used as a marker for the selection of plant cells grown in the presence of one or more herbicides that inhibit GPPD.

Additionally provided are methods for identifying GPPD polypeptides with herbicide resistance activity against a GPPD inhibitor.

The compositions and methods of the invention are useful for creating organisms with enhanced resistance to herbicides that inhibit HPPD. These organisms and compositions containing the organisms are suitable for agricultural purposes. Plants or seeds containing a nucleic acid sequence encoding a GPPD polypeptide in accordance with the invention can be grown in the field and harvested to obtain a plant product. The compositions of the invention are also useful for detecting the presence of GPPD-inhibiting herbicide-resistant polypeptides or nucleic acids in products or organisms.

Brief description of the figures

In fig. 1 is a simplified diagram of a coupled HPPD activity assay used in the present invention to determine the enzymatic activity of exemplary HPPD polypeptides.

In fig. Figure 2 shows the approximate kinetic changes in absorbance at 320 nm (Abs320) in samples of untreated wild-type and knockout GPPD polypeptide extracts observed with 200 µM GPPD and 0, 4 or 13 µM comp. 1 (2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4(trifluoromethyl)benzamide) according to Example 3 in the related HPPD activity assay. The GPPD knockout polypeptide was prepared by replacing histidine with alanine at the amino acid position corresponding to amino acid position 162 of SEQ ID NO: 1. This position is well known for its importance due to its involvement in the coordinate binding of the iron atom in the active site of the GPPD polypeptide (Serre et al. ( 1999), Structure, 7, 977-988).

In fig. 3 shows the approximate absorbance kinetic changes at 320 nm (Abs320) of the purified mutant GPPD polypeptide corresponding to SEQ ID NO: 17 in accordance with Example 3, observed at high substrate concentrations and with 0, 48, 240 or 1200 μM comp. 2 (2-methylN-(5-methyl-1,3,4-oxαduαzol-2-ul)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide) in a related GPPD activity assay. The apparent kinetic constant (k _app ) was determined as the change in signal over time (delta_Abs320/min) with a limited time interval.

In fig. 4 depicts data from an example determination of ki with a purified mutant GPPD polypeptide corresponding to SEQ ID NO: 17, with various concentrations of inhibitor and substrate (GPPD) by fitting according to the competitive inhibition model:

a) kinetic changes in absorption at 320 nm over time (delta_Abs320/min) at

- 5 045758 present 0 - 0.0012 M Conn. 2 at a given substrate concentration, in accordance with example 3;

b) kinetic changes in absorption at 320 nm over time (delta_Abs320/min) in the presence of 0 - 0.0012 M Comp. 1 (2-chloro-3-(methylsulfanyl)-N-(1 -methyl-1H-tetrazol-5-yl)-4(trifluoromethyl)benzamide) at a given substrate concentration, in accordance with example 3;

c) kinetic changes in absorption at 320 nm over time (delta_Abs320/min) in the presence of 0 - 0.0012 M MST at a given substrate concentration, in accordance with example 3;

d) kinetic changes in absorption at 320 nm over time (delta_Abs320/min) in the presence of 0 - 0.0012 M DKN at a given substrate concentration, in accordance with example 3.

All exemplary GPPD polypeptides, which are given in table. 2, 3, 4, and 5 were measured and analyzed as shown, for example, with SEQ ID NO: 17 in FIG. 3 and 4.

Detailed Description of the Invention

Hereinafter, the present invention will be described more fully with reference to the accompanying figures, which depict some, but not all, embodiments of the invention. Of course, these inventions can be embodied in many different forms and should not be construed as being limited to the embodiments given in the present invention; rather, these embodiments are presented in such a way that this disclosure satisfies applicable legal requirements. The same reference numerals refer to the same elements throughout the description.

Many modifications and other embodiments of the inventions set forth herein may occur to one skilled in the art to which these inventions relate, having benefit from the concepts presented in the above descriptions and the accompanying drawings. It is therefore understood that the inventions are not intended to be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are used in the present invention, they are used in a general and descriptive sense only and not for purposes of limitation.

Review.

Several attempts have been made to impart agronomically acceptable levels of resistance to a wide range of GPPD-inhibiting herbicides to plants, including bypassing GPPD-mediated homogentisate production (US 6,812,010), overexpressing a sensitive enzyme to produce sufficient amounts of the target enzyme in the plant relative to the herbicide (WO 96/ 38567), and mutation of GPPD to obtain a target enzyme that, while retaining its properties to catalyze the conversion of GPPD to homogentisate, is less sensitive to GPPD inhibitors than natural GPPD before the mutation.

Despite these advances in developing plants exhibiting resistance to several of the GRPD-inhibiting herbicides described above, there is still a need to develop and/or improve plant resistance to newer or to several different GPPD-inhibiting herbicides belonging to different chemical classes, in especially for GPPD-inhibiting herbicides belonging to classes such as triketones (e.g., benzobicyclone, sulcotrione mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione), diketonitriles, isoxazoles (e.g., isoxaflutole), hydroxypyrazoles (e.g., pyrazoxyphene, benzophenap, pyrazolinate, pyrasulfotol , topramesone, tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides, N-(1,3,4-oxαduαzol-2yl)benzamides (for example, 2-methyl-N-(5-methyl -1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), N-(tetrazol-5-yl)- or N(triazole -5-yl)arylcarboxamides (e.g. 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1 -methyl-1Htetrazol-5-yl)benzamide), 4-(difluoromethyl)-2-methoxy-3 -(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5yl)benzamide); 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1Htetrazol-5-yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, triketones, N-(triazol-2-yl) arylcarboxamides, triazinones and pyrazolones.

Thus, the present invention provides improved compositions and methods for controlling herbicidal resistance to a GPPD inhibitor. GPPD inhibitory herbicides such as triketone herbicides (e.g. benzobicyclone, sulcotrione mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione), diketonitriles, isoxazoles (e.g. isoxaflutole), hydroxypyrazoles (e.g. pyrazoxyphene, benzophenap, pyrazolinate, pyrasulfotol, topramesone , tolpyralate), N-(1,2,5-oxaduαzol-3-yl)benzamides, N-(1,3,4-oxadiazol-2-yl)benzamides (for example, 2-methyl-N(5-methyl- 1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), N-(tetrazol-5-yl)- or N-( triazol-5-yl)arylcarboxamides (e.g. 2chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy3-( methylsulfonyl)-N-(1 -methyl-1H-tetrazol-5-yl)benzamide; 2-chloro-3-(methylsulfanyl)-M-(1 -methyl-1Htetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, derivatives pyridazinone, oxoprazine derivatives, K-(triazol-2-yl)arylcarboxamides, triazinones and pyrazolones have excellent herbicidal effectiveness against a wide range of economically important monocotyledonous and dicotyledonous annual weeds. The active substances also have a good ability to control perennial pests that are difficult to control and spread from rhizomes, root taps or other perennial organs. In the context of the present invention, the term herbicide should be understood as a herbicidal active substance by itself or such a substance that is combined with an additive that modifies its effectiveness, such as, for example, an agent that increases its activity (synergistic agent), or an agent limiting its activity (safener). ). The herbicide may also contain solid or liquid adjuvants or carriers commonly used in formulation technology (for example, natural or regenerated minerals, solvents, dispersants, wetting agents, tackifying agents, emulsifiers, growth promoting agents, etc.), and also one or more other herbicides and/or one or more pesticides (for example, insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bactericides, nematicides, molluscicides, etc.).

The methods involve transforming organisms with nucleotide sequences encoding a GPPD-inhibiting herbicide resistance gene in accordance with the invention or otherwise introducing such GPPD-inhibiting herbicide resistance genes into organisms that do not contain them (for example, by mating, cell fusion, or crossing of organisms containing the introduced a gene for resistance to a herbicide that inhibits GPPD in accordance with the invention with organisms that do not contain it and obtaining offspring containing such a gene). The nucleotide sequences according to the invention are suitable for producing plants exhibiting increased resistance to GPPD-inhibiting herbicides, in particular increased resistance to GPPD-inhibiting herbicides from the class of triketones (preferably benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, or phenquinotrione), diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxyphene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, or tolpyralate), K-(1,2,5-oxadiazol-3yl)benzamides, K-(1,3, 4-oxadiazol-2-yl)benzamides (preferably 2-methyl-N-(5-methyl-1,3,4oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), H-(tetrazol-5-yl)- or K-(triazol-5-yl)arylcarboxamides (preferably 2-chloro-3ethoxy-4-(methylsulfonyl)-K-(1-methyl-1H- tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide, 2-chloro-3-(methylsulfanyl)- M-(1-methyl-1Htetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2(methoxymethyl)-3-(methylsulfinyl)-K-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, Ch-(triazol-2-yl)arylcarboxamides , triazinones and pyrazolones.

Expression of a GPPD-inhibiting herbicide resistance gene according to the invention can also lead to resistance to coumaron-derived herbicides (described in WO 2009/090401, WO 2009/090402, WO 2008/071918, WO 2008/009908). In this regard, any of the GPPD-inhibiting herbicide resistance genes according to the invention can also be expressed in a plant also expressing a chimeric homogentisate solanesyltransferase (HST) gene or a mutated HST gene, as described in WO 2011/145015, WO 2013/064987, WO 2013/064964, or WO 2010/029311, for the production of plants resistant to HST-inhibiting herbicides. As used herein, the coumaron derivative herbicide or HST inhibitory herbicide covers compounds that fall within the IUPAC nomenclature 5H-thiopyrano[4,3-b]pyridin-8-ol, 5Hthiopyrano[3,4-b]pyrazin-8-ol , oxathiino[5,6-b]pyridin-4-ol, and oxathiino [5,6-b]pyrazin-4-ol.

Thus, by a GPPD-inhibiting herbicide resistance gene in accordance with the invention is meant a gene encoding a polypeptide that confers on a cell or organism the ability to tolerate higher concentrations of a GPPD-inhibiting herbicide than the same cell or organism that does not express the protein, or tolerate a certain concentration of a herbicide that inhibits GPPD for a longer period of time than the same cell or organism that does not express the protein, or that gives the cell or organism the ability to photosynthesize, grow, and/or reproduce with less damage or growth inhibition, than those observed in the same cell or organism that does not express a similar protein.

A GPPD-inhibiting herbicide resistance polypeptide includes a polypeptide that confers on a cell or organism the ability to tolerate higher concentrations of GPPD-inhibiting herbicides than the same cell or organism that does not express the protein, or to tolerate some concentration of GPPD-inhibiting herbicides for a longer period. period of time than the same cell or organism that does not express the polypeptide, or which confers on the cell or organism the ability to photosynthesize, grow, and/or reproduce with less damage or growth inhibition than is observed in the same cell or organism that does not express similar polypeptide.

- 7 045758

The term polypeptide includes proteins such as enzymes, antibodies and polypeptides of medium length and short peptides up to an amino acid sequence length of less than ten.

The term enzyme as used herein means any polypeptide that catalyzes the reaction in which para-hydroxyphenylpyruvate is converted to a homogentisate. It includes natural enzymes as well as enzyme variants and their derivatives. It also contains any fragment of such an enzyme, as well as variants engineered by insertion, deletion, recombination and/or any other method, resulting in enzymes that differ in amino acid sequence from the naturally occurring enzyme or enzyme variants. It also contains protein molecules with post-translational and/or chemical modifications, such as glycosylation, gammacarboxylation and acetylation, any molecular complex or fusion protein containing one of the above-mentioned proteins.

The terms variant polypeptide or mutant polypeptide mean any polypeptide molecule obtained by mutagenesis, preferably by site-directed or random mutagenesis, with an altered amino acid sequence compared to the corresponding wild-type sequence. Tolerate, tolerant, or resistant is meant to mean either the ability to withstand a particular application of a GPPD-inhibiting herbicide, or the ability to perform essential cellular functions such as photosynthesis, protein synthesis, or respiration and reproduction in a manner that is not demonstrably different from untreated cells or organisms, or same ability to have no significant difference in yield or even have improved yield in plants treated with a GPPD-inhibiting herbicide compared to the same plants not treated with the same herbicide (but where weeds have been removed or prevented by a mechanism different from application of a GPPD-inhibiting herbicide , such as the methods described in WO 2011/100302, which is incorporated herein by reference in its entirety).

In addition to imparting resistance to a cell to a herbicide that inhibits GPPD, the GPPD nucleic acid sequences according to the invention encode polypeptides having GPPD activity, i.e. catalyzing the reaction in which para-hydroxyphenylpyruvate (HPP) is converted to a homogentisate. The catalytic activity of a HPPD polypeptide can be determined by various methods known in the art. Applications WO 2009/144079 and WO 2014/043435 describe various suitable screening methods.

The enzymatic activity of GPPD polypeptides can be measured by any method that allows either to determine the reduction in the amount of GPPD or O ₂ substrates, or to measure the accumulation of any products obtained from the enzymatic reaction, i.e. homogentisate or CO ₂ . In particular, GPPD activity can be measured by the method described in WO 2009/144079; Garcia et al. (1997), Biochem. J. 325, 761-769; Garcia et al. (1999), Plant Physiol. 119, 1507-1516; or WO 2012/021785, which are incorporated herein by reference in their entirety.

For purposes of the present invention, a reference GPPD polypeptide (or a GPPD gene encoding such a polypeptide) is any GPPD polypeptide or nucleic acid to which a GPPD polypeptide or GPPD nucleic acid is compared in accordance with the invention. For purposes of describing the HPPD polypeptides of the present invention, the terms protein and polypeptide are used interchangeably. This reference GPPD polypeptide may be a natural plant, bacterial or animal GPPD, or may be a mutated GPPD polypeptide known in the art, such as the mutants PfP215L and PfG336F, International Application Publication WO 2009/144079, or may be any of the proteins PfHPPDevo33, PfHPPDevo36, PfHPPDevo37, PfHPPDevo40, or PfHPPDevo41 from WO 2014/043435; PfHPPDevo41 is set forth herein as SEQ ID NO: 2. Such a HPPD reference polypeptide can be used to determine whether a HPPD polypeptide or nucleic acid of the invention has a particular relevant property (e.g., improved, comparable, or reduced resistance to a HPPD inhibitory herbicide or enzymatic activity of the GPPD polypeptide; improved, comparable or reduced expression in the host cell; improved, comparable or reduced protein stability, etc.).

In various embodiments of the present invention, the herbicide-resistant GPPD inhibitor polypeptide encoded by a nucleic acid (including isolated, recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing the nucleic acid thereof, GPPD polypeptides and compositions encoded by nucleic acid, as well as methods of using a polypeptide encoded by the nucleic acid to increase plant resistance to herbicides that inhibit GPPD, in particular increasing resistance to herbicides that inhibit GPPD from the class of triketones (preferably benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinothrone) , diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxyphene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides, N-(1,3,4 -oxαduαzol-2yl)benzamides (preferably 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4- 8 045758 (trifluoromethyl)benzamide (also mentioned in hereinafter referred to as Conn. 2), N-(tetrazol-5-yl)- or N(triazol-5-yl)arylcarboxamides (preferably 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1Htetrazol-5- yl)benzamide, 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5yl)benzamide, 2-chloro-3-(methylsulfanyl)-N-(1- methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1Htetrazol-5 -yl)-4-(trifluoromethyl)benzamide), pyridazinone derivatives, oxoprazine derivatives, N(triazol-2-yl)arylcarboxamides, triazinones and pyrazolones) have (a) a proline at the amino acid position corresponding to amino acid position 335 SEQ ID NO: 1 , (b) a histidine or aspartic acid at a position corresponding to amino acid position 336 of SEQ ID NO: 1, and (c) a serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1 and, optionally, one or more additional amino acid substitutions at positions , corresponding to amino acid positions 204, 213, 264, 268, 270, 310, 315, 330, 331, 338, 339, 340, 344, 345 SEQ ID NO: 1, including GPPD proteins set forth in any SEQ ID NOs: 3- 108. By relevant is meant the position of a nucleotide or amino acid relative to that position in SEQ ID NO: 1 when two (or more) sequences are aligned using standard alignment algorithms. The term position in a polynucleotide or polypeptide refers to specific single bases or amino acids in the sequence of a polynucleotide or polypeptide, respectively. The term site in a polynucleotide or polypeptide refers to a specific position or region in the sequence of a polynucleotide or polypeptide, respectively. The term polynucleotide corresponds to any genetic material of any length and any sequence containing single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural genes, gene groups, plasmids, whole genomes and their fragments.

In one embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) consists of an amino acid sequence containing (a) proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (b) histidine or aspartic acid at the position corresponding to amino acid position 336 of SEQ ID NO: 1, and (c) serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1, and wherein said GPPD polypeptide is resistant to one or more GPPD-inhibiting herbicides.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ), which is resistant to one or more GPPD-inhibiting herbicides, consists of an amino acid sequence containing (a) proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (b) histidine or aspartic acid at the amino acid position position 336 of SEQ ID NO: 1, and (c) a serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1, and additionally containing

i) methionine, threonine, serine or leucine at an amino acid position corresponding to amino acid position 204 of SEQ ID NO: 1; and/or ii) lysine or leucine at an amino acid position corresponding to amino acid position 213 of SEQ ID NO: 1; and/or iii) arginine, lysine, glutamine, or leucine at an amino acid position corresponding to amino acid position 264 of SEQ ID NO: 1; and/or iv) arginine, glycine or serine at an amino acid position corresponding to amino acid position 268 of SEQ ID NO: 1; and/or

v) arginine, leucine, glutamic acid, proline or serine at an amino acid position corresponding to amino acid position 270 SEQ ID NO: 1; and/or vi) a serine, histidine or lysine at an amino acid position corresponding to amino acid position 310 of SEQ ID NO: 1; and/or vii) arginine, methionine or histidine at an amino acid position corresponding to amino acid position 315 of SEQ ID NO: 1; and/or viii) histidine, alanine, phenylalanine, valine or glycine at an amino acid position corresponding to amino acid position 330 SEQ ID NO: 1; and/or

- 9 045758 ix) proline, histidine, serine, isoleucine or leucine at the amino acid position corresponding to amino acid position 331 SEQ ID NO: 1; and/or

x) valine at the amino acid position corresponding to amino acid position 338 SEQ

ID NO: 1; and/or xi) glutamic acid, arginine, alanine or threonine at an amino acid position corresponding to amino acid position 339 of SEQ ID NO: 1; and/or xii) arginine, glutamine, methionine, glutamic acid, glycine, leucine, or valine at an amino acid position corresponding to amino acid position 340 of SEQ ID NO: 1; and/or xiii) glutamine, proline, or arginine at an amino acid position corresponding to amino acid position 344 of SEQ ID NO: 1; and/or xiv) lysine, arginine, methionine, alanine or valine at an amino acid position corresponding to amino acid position 345 of SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) that is resistant to one or more herbicides that inhibit GPPD consists of an amino acid sequence containing (a) proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (b) histidine or aspartic acid at the position corresponding to amino acid position 336 SEQ ID NO: 1, and (c) a serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1, and additionally containing

i) leucine or lysine at an amino acid position corresponding to amino acid position 213 of SEQ ID NO: 1; and/or ii) arginine or leucine at an amino acid position corresponding to amino acid position 264 of SEQ ID NO: 1; and/or iii) arginine, glycine or serine at an amino acid position corresponding to amino acid position 268 of SEQ ID NO: 1; and/or iv) glutamic acid or serine at an amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1; and/or

v) arginine or methionine at an amino acid position corresponding to amino acid position 315 of SEQ ID NO: 1; and/or vi) a histidine at an amino acid position corresponding to amino acid position 330 of SEQ ID NO: 1; and/or vii) valine at an amino acid position corresponding to amino acid position 338 of SEQ ID NO: 1; and/or viii) arginine or valine at an amino acid position corresponding to amino acid position 340 of SEQ ID NO: 1; and/or ix) glutamine at an amino acid position corresponding to amino acid position 344 of SEQ ID NO: 1; and/or

x) lysine, valine or methionine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) that is resistant to one or more herbicides that inhibit GPPD consists of an amino acid sequence containing (a) proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (b) histidine or aspartic acid at the position corresponding to amino acid position 336 SEQ ID NO: 1, and (c) a serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1 and additionally containing

i) lysine at an amino acid position corresponding to amino acid position 213 of SEQ ID NO: 1; and/or ii) arginine or leucine at an amino acid position corresponding to amino acid position 264 of SEQ ID NO: 1; and/or iii) glycine or arginine at an amino acid position corresponding to amino acid position 268 of SEQ ID NO: 1; and/or

- 10 045758 iv) glutamic acid at the amino acid position corresponding to amino acid position 270 SEQ ID NO: 1; and/or

v) arginine at an amino acid position corresponding to amino acid position 315 of SEQ

ID NO: 1; and/or vi) a histidine at an amino acid position corresponding to amino acid position 330 of SEQ ID NO: 1; and/or vii) valine at an amino acid position corresponding to amino acid position 338 of SEQ ID NO: 1; and/or viii) arginine or valine at an amino acid position corresponding to amino acid position 340 of SEQ ID NO: 1; and/or ix) glutamine at an amino acid position corresponding to amino acid position 344 of SEQ ID NO: 1; and/or

x) lysine, valine or methionine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) that is resistant to one or more herbicides that inhibit GPPD consists of an amino acid sequence containing (a) proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (b) histidine or aspartic acid at the position corresponding to amino acid position 336 SEQ ID NO: 1, and (c) serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1 and additionally containing (i) glycine or arginine at an amino acid position corresponding to amino acid position 268 of SEQ ID NO: 1, (ii) glutamic an acid at an amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1, (iii) valine at an amino acid position corresponding to amino acid position 340 of SEQ ID NO: 1; and (iv) valine at an amino acid position corresponding to amino acid position 345 of SEQ ID NO: 1,

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) that is resistant to one or more herbicides that inhibit GPPD consists of an amino acid sequence containing (a) proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (b) histidine or aspartic acid at the position corresponding to amino acid position 336 SEQ ID NO: 1, and (c) serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1 and further containing (i) lysine at an amino acid position corresponding to amino acid position 213 of SEQ ID NO: 1, (ii) glycine at amino acid position a position corresponding to amino acid position 268 of SEQ ID NO: 1, (iii) glutamic acid at an amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1, (iv) valine at an amino acid position corresponding to amino acid position 340 of SEQ ID NO: 1; and (v) valine at an amino acid position corresponding to amino acid position 345 of SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ), which is resistant to one or more GPPD-inhibiting herbicides, consists of an amino acid sequence containing

- 11 045758 (a) proline at amino acid position corresponding to amino acid position 335 SEQ

ID NO: 1, (b) histidine or aspartic acid at a position corresponding to amino acid position 336 of SEQ ID NO: 1, and (c) serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1 and additionally containing (i) lysine at amino acid position corresponding to amino acid position 213 of SEQ ID NO: 1, (ii) leucine at an amino acid position corresponding to amino acid position 264 of SEQ ID NO: 1, (iii) glycine at an amino acid position corresponding to amino acid position 268 of SEQ ID NO: 1, ( iv) glutamic acid at an amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1, (v) valine or arginine at an amino acid position corresponding to amino acid position 340 of SEQ ID NO: 1;

(vi) glutamine at an amino acid position corresponding to amino acid position 344 of SEQ ID NO: 1, and (vii) methionine at an amino acid position corresponding to amino acid position 345 of SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) that is resistant to one or more herbicides that inhibit GPPD consists of an amino acid sequence containing (a) proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, (b) histidine or aspartic acid at the position corresponding to amino acid position 336 SEQ ID NO: 1, and (c) serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1 and further containing (i) leucine at an amino acid position corresponding to amino acid position 264 of SEQ ID NO: 1, (ii) arginine at amino acid position position corresponding to amino acid position 268 of SEQ ID NO: 1, (iii) glutamic acid at amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1, (iv) arginine at amino acid position 315 corresponding to amino acid position of SEQ ID NO: 1, ( v) valine at the amino acid position corresponding to amino acid position 340 SEQ ID NO: 1;

(vi) glutamine at an amino acid position corresponding to amino acid position 344 of SEQ ID NO: 1, and (vii) methionine at an amino acid position corresponding to amino acid position 345 of SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) that is resistant to one or more herbicides that inhibit GPPD consists of an amino acid sequence containing

a) proline at an amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1, and

b) a histidine at a position corresponding to amino acid position 336 of SEQ ID NO: 1, or

c) aspartic acid at a position corresponding to amino acid position 336 of SEQ ID NO: 1, and

d) serine at a position corresponding to amino acid position 337 SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) consists of an amino acid sequence containing:

a) proline at the amino acid position corresponding to amino acid position 335 SEQ

ID NO: 1;

b) histidine at a position corresponding to amino acid position 336 of SEQ ID NO: 1 or aspartic acid at a position corresponding to amino acid position 336 of SEQ ID NO: 1;

c) serine at a position corresponding to amino acid position 337 SEQ ID NO: 1; And

d) histidine at a position corresponding to amino acid position 330 SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) that is resistant to one or more herbicides that inhibit GPPD consists of an amino acid sequence containing:

a) proline at an amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1;

b) histidine at a position corresponding to amino acid position 336 of SEQ ID NO: 1, or aspartic acid at a position corresponding to amino acid position 336 of SEQ ID NO: 1;

c) serine at a position corresponding to amino acid position 337 SEQ ID NO: 1; And

d) valine at a position corresponding to amino acid position 340 SEQ ID NO: 1.

In another embodiment, a GPPD polypeptide in accordance with the present invention (including a nucleotide sequence encoding it, and recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleotide sequence encoding a GPPD polypeptide in accordance with the invention ) that is resistant to one or more herbicides that inhibit GPPD consists of an amino acid sequence containing:

a) proline at an amino acid position corresponding to amino acid position 335 SEQ ID NO: 1

b) histidine at a position corresponding to amino acid position 336 of SEQ ID NO: 1 or aspartic acid at a position corresponding to amino acid position 336 of SEQ ID NO: 1;

c) serine at a position corresponding to amino acid position 337 SEQ ID NO: 1; And

d) valine at a position corresponding to amino acid position 345 SEQ ID NO: 1.

In table 1 shows the corresponding amino acid positions compared to the reference wild-type Pseudomonas fluorescens HPPD polypeptide (SEQ ID NO: 1), wherein variants of the HPPD polypeptide according to the invention contain three or more amino acid substitutions. Unless explicitly stated otherwise, exchanges at the corresponding amino acid positions always refer to the reference wild-type Pseudomonas fluorescens GPPD polypeptide corresponding to SEQ ID NO: 1.

Table 1 Overview of approximate amino acid exchanges relative to the polypeptide

HFPD corresponding to SEQ ID NO: 1

Amino acid position relative to SEQ ID NO: 1 Approximate amino acid exchanges 204 M, T, S, L 213 L, K 264 R, K, Q, L 268 G, S, R 270 R, L, Ε, Р, S 310 S, n, k 315 R, Μ, N 330 H, A, F, V, G

- 13 045758

331 I, Η, P, S, L 335 P 336 D,H 337 s 338 V 339 E, R, A, T 340 G, R, E, V, Q, M, L 344 Q, P, R 345 V, K, M, R, A

Amino acids are referred to in the present invention using the amino acid name, a three-letter abbreviation, or a one-letter abbreviation. The table below shows the list

standard amino acids along with their abbreviations. Alanin A Ala Cysteine WITH Cys Aspartic D Asp acid Glutamic acid E Glu Phenylalanine F Phe Glycine G Gly Histidine N His Isoleucine I He Lysine TO Lys Leucine L Leu Methionine M Met Asparagine N Asn Proline R Pro Glutamine Q Gin Arginine R Arg Serin S Ser Threonine T Thr Valin V Vai Tryptophan W Trp Tyrosine Y Tyr Cysteine C Cys

It is well known to one skilled in the art that the genetic code is degenerate, that is, more than one codon triplet can code for the same amino acid. Therefore, the amino acid sequences presented here can be generated by alternative sequences that use different codons to encode the same amino acid sequence.

In another embodiment, the sequence of the GPPD polypeptides in accordance with the invention is at least 53%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, according to at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth herein as SEQ ID NO: 1.

Exemplary HPPD sequences that may be modified in accordance with the present invention include sequences from bacteria, in particular Pseudomonas spp. type, more preferably from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas testosteroni (Comamonas testosteroni).

For the purposes of the present invention, the GPPD polypeptide in accordance with the invention may also include other modifications, for example, in which certain amino acids (for example, amino acids 1-17) have been replaced, added or deleted for cloning purposes, to create a transit fusion

- 14 045758 peptide, etc., which retains the activity of GPPD, i.e. the ability to catalyze the conversion of para-hydroxyphenylpyruvate to a homogentisate, or can be any HPPD polypeptide that can be further improved. For example, a HPPD polypeptide that may be further improved by the modifications described herein may be a variant of HPPD derived from Pseudomonas fluorescens, set forth herein as any of SEQ ID NOs: 3-108.

In a preferred embodiment, the GPPD polypeptides of the present invention that are resistant to one or more GPPD inhibitory herbicides are equivalent to SEQ ID NO: 1 (Pseudomonas fluorescens) in addition to the amino acids substituted in accordance with the present invention, i.e. the corresponding GPPD polypeptide is identical to SEQ ID NO: 1, but having (a) a proline at amino acid position 335 of SEQ ID NO: 1, (b) a histidine or aspartic acid at amino acid position 336 of SEQ ID NO: 1, and (c) a serine at amino acid position 337 SEQ ID NO: 1.

In another preferred embodiment, GPPD polypeptides of the present invention that are resistant to one or more GPPD inhibitory herbicides are equivalent to SEQ ID NO: 1 (Pseudomonas fluorescens) in addition to the amino acids substituted in accordance with the present invention, i.e. , the corresponding GPPD polypeptide is identical to SEQ ID NO: 1, but has one or more substitutions at the corresponding amino acid position according to the above table. 1, provided that proline exists at position 335 of SEQ ID NO: 1, histidine or aspartic acid exists at position 336 of SEQ ID NO: 1, and serine exists at position 337 of SEQ ID NO: 1. In another preferred embodiment, HFPD polypeptides in accordance with the present invention that are resistant to one or more herbicides that inhibit GPPD are equivalent to SEQ ID NO: 1 (Pseudomonas fluorescens) in addition to the amino acids replaced in accordance with the present invention, i.e., the corresponding GPPD polypeptide is identical to SEQ ID NO: 1, but has amino acid substitutions at the corresponding amino acid position(s) as defined in table. 2 (below) in lines SEQ ID NO: 7 - SEQ ID NO: 19 and SEQ ID NO: 21 - SEQ ID NO: 108.

In some embodiments, a nucleotide sequence in accordance with the invention (including isolated, recombinant and chimeric genes thereof, vectors, host cells, plants, plant parts and seeds containing a nucleic acid sequence, amino acid sequences and compositions thereof encoded by the nucleic acid sequence, as well as methods of using a nucleic acid sequence to increase plant resistance to herbicides that inhibit GPPD, in particular, increasing resistance to herbicides that inhibit GPPD from the class of triketones (preferably benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrion), diketonitriles, isoxazoles (preferably isoxaflutole) hydroxypyrazoles (preferably pyrazoxifene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate), N-(1,2,5-oxaduαzol-3-ul)benzamides, N-(1,3,4-oxadiazol-2- yl)benzamides (preferably 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), N-(tetrazol-5-yl)- or N(triazol-5-yl)arylcarboxamides (preferably 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1Htetrazol-5- yl)benzamide, 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5yl)benzamide, 2-chloro-3-(methylsulfanyl)-N-(1- methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1Htetrazol-5 -yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, N(triazol-2-yl)arylcarboxamides, triazinones and pyrazolones, encodes the amino acid sequence set forth in any of SEQ ID NOs: 3-108, and fragments thereof and variants that encode a GPPD polypeptide that inhibits herbicide resistance.

A. Methods for measuring resistance to a GPPD inhibitor.

To evaluate GPPD polypeptides in accordance with the invention, any suitable method for measuring resistance to GPPD-inhibiting herbicides can be used. Resistance may be measured by monitoring the ability of a cell or organism to overcome application of a particular GPPD-inhibiting herbicide, or the ability to perform essential cellular functions such as photosynthesis, protein synthesis, respiration, or reproduction in a manner that is not demonstrably different from untreated cells or organisms, or the ability to have no significant difference in yield or even have improved yield in plants treated with a GPPD-inhibiting herbicide compared to the same plants not treated with the same herbicide (but where weeds have been removed or prevented by a mechanism different from application of a GPPD-inhibiting herbicide) . In some embodiments, resistance can be measured by the visible indicator phenotype of a cell or organism transformed with a nucleic acid containing a gene encoding the corresponding GPPD polypeptide, or by an in vitro assay of a GPPD polypeptide, in the presence of varying concentrations of different GPPD inhibitory herbicides. Reactions

- 15 045758 on doses and relative shifts in dose responses associated with such indicator phenotypes (browning, growth inhibition, discoloration, herbicidal effect, etc.) are for convenience expressed, for example, by GR50 values (concentration causing 50% growth reduction) or MIC (minimum inhibitory concentration) wherein increasing values correspond to increasing resistance inherent in the HPPD polypeptide, in the usual manner, based on plant damage, meristem bleaching symptoms, etc. over a range of different herbicide concentrations. These data can be expressed, for example, by GR50 values obtained from dose/response curves, where dose is plotted on the x-axis and mortality percentage, herbicidal effect, number of green plants emerged, etc. are plotted on the y-axis, where increased GR50 values correspond to increased levels of resistance inherent in the expressed GPPD polypeptide. The herbicides can be suitably applied before or after the plants have emerged.

In various embodiments, the level of stability of the nucleic acid or gene encoding a GPPD polypeptide in accordance with the invention or a GPPD polypeptide in accordance with the invention can be tested through gene transfer, regeneration, propagation and spray tests of a test plant, such as tobacco, or a crop plant, such as soybeans, corn or cotton. According to the results of such testing, such plants are more resistant, preferably tolerate a dose of at least 2 times the normal dose recommended for field use, even more preferably tolerate a dose up to 4 times the normal dose recommended for field use, to GPPD-inhibiting herbicides (e.g., GPPD-inhibiting herbicides from the triketone class (preferably benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinothrion), diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxyphene, benzophenap, pyrazolinate, pyrasulf otal , topramesone, tolpiralate), N-(1,2,5-oxadiazol-3yl)benzamides, N-(1,3,4-oxadiazol-2-yl)benzamides (preferably 2-methyl-N-(5-methyl- 1,3,4oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), K-(tetrazol-5-yl)- or K-(triazol- 5-yl)arylcarboxamides (preferably 2-chloro-3ethoxy-4-(methylsulfonyl)-N-(1-methyl-N-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3(methylsulfonyl) -K-(1-methyl-1H-tetrazol-5-yl)benzamide; 2-chloro-3-(methylsulfanyl)-C-(1-methyl-Stetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2(methoxymethyl)-3-(methylsulfinyl)-K-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, K-(triazol-2-yl)arylcarboxamides , triazinones and pyrazolones than such plants containing no exogenous gene encoding the GPPD polypeptide, or plants containing a gene including a reference DNA encoding the GPPD polypeptide, for example, Pseudomonas fluorescens DNA encoding GPPD, under the control of the same promoter as the nucleic acid encoding the GPPD polypeptide of the present invention. Accordingly, the term capable of increasing the resistance of a plant to at least one herbicide affecting GPPD means the resistance of a plant expressing GPPD in accordance with the invention to a dose of at least 1, 2, or 3, or 4 or more times higher normal field dose of a GPPD inhibitory herbicide compared to a plant expressing only its own endogenous GPPD or a plant expressing a reference GPPD polypeptide. In this regard, the term GPPD herbicide is not limited to substances known and/or used as herbicides, but refers to any substances that inhibit the catalytic activity of GPPD polypeptides.

The term herbicide resistance, herbicide resistance or herbicide insensitivity also refers to the ability of an enzyme to carry out its respective catalytic reaction in the presence of an inhibitor/herbicide or after exposure to an inhibitor/herbicide. Herbicidal resistance of enzymes, i.e. their ability to counteract the inhibitory effect of the herbicide can be expressed qualitatively and quantitatively. Qualitatively, enzymes that tolerate different categories or even different classes of inhibitors are highly resistant and vice versa. In quantitative terms, the stability of an enzyme compared to a single herbicide can be expressed as the corresponding residual activity or residual turnover observed in one sample of that enzyme, calculated as the activity ratio (kapp, kinetic measure) or total substrate turnover (signal change, endpoint measurement) in the absence and presence of one inhibitor (Bergmeyer, HU: Methods of enzymatic analysis, 1974). In various embodiments, to determine residual activity, the apparent kinetic constant (kapp) of a specific substrate conversion can be measured as the kinetic changes in absorbance at 320 nm in a coupled assay in that the homogentisate (HH) formed by HPPD from HFP is directly converted to a highly absorbent molecule maleylacetoacetate (MAA) by a second enzyme, homogentisate dioxygenase (HGD), used in excess continuously in all assays. The ratio k _cat /k _M of enzyme activity is proportional to the apparent kinetic constant kapp and proportional to k _cat /k _M *[E] ([E] = enzyme concentration). A competitive inhibitor exhibits an apparent increase in k _M and hence an inverse decrease in kapp at nonsaturable substrate concentrations.

- 16 045758

Since both measurements of k _app in the presence and absence of inhibitor are made using an identical enzyme sample, crude or purified, and thus at the same enzyme concentration, the enzyme concentration is excluded from the calculation of residual activity and the ratio of both k _app directly indicates the change in k _M due to inhibition. Notably, this concept refers to enzyme/inhibitor pairs interacting with competitive inhibition, likely true for almost all polypeptide variants and inhibitors described. The inhibition constant ki for an enzyme and its corresponding inhibitor describes the strength of binding of the inhibitor to that enzyme. Increased resistance is given for ratios of 1.5, 2, 3, 4, 5, 7, 10, 20, 30, 40, 50, 100, 200 or higher and compared to the GPPD reference sequence in the presence or absence of any corresponding GPPD inhibitory herbicide .

A specific, although not limiting, type of assay that can be used to evaluate GPPD polypeptide sequences in accordance with the invention is a colorimetric assay (as described, for example, see US 6,768,044). In this assay, for example, E. Coli cells containing the vector pSE420-HPPDx (HPPDx means any gene encoding a putative HPPD polypeptide; the core vector pSE420 was obtained from Invitrogen Karlsruhe, Germany) or a modified version of pSE420 (pSE420(RI)NX)-HPPDx produce soluble melanin-like pigments from tyrosine catabolism when the overexpressed GPPD polypeptide is active. These melanin-like pigments are analyzed in liquid culture or by plating a culture of E. coli onto solid agar such as LB broth. After 16 hours to 8 days at 20-30°C, culture media or agar wells that have been inoculated with an E. coli culture containing the empty vector pSE420 do not change the color of the media or those plated with an E. coli culture. containing the pSE420HPPDx vector containing the gene encoding the inactive HPPD also does not change the color of the medium, while wells inoculated with an E. coli culture containing the pSE420-HPPDx vector encoding the active HPPD are brownish. In the presence of a GPPD inhibitory herbicide, this pigment production can be inhibited and the crop will not change the color of the medium unless a GPPD polypeptide resistant to the inhibitory herbicide is expressed and active. This test has previously been demonstrated to reflect HFPD activity and resistance to HFPD-inhibiting herbicides, regardless of the origin of this activity, and allows identification of HFPD activity (US 6,768,044), i.e. at a quality level.

B. Methods for introducing mutations into the GPPD sequence.

In the mutated HPPD polypeptides encoded by the nucleic acid of the invention, at least three amino acids have been replaced as defined above.

Substitution may be made in the nucleic acid sequence encoding the reference GPPD polypeptide, as defined above, by any means suitable for replacing, in said sequence, a codon that encodes the amino acid to be replaced by a codon that corresponds to the amino acid that is to replace it, said codons are widely described in the literature and are well known to those skilled in the art.

Several molecular biology techniques can be used to achieve this replacement. A suitable method for producing a mutated nucleic acid sequence in accordance with the invention and the corresponding protein involves performing site-directed mutagenesis at codons encoding one or more preselected amino acids. Methods for obtaining such site-directed mutations are well known to the skilled person and are widely described in the literature (in particular: Directed Mutagenesis: A Practical Approach, 1991, Edited by M.J. McPHERSON, IRL PRESS), or are methods for which it is possible to use commercial kits (for example , QUIKCHANGEfff lightening mutagenesis kit from Qiagen or Stratagene). After site-directed mutagenesis, it is convenient to select cells containing mutated GPPD that are less sensitive to the GPPD inhibitor using an appropriate screening tool. Appropriate screening methods to achieve this have been described above.

Alternatively, the DNA sequence encoding the reference GPPD polypeptide can be modified in silico to encode a GPPD polypeptide having one or more of the substitutions listed in the present invention and then synthesized de novo. This method is also well known in the prior art and is described in the literature. The nucleotide sequence encoding the mutated GPPD polypeptide can be introduced into a host cell as described elsewhere in the present invention.

C. Isolated polynucleotides and their variants and fragments.

In some embodiments, the present invention includes isolated or recombinant polynucleotides. The term polynucleotide corresponds to any genetic material of any length and any sequence containing single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural genes, gene groups, plasmids, whole genomes and their fragments. The recombinant polynucleotide or polypeptide/protein, or biologically active portion thereof, as defined herein, is no longer present in its original, native organism, for example when contained in a heterologous host cell or in a transgenic

- 17 045758 plant cell, seed or plant. In one embodiment, the recombinant polynucleotide does not contain sequences (eg, protein coding or regulatory sequences) that naturally flank the nucleic acid (ie, sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which polynucleotide is obtained. The term recombinant covers polynucleotides or polypeptides that have been manipulated with respect to a naturally occurring polynucleotide or polypeptide such that the polynucleotide or polypeptide differs (eg, in chemical composition or structure) from what occurs naturally. In another embodiment, the recombinant polynucleotide does not contain internal sequences (ie, introns) that occur naturally in the genomic DNA of the organism from which the polynucleotide is derived. A typical example of such a polynucleotide is the so-called complementary DNA (cDNA). For example, in various embodiments, an isolated polynucleotide encoding resistance to a GPPD-inhibiting herbicide may contain less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flanks a polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Nucleic acid molecules in accordance with the invention include those that encode GPPD in accordance with the invention. In some embodiments, a nucleic acid molecule in accordance with the invention is operably linked to a promoter capable of directing expression of the nucleic acid molecule in a host cell (eg, a plant host cell or a bacterial host cell).

In addition, the present invention provides exemplary variants and fragments of any nucleic acid sequence encoding the amino acid sequences set forth in any of SEQ ID NOs: 3-108. The polynucleotide fragment may encode a biologically active portion of the polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed elsewhere in the present invention. Polynucleotides that are fragments of a polynucleotide include at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 , 1000, 1050, 1100, 1150 contiguous nucleotides, or up to the number of nucleotides present in the full-length polynucleotide described herein, depending on the intended purpose (for example, the HFPD nucleic acid described herein). By contiguous nucleotides we mean nucleotide residues located directly next to each other.

The polynucleotide fragments of the present invention will generally encode polypeptide fragments that retain the biological activity of the full-length GPPD-inhibiting herbicide resistance protein; i.e., herbicide resistance activity. By maintaining herbicide resistance activity, it is meant that the fragment will have at least about 30%, at least about 50%, at least about 70%, at least about 80, 85, 90, 95, 100, 110, 125, 150, 175, 200, 250%, at least about 300% or more of the herbicide resistance activity of the full-length GPPD-inhibiting herbicide resistance protein set forth herein as SEQ ID NOs: 3-108. Methods for measuring herbicide resistance activity are well known in the art and exemplary methods are described in the present invention. In a non-limiting example, a fragment according to the invention will be resistant to the same dose of GPPD inhibitory herbicide, or resistant to 1x, 2x, 3x, 4x or an even higher dose of GPPD inhibitory herbicide, or the fragments will be the same or more resistant based on ki between fragment and SEQ ID NOs: 3-108.

A polynucleotide fragment that encodes the biologically active portion of a polypeptide in accordance with the invention will encode at least about 150, 175, 200, 250, 300, 350 contiguous amino acids, or up to the total number of amino acids present in a full-length polypeptide in accordance with the invention. In a non-limiting example, a polynucleotide fragment that encodes a biologically active portion of a HPPD polypeptide has (a) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO: 1 and (b) a histidine or aspartic acid at a position corresponding to amino acid position 336 of SEQ ID NO: 1. 1 and (c) serine at a position corresponding to amino acid position 337 of SEQ ID NO: 1 and, optionally, one or more additional amino acid substitutions at positions corresponding to amino acid positions 204, 213, 264, 268, 270, 310, 315, 330, 331, 338, 339, 340, 344, 345 SEQ ID NO: 1, including the GPPD protein set forth in any of SEQ ID NOs: 3-108.

The invention also covers polynucleotide variants as described above. Polynucleotide variants also include those sequences that encode GPPD in accordance with the invention, but are conservatively different due to degeneracy of the genetic code, as well as those that are sufficiently identical. Variants of the present invention will retain GPPD polypeptide activity and resistance to GPPD inhibitory herbicide. The term “substantially identical” means a polypeptide or polynucleotide sequence that has at least about 53%, at least about 60 or 65% sequence identity, about 70 or 75% sequence identity, about

- 18 045758 but 80 or 85% sequence identity, approximately 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity compared to a reference sequence, using one of the alignment programs using standard parameters. One skilled in the art will appreciate that these values can be suitably adjusted to determine the respective identity of the polypeptides encoded by the two polynucleotides, taking into account codon degeneracy, amino acid similarity, reading frame position, and the like.

Bacterial genes very often have numerous methionine initiation codons close to the start of the open reading frame. Often, initiation of translation at one or more of these start codons results in the formation of a functional protein. Such start codons may include ATG codons. However, bacteria such as Bacillus sp. also recognize the GTG codon as a start codon, and proteins that initiate translation at GTG codons contain methionine as the first amino acid. Moreover, it is often not possible to determine a priori which of these codons is naturally used in a bacterium. Thus, it should be understood that the use of one of the alternative methionine codons may result in variants conferring herbicide resistance. These herbicide resistance proteins are covered by the present invention and can be used in the methods in accordance with the present invention. Naturally occurring allelic variants can be determined using well-known molecular biology techniques such as polymerase chain reaction (PCR) and hybridization techniques, as described below. Variant polynucleotides also include polynucleotides of synthetic origin, formed, for example, using site-directed or other mutagenesis strategies, but which still encode a polypeptide having the desired biological activity.

In addition, one skilled in the art will understand that changes can be introduced by further mutation of the polynucleotides in accordance with the invention, thereby leading to further changes in the amino acid sequence of the encoded polypeptides, without changing the biological activity of the polypeptides. Thus, variants of isolated polynucleotides can be created by introducing one or more additional nucleotide substitutions, additions or deletions into the corresponding polynucleotide encoding GPPD in accordance with the invention, such that 3-5, 1-7, 1-9, 1-11, 1-13, 1-15, or 1-17 amino acid substitutions, additions or deletions, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acid substitutions, additions or deletions. Other mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis, or gene shuffling techniques. Such polynucleotide variants are also covered by the invention.

Polynucleotide variants can be created by introducing mutations in a random order throughout all or part of the coding sequence, such as saturation mutagenesis or permutation mutagenesis, and the resulting mutants can be screened for the ability to confer herbicide resistance activity to identify mutants that retain activity.

Additional methods for generating variants include subjecting a cell expressing a protein (or a library thereof) of the invention to special conditions that challenge the activity of the protein. Special conditions may include, but are not limited to, changes in temperature, changes in pH, changes in concentrations of substrates or inhibitors, and changes in the buffer composition or concentrations thereof. The protein library may be subjected to these conditions during expression of the protein (eg, in E. coli or other host) or after the creation of a protein extract, or after purification of the protein.

The functional or enzymatic activity of a library of proteins subjected to stress conditions can then be compared with a reference protein to identify proteins with improved properties. This comparison of activities can be performed as part of a growth screen or alternatively as part of an enzymatic assay that quantifies protein activity. Properties that may be identified as improved may include resistance to herbicides that inhibit HPPD, changes in kinetic constants (including KM, Ki, k _cat ), protein stability, protein thermal stability, or optimal temperature and pH for the protein.

D. Isolated proteins and their variants and fragments.

Herbicide resistance polypeptides are also covered by the present invention. Herbicide resistance polypeptide includes preparations of polypeptides containing less than about 30, 20, 10 or 5% (by dry weight) of herbicide resistance polypeptide (hereinafter also referred to as contaminant protein). In the context of the present invention, the herbicide resistance protein means the HPPD polypeptide described in the present invention. Also provided are fragments, biologically active portions and variants thereof which may be used in the practice of the methods of the present invention.

The fragments or biologically active portions include polypeptide fragments containing a portion of the amino acid sequence encoding a herbicide resistance protein and retaining herbicide resistance activity. The biologically active portion of the herbicide resistance protein may be a polypeptide of, for example, 10, 25, 50, 100 or more amino acids in length. Such biologically active moieties can be obtained using recombination techniques and evaluated for herbicide resistance activity.

By variant is meant a protein or polypeptide whose amino acid sequence is at least about 53%, 60%, 65%, about 70%, 75%, about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% identical to any of exemplary SEQ ID NOs: 3-108, wherein the variant has GPPD polypeptide activity and resistance to GPPD inhibitory herbicides. One skilled in the art will appreciate that these values can be suitably adjusted to determine the respective identity of the polypeptides encoded by the two polynucleotides, taking into account codon degeneracy, amino acid similarity, reading frame position, and the like.

For example, conservative amino acid substitutions may be made at one or more non-essential amino acid residues. A nonessential amino acid residue is a residue that can be changed in the reference polypeptide sequence without changing biological activity, while an essential amino acid residue is required for biological activity. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by a residue having a similar side chain. Families of amino acids with similar side chains are defined in the prior art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine , tryptophan, histidine). Amino acid substitutions can be made at non-conserved sites that retain their function. Typically, such substitutions are not made for conserved amino acid residues or for amino acid residues within a conserved motif when such residues are essential for the activity of the polypeptide. However, one skilled in the art will appreciate that functional variants may have minor conservative or non-conservative changes to conserved residues.

Also included in the present invention are antibodies to GPPD according to the present invention, or to variants or fragments thereof. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; US Patent No. 4,196,265).

Thus, one aspect of the invention relates to antibodies, single chain antigen binding molecules or other proteins that specifically bind to one or more protein or peptide molecules of the invention and homologs, chimeras or fragments thereof. In a particularly preferred embodiment, the antibody specifically binds to a protein whose amino acid sequence is set forth in SEQ ID NOs: 1-108 or a fragment thereof.

Antibodies in accordance with the invention can be used to quantitatively or qualitatively detect peptide or protein molecules in accordance with the invention, or to detect post-translational modifications of proteins. As used in the present invention, an antibody or peptide is considered to specifically bind to a protein or peptide molecule in accordance with the invention unless such binding is competitively inhibited by the presence of unrelated molecules.

E. Gene accumulation.

In commercial crop production, it is desirable to eradicate unwanted plants (i.e., weeds) from crop fields through sound pesticide control. The ideal treatment would be one that could be applied to the entire field but would only kill unwanted plants without affecting crop plants. One such treatment system involves the use of herbicide-resistant crops so that when herbicide is sprayed over a field of herbicide-resistant crops, they continue to grow while non-herbicide-resistant weeds are killed or seriously damaged. Ideally, such treatment systems would take advantage of the variation in herbicide properties so that weed control would be the best possible combination of flexibility and economy. For example, individual herbicides have different periods of action in the field, and some herbicides persist and are effective for a relatively long time after application in the field, while other herbicides quickly break down into different and/or inactive compounds. An ideal treatment system would allow the use of different herbicides so that growers could tailor herbicide selection to a specific situation.

While a number of herbicide-tolerant crops are commercially available today, a problem with many commercial herbicides and herbicide/crop combinations is that individual herbicides typically have incomplete

- 20 045758 spectrum of activity in relation to common types of weeds. For most individual herbicides that have been in use for some time, populations of herbicide-resistant weed species and biotypes have become dominant (see, for example, Tranel and Wright (2002) Weed Science 50: 700-712; Owen and Zelaya (2005) Pest Manag. Sci. 61: 301-311). Transgenic plants resistant to more than one herbicide have been described (see, for example, WO 2005/012515). However, improvements are continually required in every aspect of crop production, weed control capabilities, extending residual weed control, and improving crop yields.

The GPPD protein or nucleotide sequence according to the invention is advantageously combined in plants with other genes encoding proteins or RNA that impart beneficial agronomic properties to such plants. Among the genes encoding proteins or RNA that confer beneficial agronomic properties on transformed plants are DNA sequences encoding proteins that confer resistance to one or more herbicides that differ in their chemical structure from herbicides that inhibit GPPD, and others that confer resistance to certain herbicides. insects, conferring resistance to certain diseases, DNA encoding RNA, ensuring the fight against nematodes or insects, etc.

Such genes are specifically described in PCT Publication Applications WO 91/02071 and WO 95/06128, as well as US Patent No. 7,923,602 and US Patent Application Publication No. 20100166723, each of which is incorporated herein by reference in its entirety.

Among the DNA sequences encoding proteins that confer resistance to certain herbicides to transformed plant cells and plants, mention may be made of the bar or PAT gene or the Streptomyces coelicolor gene described in WO 2009/152359 conferring resistance to glufosinate herbicides, a gene encoding a suitable EPSPS conferring resistance to herbicides that target EPSPS, such as glyphosate and its salts (US 4,535,060, US 4,769,061, US 5,094,945, US 4,940,835, US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5, 312,910, US 5,627,061, US 5,633,435), gene , encoding glyphosate n-acetyltransferase (e.g. US 8,222,489, US 8,088,972, US 8,044,261, US 8,021,857, US 8,008,547, US 7,999,152, US 7,998,703, US 7,863,503, US 7,714 ,188, US 7,709,702, US 7,666,644, US 7,666,643, US 7,531,339, US 7,527,955, and US 7,405,074), or the gene encoding glyphosate oxidoreductase (for example, US 5,463,175).

Among the DNA sequences encoding a useful EPSPS conferring resistance to herbicides targeting EPSPS, mention may be made more specifically of a gene encoding a plant EPSPS, in particular maize EPSPS, in particular maize EPSPS, containing two mutations, in particular a mutation at amino acid position 102 and mutation at amino acid position 106 (WO 2004/074443), and which is described in patent application US 6566587, hereinafter referred to as double mutant maize EPSPS or 2mEPSPS, or the gene encoding EPSPS isolated from Agrobacterium and described by sequence ID No. 2 and sequence ID No. 3 US Patent 5,633,435, also designated CP4.

Among the DNA sequences encoding a useful EPSPS conferring resistance to herbicides targeting EPSPS, mention may be made more specifically of the gene encoding the EPSPS GRG23 from Arthrobacter globiformis, as well as mutant GRG23 ACE1, GRG23 ACE2, or GRG23 ACE3, in particular mutants or variants GRG23 as described in WO 2008/100353, such as GRG23(ace3)R173K SEQ ID No. 29 in WO 2008/100353.

In the case of DNA sequences encoding EPSPS and in particular those encoding the above genes, the encoding sequence for these enzymes is advantageously preceded by a sequence encoding a transit peptide, in particular the optimized transit peptide described in US patent 5,510,471 or 5,633,448.

Exemplary herbicide resistance traits that may be combined with the nucleic acid sequence of the invention also include at least one ALS inhibitor (WO 2007/024782); a mutated Arabidopsis ALS/AHAS gene (US Pat. No. 6,855,533); genes encoding 2,4-D-monooxygenases conferring resistance to 2,4-D (2,4-dichlorophenoxyacetic acid) by metabolization (U.S. Patent 6,153,401); and genes encoding dicamba monooxygenases conferring resistance to dicamba (3,6-dichloro- 2-methoxybenzoic acid) by metabolization (US 2008/0119361 and US 2008/0120739).

In various embodiments, the HFPD of the invention is assembled with one or more herbicide resistance genes, including one or more additional resistance genes to HFPD-inhibiting herbicides and/or one or more glyphosate and/or glufosinate resistance genes. In one embodiment, the GPPD according to the invention is combined with 2mEPSPS and bar.

Among the DNA sequences encoding proteins related to insect resistance properties, it is worth mentioning the Bt proteins, widely described in the literature and well known to specialists in this field. Also worth mentioning are proteins isolated from bacteria such as Photorhabdus (WO 97/17432 and WO 98/08932).

- 21 045758

Among such DNA sequences encoding target proteins that confer new properties of resistance to insects, it is worth mentioning more specifically the Bt Cry or VIP proteins, widely described in the literature and well known to specialists in this field. These include the Cry1F protein or fusions derived from the Cry1F protein (for example, the Cry1A-Cry1F fusion proteins described in US 6,326,169; US 6,281,016; US 6,218,188, or toxic fragments thereof), Oy1A type proteins or toxic fragments thereof, preferably the Oy1Ac protein or hybrids, derived from the Cry1Ac protein (for example, the Cry1Ab-Cry1Ac fusion protein described in US 5,880,275) or the Cry1Ab or Bt2 protein or insecticidal fragments thereof, as described in EP451878, the Cry2Ae, Cry2Af or Cry2Ag proteins, as described in WO 2002/057664 or toxic thereof fragments, Cry1A.105 protein described in WO 2007/140256 (SEQ ID No. 7) or a toxic fragment thereof, VIP3Aa19 protein under NCBI catalog number ABG20428, VIP3Aa20 protein under catalog number ABG20429 (SEQ ID No. 2 in WO 2007 /142840), VIP3A proteins produced in cotton events COT202 or COT203 (WO 2005/054479 and respectively WO 2005/054480), Cry proteins as described in WO2001/47952, VIP3Aa protein or a toxic fragment thereof, as described in Estruch et al . (1996), Proc Natl Acad Sci U S A. 28;93(11):5389-94 and US 6,291,156, insecticidal proteins from strains of Xenorhabdus species (as described in WO 98/50427), Serratia (particularly from S. entomophila) or Photorhabdus species, such as the Tc proteins from Photorhabdus as described in WO98/08932 (eg, Waterfield et al., 2001, Appl Environ Microbiol. 67(11):5017-24; Ffrench-Constant and Bowen, 2000, Cell Mol Life Sci.;57(5):828-33). Also included in the present invention are any variants and mutants of any of these proteins that differ by a few (1-10, preferably 1-5) amino acids from any of the above sequences, especially the sequence of a toxic fragment thereof, or that are fused to a transit peptide such as plastid transit peptide or any other protein or peptide.

In various embodiments, the GPPD sequence according to the invention can be combined in plants with one or more genes conferring a desired trait, such as herbicide tolerance, insect resistance, drought tolerance, nematode control, water use efficiency, water use efficiency nitrogen, improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality, stress resistance, improved reproduction, etc.

Particularly suitable transgenic events that can be combined with genes in accordance with the present invention in plants of the same species (for example, by crossing or retransforming a plant containing another transgenic event with a chimeric gene in accordance with the invention) include event 531/PV- GHBK04 (cotton, insect control, described in WO 2002/040677), event 1143-14A (cotton, insect control, not deposited, described in WO 2006/128569); event 1143-51B (cotton, insect control, not deposited, described in WO 2006/128570); event 1445 (cotton, herbicide resistance, not deposited, described in US-A 2002-120964 or WO 2002/034946 Event 17053 (rice, herbicide resistance, deposited as PTA9843, described in WO 2010/117737); event 17314 (fig, herbicide resistance, deposited as PTA-9844, described in WO 2010/117735); event 281-24-236 (cotton, insect control - herbicide resistance, deposited as PTA-6233, described in WO 2005/103266 or US- A 2005-216969); event 3006-210-23 (cotton, insect control - herbicide resistance, deposited as PTA-6233, described in US-A 2007-143876 or WO 2005/103266); event 3272 (corn, traits quality, deposited as PTA-9972, described in WO 2006/098952 or US-A 2Oo6-230473); event 33391 (wheat, herbicide resistance, deposited as PTA-2347, described in WO 2002/027004), event 40416 (corn , insect control - herbicide resistance, deposited as ATCC PTA11508, described in WO 11/075593); event 43A47 (corn, insect control - herbicide resistance, deposited as ATCC PTA-11509, described in WO 2011/075595); event 5307 (corn, insect control, deposited as ATCC PTA-9561, described in WO 2010/077816); event ASR-368 (bentgrass, herbicide resistance, deposited as ATCC PTA-4816, described in US-A 2006-162007 or WO 2004/053062); event B16 (corn, herbicide resistance, not deposited, described in US-A 2003-126634); event BPS-CV127-9 (soybean, herbicide resistance, deposited as NCIMB No. 41603, described in WO 2010/080829); event BLR1 (oilseed rape, restoration of male sterility, deposited as NCIMB 41193, described in WO 2005/074671); event CE43-67B (cotton, insect control, deposited as DSM ACC2724, described in US-A 2009-217423 or WO 2006/128573); event CE44-69D (cotton, insect control, not deposited, described in US-A 2010-0024077); event CE44-69D (cotton, insect control, not deposited, described in WO 2006/128571); event CE46-02A (cotton, insect control, not deposited, described in WO 2006/128572); event COT102 (cotton, insect control, not deposited, described in USA 2006-130175 or WO 2004/039986); event COT202 (cotton, insect control, not deposited, described in US-A 2007-067868 or WO 2005/054479); event COT203 (cotton, insect control, not deposited, described in WO 2005/054480); event DAS21606-3/1606 (soybean, herbicide resistance, deposited as PTA-11028, described in WO 2012/033794), event DAS40278 (corn, herbicide resistance, deposited as ATCC PTA-10244, described in WO 2011/022469 );

- 22 045758 event DAS-44406-6/pDAB8264.44.06.1 (soybeans, herbicide resistance, deposited as PTA-11336, described in WO 2012/075426), event DAS-14536-7/pDAB8291.45.36.2 ( soybean, herbicide resistance, deposited as PTA-11335, described in WO 2012/075429), event DAS-59122-7 (corn, insect control - herbicide resistance, deposited as ATCC PTA 11384, described in US-A 2006 -070139); event DAS-59132 (corn, insect control - herbicide resistance, not deposited, described in WO 2009/100188); event DAS68416 (soybean, herbicide resistance, deposited as ATCC PTA-10442, described in WO 2011/066384 or WO 2011/066360); event DP-098140-6 (corn, herbicide resistance, deposited as ATCC PTA-8296, described in US-A 2009-137395 or WO 08/112019); event DP-305423-1 (soybeans, quality attributes, not deposited, described in US-A 2008-312082 or WO 2008/054747); event DP-32138-1 (maize, hybridization system, deposited as ATCC PTA-9158, described in US-A 2009-0210970 or WO 2009/103049); event DP-356043-5 (soybean, herbicide resistance, deposited as ATCC PTA-8287, described in US-A 2010-0184079 or WO 2008/002872); event EE-1 (eggplant, insect control, not deposited, described in WO 07/091277); event FI117 (corn, herbicide resistance, deposited as ATCC 209031, described in US-A 2006-059581 or wO 98/044140); event FG72 (soybean, herbicide resistance, deposited as PTA-11041, described in WO 2011/063413), event GA21 (corn, herbicide resistance, deposited as ATCC 209033, described in US-A 2005-086719 or WO 98/ 044140); event GG25 (corn, herbicide resistance, deposited as ATCC 209032, described in US-A 2005-188434 or WO 98/044140); event GHB119 (cotton, insect control - herbicide resistance, deposited as ATCC PTA-8398, described in WO 2008/151780); event GHB614 (cotton, herbicide resistance, deposited as ATCC PTA-6878, described in US-A 2010-050282 or WO 2007/017186); event GJ11 (corn, herbicide resistance, deposited as ATCC 209030, described in US-A 2005-188434 or WO 98/044140); GM event RZ13 (sugar beet, virus resistance, deposited as NCIMB-41601, described in WO 2010/076212); event H7-1 (sugar beet, herbicide resistance, deposited as NCIMB 41158 or NCIMB 41159, described in US-A 2004-172669 or WO 2004/074492); event JOPLIN1 (wheat, disease resistance, not deposited, described in US-A 2008-064032); event LL27 (soybean, herbicide resistance, deposited as NCIMB41658, described in WO 2006/108674 or US-A 2008-320616); event LL55 (soybean, herbicide resistance, deposited as NCIMB 41660, described in WO 2006/108675 or US-A 2008-196127); event LLcotton25 (cotton, herbicide resistance, deposited as ATCC PTA-3343, described in WO 2003/013224 or US-A 2003-097687); event LLRICE06 (rice, herbicide resistance, deposited as ATCC 203353, described in US 6,468,747 or WO 2000/026345); event LLRice62 (rice, herbicide resistance, deposited as ATCC 203352, described in WO 2000/026345), event LLRICE601 (rice, herbicide resistance, deposited as ATCC PTA-2600, described in US-A 2008-2289060 or WO 2000/ 026356); event LY038 (corn, quality attributes, deposited as ATCC PTA-5623, described in US-A 2007-028322 or WO 2005/061720); event MIR162 (corn, insect control, deposited as PTA-8166, described in US-A 2009-300784 or WO 2007/142840); event MIR604 (corn, insect control, not deposited, described in US-A 2008-167456 or WO 2005/103301); event mOn15985 (cotton, insect control, deposited as ATCC PTA-2516, described in US-A 2004-250317 or WO 2002/100163); event MON810 (corn, insect control, not deposited, described in US-A 2002-102582); event MON863 (corn, insect control, deposited as ATCC PTA-2605, described in WO 2004/011601 or US-A 2006-095986); event MON87427 (maize, pollination control, deposited as ATCC PTA-7899, described in WO 2011/062904); event MON87460 (corn, stress tolerance, deposited as ATCC PTA-8910, described in WO 2009/111263 or US-A 2011-0138504); event MON87701 (soybean, insect control, deposited as ATCC PTA-8194, described in US-A 2009-130071 or WO 2009/064652); event MON87705 (soybeans, quality traits - herbicide resistance, deposited as ATCC PTA-9241, described in US-A 2010-0080887 or WO2010/037016); event MON87708 (soybean, herbicide resistance, deposited as ATCC PTA-9670, described in WO 2011/034704); event MON87712 (soybeans, yield, deposited as PTA-10296, described in WO 2012/051199), event MON87754 (soybeans, quality attributes, deposited as ATCC PTA-9385, described in WO 2010/024976); event MON87769 (soybeans, quality attributes, deposited as ATCC PTA-8911, described in US-A 2011-0067141 or WO 2009/102873); event MON88017 (corn, insect control - herbicide resistance, deposited as ATCC PTA5582, described in US-A 2008-028482 or WO 2005/059103); event MON88913 (cotton, herbicide resistance, deposited as ATCC PTA-4854, described in WO 2004/072235 or US-A 2006059590); event MON88302 (oilseed rape, herbicide resistance, deposited as PTA10955, described in WO 2011/153186), event MON88701 (cotton, herbicide resistance, deposited as PTA-11754, described in WO 2012/134808), event MON89034 (corn, insect control, deposited as ATCC PTA-7455, described in WO 07/140256 or US-A 2008-260932); event MON89788 (soybean, herbicide resistance, deposited as ATCC PTA-6708, described in USA 2006-282915 or WO 2006/130436); event MS11 (oilseed rape, pollination control - sustainable

- 23 045758 herbicide sensitivity, deposited as ATCC RTA-850 or RTA-2485, described in WO 2001/031042); event MS8 (oilseed rape, pollination control - herbicide resistance, deposited as ATCC PTA-730, described in WO 2001/041558 or US-A 2003-188347); event NK603 (corn, herbicide resistance, deposited as ATCC PTA-2478, described in US-A 2007-292854); event PE-7 (rice, insect control, not deposited, described in WO 2008/114282); event RF3 (oilseed rape, pollination control - herbicide resistance, deposited as ATCC PTA-730, described in WO 2001/041558 or US-A 2003-188347); event RT73 (oilseed rape, herbicide resistance, not deposited, described in WO 2002/036831 or US-A 2008-070260); event SYHT0H2 / SYN-000H2-5 (soybeans, herbicide resistance, deposited as PTA-11226, described in WO 2012/082548), event T227-1 (sugar beet, herbicide resistance, not deposited, described in WO 2002/ 44407 or US-A 2009-265817); event T25 (corn, herbicide resistance, not deposited, described in US-A 2001-029014 or WO 2001/051654); event T304-40 (cotton, insect control - herbicide resistance, deposited as ATCC PTA-8171, described in US-A 2010-077501 or WO 2008/122406); event T342-142 (cotton, insect control, not deposited, described in WO 2006/128568); event TC1507 (corn, insect control - herbicide resistance, not deposited, described in US-A 2005-039226 or WO 2004/099447); event VIP1034 (corn, insect control - herbicide resistance, deposited as ATCC PTA-3925., described in WO 2003/052073), event 32316 (corn, insect control - herbicide resistance, deposited as PTA-11507, described in WO 2011/084632), Event 4114 (corn, insect control - herbicide resistance, deposited as PTA-11506, described in WO 2011/084621), Event EE-GM3/FG72 (soybeans, herbicide resistance, ATCC accession number PTA-11041) optionally collected with event EE-GM1/LL27 or event EE-GM2/LL55 (WO 2011/063413 A2), event DAS-68416-4 (soybeans, herbicide resistance, ATCC accession number PTA-10442, WO 2011/066360A1), event DAS-68416-4 (soybeans, herbicide resistance, ATCC registration number PTA-10442, WO 2011/066384 A1), event DP-040416-8 (corn, insect control, ATCC registration number PTA11508 , WO 2011/075593 A1), event DP-043A47-3 (corn, insect control, ATCC registration number PTA-11509, WO 2011/075595 A1), event DP-0o4114-3 (corn, insect control, ATCC registration number RTA-11506, WO2011/084621 A1), event DP-032316-8 (corn, insect control, ATCC registration number RTA-11507, WO 2011/084632 A1), event MON-88302-9 (oilseed rape, resistance to herbicides, ATCC accession number PTA-10955, WO 2011/153186 A1), event DAS-21606-3 (soybeans, herbicide resistance, ATCC accession number PTA-11028, WO 2012/033794 A2), event MON-87712-4 (soybeans, quality attributes, ATCC registration number. PTA-10296, WO2012/051199 A2), event DAS-44406-6 (soybeans, accumulated resistance to herbicides, ATCC accession number. PTA-11336, WO2012/075426A1), event DAS-14536-7 (soybeans, accumulated resistance to herbicides, ATCC registration number PTA-11335, WO 2012/075429A1), event SYN000H2-5 (soybeans, herbicide resistance, ATCC registration number PTA-11226, WO 2012/082548 A2), event DP-061061-7 ( oilseed rape, herbicide resistance, deposit number not available, WO 2012071039 A1), event DP-073496-4 (oilseed rape, herbicide resistance, deposit number not available, US 2012131692), event 8264.44.06.1 (soybeans, accumulated herbicide resistance , registration number PTA-11336, WO 2012075426 A2), event 8291.45.36.2 (soybeans, accumulated herbicide resistance, registration number PTA-11335, WO 2012075429 A2), event SYHT0H2 (soybeans, ATCC registration number PTA-11226 , WO 2012/082548 A2), event MON88701 (cotton, ATCC registration number PTA-11754, WO 2012/134808 A1), event KK179-2 (alfalfa, ATCC registration number PTA-11833, WO 2013/003558 A1), event pDAB8264 .42.32.1 (soybeans, accumulated herbicide resistance, ATCC accession number PTA-11993, WO 2013/010094 A1), event MZDT09Y (corn, ATCC accession number PTA-13025, WO 2013/012775 A1), event 4114 (maize , insect control, ATCC registration number PTA-11506) WO 201314901, event MON87411 (maize, ATCC registration number PTA-12669) WO 2013169923, event A26-5 (cotton, insect control) WO 2013170398, event A2-6 (cotton , insect control) WO2013170399, event 9582.816.15.1 (soybeans, insect control, herbicide resistance), ATCC accession number PTA-12588) WO 2014004458, event 33121 (maize, insect control, herbicide resistance, ATCC accession number PTA13392) WO 2014116854, event 32218 (maize insect control, herbicide resistance, ATCC accession number PTA-13391) WO 2014116989, event SPT-7R-949D SPT-7R-1425D (rice male sterility) WO 2014154115, event MON87751 (soy beans, ATCC registration number. PYA-120166) WO 2014201235, event Рр009-401 Рр009-415 Рр009-469 (turf, ATCC registration number RTA-120354, RTA-120353, RTA-120355) WO 2015006774, event Bs2-X5 (tomato, ATCC) W O 2015017637, event MON87403 (maize, grain harvest, ATCC registration number RTA-13584 WO 2015053998, event 32218 (maize, insect control, ATCC registration number RTA-13391) WO 2015112182.

F. Polynucleotide constructs.

Polynucleotides encoding the GPPD polypeptides of the present invention can be modified to obtain or enhance expression in plant cells. Polynucleotides encoding the polypeptides identified in the present invention may provide

- 24 045758 baked in expression cassettes for expression in the corresponding plant. A plant expression cassette includes a DNA construct, including a recombinant DNA construct, capable of resulting in expression of a polynucleotide in a plant cell. The cassette may include, in the 5'-3' direction of transcription, a transcription initiation region (i.e., a promoter, in particular a heterologous promoter) operably linked to one or more corresponding polynucleotides, and/or a translation and transcription termination region (i.e., a termination region ), which function in plants. The cassette may also further contain at least one additional polynucleotide administered to the body, such as a selectable marker gene. Alternatively, additional polynucleotide(s) may be provided on multiple expression cassettes. Such an expression cassette is provided with multiple restriction sites for insertion of polynucleotide(s) so that it is subject to transcriptional regulation in regulatory regions.

In another embodiment, the present invention provides a chimeric gene comprising a coding sequence that includes a heterologous nucleic acid according to the invention operably linked to a plant-expressed promoter and optionally a transcription termination and polyadenylation site. Heterologous generally refers to a polynucleotide or polypeptide that is not endogenous to a cell or not endogenous to a location in the natural genome in which it is present, and is added to a cell by infection, transfection, microinjection, electroporation, microprojection, or the like. By functional linkage we mean a functional link between two polynucleotides. For example, when a promoter is operably linked to a DNA sequence, the promoter sequence initiates and intermediates the transcription of the DNA sequence. It is recognized that operably linked polynucleotides may or may not be contiguous, and where the term is used to refer to the junction of two polypeptide coding regions, the polypeptides are expressed in the same reading frame.

A promoter can be any polynucleotide sequence that exhibits transcriptional activity in selected plant cells, plant parts or plants. The promoter may be natural or similar, or foreign and heterologous to the host plant and/or DNA sequence of the invention. If the promoter is natural or similar to the host plant, then it is implied that the promoter is present in the natural plant into which it is introduced. If a promoter is foreign or heterologous to a DNA sequence in accordance with the invention, then it is meant that the promoter is not a natural or naturally occurring promoter for the DNA sequence in accordance with the invention operably associated with it. The promoter can be inducible or constitutive. It may occur naturally, be composed of parts of various naturally occurring promoters, or may be partially or completely synthetic. Guidance for promoter design is provided by studies of promoter structure such as Harley and Reynolds (1987) Nucleic Acids Res. 15:2343–2361. In addition, the location of the promoter relative to the transcription start can be optimized. See, for example, Roberts et al. (1979) Proc. Natl. Acad. Sci. USA 76:760-764. For use in plants, many suitable promoters are well known in the art.

For example, suitable constitutive promoters for use in plants include: plant virus promoters, such as the peanut chlorotic streak caulimovirus (PC1SV) promoter (US Pat. No. 5,850,019); cauliflower mosaic virus (CaMV) 35S promoter (Odell et al. (1985) Nature 313:810-812); Chlorella virus methyltransferase gene promoters (U.S. Patent No. 5,563,328) and the full-length mosaic mosaic virus (FMV) transcription promoter (U.S. Patent No. 5,378,619); promoters of genes such as rice actin (McElroy et al. (1990) Plant Cell 2:163-171 and US Pat. No. 5,641,876); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730 and US Patent No. 5,510,474); maize histone H3 (Lepetit et al. (1992) Mol. Gen. Genet. 231:276-285 and Atanassova et al. (1992) Plant J. 2(3):291-300); Brassica napus ALS3 (PCT application WO 97/41228); plant ribulose biscarboxylase/oxygenase small subunit gene (RuBisC; circovirus (AU 689 311) or cassava vein mosaic virus (CsVMV, US 7,053,205); as well as promoters of various Agrobacterium genes (see US Pat. Nos. 4,771,002; 5,102,796; 5,182.2 00; and 5,428,147 ).

Suitable inducible promoters for use in plants include: the ACE1 copper responsive promoter (Mett et al. (1993) PNAS 90:4567-4571); the maize In2 gene promoter responsive to benzenesulfonamide herbicide safeners (Hershey et al. (1991) Mol. Gen. Genetics 227:229-237 and Gatz et al. (1994) Mol. Gen. Genetics 243:32-38); as well as the Tet repressor promoter from Tn10 (Gatz et al. (1991) Mol. Gen. Genet. 227:229-237). Another inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond. An example of an inducible promoter of this type is the inducible promoter of a steroid hormone gene, the transcriptional activity of which is induced by the hormone

- 25 045758 glucocorticosteroid (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421) or the recent use of a chimeric transcription activator, XVE, for use in an estrogen receptor-based inducible expression system in plants activated by estradiol ( Zuo et al (2000) Plant J, 24:265-273). Other inducible promoters for use in plants are described in EP 332104, PCT WO 93/21334 and PCT WO 97/06269, which are incorporated herein by reference in their entirety. Promoters composed of parts of other promoters and partially or completely synthetic promoters can also be used. See, for example, Ni et al. (1995) Plant J. 7:661-676 and PCT WO 95/14098 describing such promoters for use in plants.

In one embodiment of the present invention, a promoter sequence specific to particular plant sites or tissues, such as seed-specific promoters (Datla, R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), especially napin promoter (EP 255 378 A1), phaseolin promoter, glutenin promoter, helianthin promoter (WO 92/17580), albumin promoter (WO 98/45460), oleosin promoter (WO 98/45461) , SAT1 promoter or SAT3 promoter (PCT/US98/06978).

An inducible promoter can also be used, preferably selected from phenylalanine ammonia lyase (PAL), HMG-KoA reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI), PR1 family gene, nopaline synthase (nos) and vspB promoters (US 5 670 349, table 3), the HMG2 promoter (US 5 670 349), the apple beta-galactosidase promoter (ABG1) and the apple aminocyclopropane carboxylate synthase (ACC synthase) promoter (WO 98/45445). Numerous promoters can be used in the constructs of the invention, including ones sequential to each other.

A promoter may include, or be modified to include, one or more enhancer elements. In some embodiments, a promoter may include multiple enhancer elements. Promoters containing enhancer elements provide higher levels of transcription compared to promoters without them. Suitable enhancer elements for use in plants include the PC1SV enhancer element (U.S. Patent No. 5,850,019), the CaMV 35S enhancer element (U.S. Patent Nos. 5,106,739 and 5,164,316), and the FMV enhancer element (Maiti et al. (1997) Transgenic Res. 6:143 -156); translation activator of tobacco mosaic virus (TMV), described in the application WO 87/07644, or tobacco etch virus (TEV), described by Carrington & Freed 1990, J. Virol. 64: 1590-1597, for example, or introns such as maize adh1 intron or rice actin intron 1. See also PCT WO96/23898, WO 2012/021794, WO 2012/021797, WO 2011/084370 and WO 2011/028914.

Often such constructs may contain 5' and 3' untranslated regions. Such constructs may contain a signal sequence or leader sequence to facilitate co-translational or post-translational transport of the peptide in question to certain intracellular structures, such as chloroplasts (or other plastids), endoplasmic reticulum or Golgi apparatus, or be secreted. For example, the construct may be designed to contain a signal peptide to facilitate passage of the peptide into the endoplasmic reticulum. By signal sequence is meant a sequence that is known or suspected to result in the co-translational or post-translational transport of a peptide across the cell membrane. In eukaryotes, this usually involves secretion into the Golgi apparatus, with some resulting glycosylation. By leader sequence we mean any sequence that, when translated, provides an amino acid sequence sufficient to trigger the co-translational transport of a peptide chain into a subcellular organelle. Thus, this includes leader sequences targeted for transport and/or glycosylation through the endoplasmic reticulum, vacuoles, plastids including chloroplasts, mitochondria, and the like. It may also be preferable to construct a plant expression cassette containing an intron such that processing of the intron into mRNA is required for expression.

By 3' untranslated region is meant a polynucleotide located downstream of the coding sequence. Polyadenylation signal sequences and other sequences encoding regulatory signals that can influence the addition of polyadenylic acid sites to the 3' end of the precursor mRNA are 3' untranslated regions. By 5' untranslated region is meant a polynucleotide located upstream of the coding sequence.

Other untranslated elements upstream or downstream include enhancers. Enhancers are polynucleotides whose action enhances the expression of the promoter region. Enhancers are well known in the art and include, but are not limited to, the SV40 enhancer region and the 35S enhancer region.

The termination region may be related to the transcription initiation region, may be related to the sequence of the present invention, or may be obtained from another source. Suitable termination regions are available from the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671–674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et

- 26 045758 al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids

Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639; and European patent application EP

633 317 A1.

In one aspect of the invention, synthetic DNA sequences are designed for a given polypeptide, such as the polypeptides in accordance with the invention. Expression of the open reading frame of a synthetic DNA sequence in a cell results in the production of a polypeptide in accordance with the invention. Synthetic DNA sequences may be useful for easily eliminating unwanted restriction endonuclease sites, for facilitating DNA cloning strategies, for altering or eliminating any potential codon bias, for altering or improving GC content, for eliminating or altering alternative reading frames, and/or for eliminating or changes in intron/exon splicing recognition sites, polyadenylation sites, Shine-Dalgarno sequences, unwanted promoter elements, etc., which may be present in the native DNA sequence. It is also possible to use synthetic DNA sequences to introduce other improvements to the DNA sequence, such as introducing an intronic sequence, creating a DNA sequence that is expressed as a fusion protein to organelle-targeting sequences such as chloroplast transit peptides, apoplast/vacuole-targeting peptides, or peptide sequences leading to retention of the resulting peptide in the endoplasmic reticulum. Synthetic genes can also be synthesized using host cell-preferred codons for improved expression, or can be synthesized using codons with a host-preferred frequency. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11; US Patents No. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, US Patent Applications Published Nos. 20040005600 and 20010003849, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, incorporated herein by reference.

In one embodiment, the target polynucleotides for expression are targeted to a chloroplast. In this method, if the target polynucleotide is not directly introduced into the chloroplast, the expression cassette further contains a polynucleotide encoding a transit peptide for directing the target nucleotide to the chloroplast. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al (1987) Plant Physiol. 84:965-968; Romer et al (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al (1986) Science 233:478-481.

Target polynucleotides directed to the chloroplast can be optimized for expression in the chloroplast by taking into account the difference in codon usage in the plant nucleus and this organelle. In this manner, target polynucleotides can be synthesized using chloroplast-preferred codons. See, for example, US Patent No. 5,380,831, incorporated herein by reference.

Such a plant expression cassette can be introduced into a plant transformation vector. A transformation vector should be understood as a DNA molecule that makes cell transformation possible. Such a molecule may consist of one or more expression cassettes and may be organized into more than one vector DNA molecule. For example, binary vectors are plant transformation vectors that use two non-contiguous DNA vectors to encode all the necessary cis- and trans-acting functions to transform plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). Vector means a polynucleotide construct designed for transfer between different host cells. An expression vector means a vector having the ability to incorporate, integrate and express a heterologous DNA sequence or fragments thereof in a foreign cell.

The plant transformation vector contains one or more DNA vectors to achieve plant transformation. For example, it is common practice in the art to use plant transformation vectors containing more than one contiguous DNA segment. These vectors are often referred to as binary vectors. Binary vectors, like vectors with helper plasmids, are most often used for Agrobacterium-mediated transformation where the size and complexity of the DNA segments required to achieve efficient transformation are very large and it is advantageous to separate the functions into individual DNA molecules. Binary vectors typically contain a plasmid vector containing the cis-acting sequences required for TDNA transfer (such as a left border region and a right border region), a selectable marker designed to be expressed in a plant cell, and a target polynucleotide (a polynucleotide designed to be expressed in a plant cell for which the creation of transgenic plants is desired). This plasmid vector also contains sequences necessary for bacterial replication. The cis-acting sequences are positioned to allow efficient transfer into and expression in plant cells. For example, the selectable marker sequence and the target sequence are located between the left and right border regions. Often the second plasmid vector contains trans-acting factors that act as an intermediate in the transfer of T-DNA from Agrobacterium to plant cells. This plasmid often contains virulence functions (Vir genes) allowing infection of plant cells by Agrobacterium, and DNA transfer by cleavage at border sequences and vir-mediated DNA transfer as understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science, 5:446-451). Several types of Agrobacterium strains (eg LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. A second plasmid vector is not necessary for introducing polynucleotides into plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

G. Plant transformation

Methods in accordance with the invention involve introducing a nucleotide construct into a plant. By introduction we mean the presentation of a nucleotide construct to a plant in such a way that said construct gains access to the interior of the plant cell. The methods of the invention do not require the use of a specific method for introducing the nucleotide construct into the plant, only that the nucleotide construct gain access inside at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. See, for example, methods for plant cell transformation and plant regeneration described in: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159 A1, EP 604 662 A1, EP 672 752 A1, US 4, 945,050, US 5,036,006 , US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174 A1, EP 486 233 A1, EP 486 234 A1, EP 539 563 A1, EP 674 725 A1, WO9 1/02071, WO 95/06128, WO 2011/095460, WO 2012006439 A2, WO 2012006443 A2, WO 2012015039 A 1, WO 2012019660 A1, WO 2012021494A1, WO 2012064827 A1, WO 2012075562A1, WO 2012077664A1, WO 2012083137A1, WO 2012084962A1, WO 2012092577 A1, WO 2012109947 A1, WO 2012129443 A 2, WO 2012138629 A2, WO 2012139416 A1, WO 2012149011 A1,

WO 2013014585 A1, WO 2013025670 A1, WO 2013033308 A2, WO 2013066007 A1, WO 2013077420 A1,

WO 2013090734 A1, WO 2013149726 A1, WO 2013180311 A1, WO 2014029044 A1, WO 2014029045 A1,

WO 2014062036 A1, WO 2014065857 A1, WO 2014100234 A1, WO 2014100406 A1, WO 2014123208 A1,

WO 2014143304 A1, WO 2014144513 A2, WO 2014157541 A1, WO 2014200842 A2, WO 2015051083 A1,

WO 2015077620 A1, WO 2015085990 A1, WO 2015099674 A1, WO 2015118640 A1, WO 2015119166 A1, each of which is incorporated herein by reference, particularly with respect to the transformation methods described.

In general, plant transformation methods involve transfer of heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by application of the maximum threshold level of a suitable selector (depending on selectable marker gene) to isolate transformed plant cells from non-transformed cell mass. Explants are usually transferred to the same fresh medium and cultured routinely. The transformed cells are then differentiated into sprouts after being placed in a regeneration medium with the addition of a maximum threshold level of the selective agent. The shoots are then transferred to a selective rooting medium to produce an established shoot or seedling. The transgenic seedlings are then grown to mature plants and produce fertile seeds (eg, Hiei et al. (1994) The Plant Journal 6:271282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Explants are usually transferred to the same fresh medium and cultured routinely. A general description of techniques and methods for creating transgenic plants can be found in Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Since the transformed material contains many cells, in any part of the target callus or tissue or group of cells being treated, both transformed and non-transformed cells are present. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often the ability to clear transformed cells is a limitation to the rapid isolation of transformed cells and the successful creation of transgenic plants. Molecular and biochemical methods can be used to confirm the presence of an integrated heterologous target gene in the genome of a transgenic plant.

The creation of transgenic plants can be accomplished by one or more methods, including, but not limited to, introducing heterologous DNA into plant cells by Agrobacterium (Agrobacterium-mediated transformation), bombarding plant cells with heterologous foreign DNA attached to particles, and various other direct or non-particle mediated DNA transfer methods (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750; Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239; Bommineni and Jauhar (1997) Maydica 42:107-120).

Methods for transforming chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. US 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913

- 28 045758

917; Svab and Maliga (1993) EMBO J. 12:601–606. The method is based on delivering DNA containing a selectable marker using a gene gun and targeting the DNA to the plastid genome through homologous recombination. Alternatively, plastid transformation can be accomplished by transactivation of a silent plastid-resistant transgene through preferential tissue expression of a nuclear-encoded plastid-directed RNA polymerase. Such a system was reported in McBride et al. (1994) Proc. Natl. Acad. Sci. US 91:7301–7305.

Transformed plant cells can be grown into plants according to standard techniques. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can be grown and pollinated with the same transformed strain or with other strains to produce a hybrid having constitutive expression of the desired phenotypic characteristic. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds may be collected to ensure that expression of the desired phenotypic characteristic is achieved. In this manner, the present invention provides a transformed seed (also referred to as a transgenic seed) containing a nucleotide construct in accordance with the invention, for example, an expression cassette in accordance with the invention, stably incorporated into the genome. In various embodiments, the seed may be coated with at least one fungicide and/or at least one insecticide, at least one herbicide, and/or at least one safener, or any combination thereof.

N. Assessment of plant transformation.

After the introduction of heterologous foreign DNA into plant cells, transformation or integration of the heterologous gene into the plant genome is confirmed by various methods, such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

A quick way to screen transformed cells, tissues, or propagules for the presence of the switched-on gene at an early stage before transplantation into soil is PCR analysis (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)) . PCR is carried out using oligonucleotide primers specific for the target gene or Agrobacterium vector background, etc.

Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell (2001) supra). In general, total DNA is extracted from the transformant, digested with suitable restriction enzymes, fractionated on an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or blot can then be probed, for example, with a radiolabeled ³² P fragment of the target DNA to confirm inclusion of the introduced gene in the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).

In Northern analysis, RNA is isolated from specific transformant tissues, fractionated on a formaldehyde-agarose gel, and applied to a nylon filter according to standard procedures routinely used in the art (Sambrook and Russell, 2001 supra). The expression of the RNA encoded by the nucleotide sequences according to the invention is then tested by hybridizing the filter with a radioactive probe obtained from the GDC by known methods (Sambrook and Russell (2001) supra). RNA can also be detected and/or quantified using reverse transcriptase PCR as known in the art (e.g., Green and Sambrook (2012) Molecular Cloning: A Laboratory Manual, ^4th edition, Cold Spring Harbor Laboratory Press, Woodbury, NY).

Transgenic plants can be analyzed by Western blots, ELISAs, droplet tests and biochemical assays to determine the presence of the protein encoded by the herbicide resistance gene by standard procedures (Sambrook and Russell (2001) supra) using antibodies that bind to one or more epitopes present on the herbicide resistance protein.

In one aspect of the invention, the GPPD genes described herein are useful as markers for assessing the transformation of bacterial or plant cells.

7. Use as a transformation marker.

The invention also relates to the use in a method of transforming plants of a nucleic acid encoding GPPD in accordance with the invention, as a marker gene or as a coding sequence making it possible to impart resistance to the plant to herbicides that are inhibitors of GPPD, and the use of one or more inhibitor ( -ov) GPPD on plants containing a nucleic acid sequence encoding GPPD in accordance with the invention. See, for example, US Pat. No. 6,791,014, which is incorporated herein by reference in its entirety.

In this embodiment, a GPPD inhibitor can be introduced into the culture medium of competent plant cells to decolorize said cells prior to transformation. The bleached competent cells are then transformed with the GPPD inhibitor resistance gene as a selectable marker, and the transformed cells that have incorporated the marker into their genome become green, allowing their isolation. Such a process can reduce the time without

- 29 045758 required for the selection of transformed cells.

Thus, one embodiment of the present invention is a method of transforming plant cells by introducing a heterologous gene into said plant cells with a gene for resistance to GPPD inhibitors as a selectable marker, the method comprising preparing and cultivating competent plant cells capable of accepting the heterologous gene to a suitable medium, and introducing a sufficient amount of GPPD inhibitor into a suitable culture medium with competent plant cells. Competent cells are then transformed with the heterologous gene and a selectable marker, and the transformed cells containing the heterologous gene are grown on a suitable medium and transformants are selected. The transformed cells can then be regenerated into a fruitful transformed plant.

J. Plants and plant parts.

By plant we mean the whole plant, plant organs (for example, leaves, stems, roots, etc.), seeds, plant cells, shoots, embryos and their offspring. Plant cells may be differentiated or undifferentiated (eg callus, cells in suspension culture, protoplasts, leaf cells, root cells, phloem cells, pollen). The present invention can be used to introduce polynucleotides into any plant species, including, but not limited to, monocots and dicots. Examples of target plants include, but are not limited to, corn (maize), sorghum, wheat, sunflowers, tomatoes, brassicas, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugar cane, tobacco, barley, oilseed rape, spp. Brassica, alfalfa, rye, millet, safflower, peanuts, sweet potatoes, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, bananas, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almonds, oats, vegetables, ornamental plants and coniferous trees.

Vegetable plants include, but are not limited to, tomatoes, lettuce, green peas, lima beans, peas and members of the genus Curcumis such as cucumber, cantaloupe and melon. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias and chrysanthemums. Crops that may also be of interest include, for example, corn, sorghum, sunflowers, tomatoes, brassicas, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugar cane, tobacco, barley, oilseed rape, etc.

This invention is suitable for any member of the monocot family, including, but not limited to, corn, rice, barley, oats, wheat, sorghum, rye, sugar cane, pineapple, yams, onions, bananas, coconut and dates.

K. Ways to increase plant productivity.

Methods for increasing plant productivity are provided. Methods include providing a plant containing a polynucleotide, or introducing into a plant or plant cell a polynucleotide comprising a nucleotide sequence encoding a GPPD in accordance with the invention, growing the plant or seed thereof in the field, and harvesting the plants or seeds. As defined in the present invention, plant yield refers to the quality and/or quantity of biomass produced by the plant. Biomass refers to any measurable plant product. An increase in biomass production is any improvement in the yield of a measurable plant product. Increasing plant yields has several commercial applications. For example, increasing plant leaf biomass can increase the yield of leafy vegetables for human and animal consumption. Additionally, increased leaf biomass can be used to increase the production of pharmaceutical or industrial products from plants. Yield improvement may include any statistically significant increase, including, but not limited to, an increase of at least 1%, an increase of at least 3%, an increase of at least 5%, an increase of at least 10%, an increase of at least 20%, at least 30%, at least 50%, at least 70%, at least 100% or greater increase.

In specific methods, a plant containing a GPPD nucleotide sequence according to the invention is treated with an effective concentration of a GPPD inhibitory herbicide, such as one or more GPPD inhibitory herbicides selected from the group consisting of GPPD inhibitory herbicides of the triketone class (preferably benzobicyclone, sulcotrione , mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione), diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxifene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate), N-(1,2,5-oxαduαzol-3-ul )benzamides, N(1,3,4-oxadiazol-2-yl)benzamides (preferably 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3(methylsulfonyl)-4 -(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), N(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably 2-chloro-3-ethoxy-4(methylsulfonyl) -N-(1-methyl-1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide; 2-chloro-3-(methylsulfanyl)-M-(1-methyl-1Htetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2- 30 045758 (methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, N(triazol-2-yl) )arylcarboxamides, triazinones and pyrazolones, and the use of the herbicide leads to an increase in plant yield.

Methods for imparting herbicide resistance to a plant or plant part are also provided. In such methods, a nucleotide sequence encoding GPPD in accordance with the invention is introduced into a plant, where expression of the polynucleotide results in resistance to herbicides that inhibit GPPD. Plants produced in this manner may be treated with an effective concentration of a herbicide (such as one or more GPPD inhibitor herbicides selected from the group consisting of GPPD inhibitor herbicides from the class of triketones (preferably benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione), diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxifene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate), N(1,2,5-oxadiazol-3-yl)benzamides, N-( 1,3,4-oxadiazol-2-yl)benzamides (preferably 2-methyl-N-(5methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide ( also referred to hereinafter as Comp. 2), N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-( 1-methyl-1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide; 2-chloro- 3 -(methylsulfanyl)-N-(1 methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, N-(triazol-2-yl)arylcarboxamides, triazinones and pyrazolones and exhibit increased herbicide resistance. An effective herbicide concentration in the present invention means an amount sufficient to slow or stop the growth of plants or plant parts that do not have inherent or acquired resistance to the herbicide.

L. Methods of controlling weeds in the field.

The present invention also relates to a method for controlling undesirable plants or regulating plant growth in crops of plants containing a HFPD nucleotide sequence in accordance with the invention, wherein one or more HFPD inhibitory herbicides, for example, one or more HFPD inhibitory herbicides are selected from a group consisting of a class such as triketones (preferably benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione), diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxifene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate), N-(1,2,5-oxadiazol-3-yl)benzamides, N-(1,3,4-oxadiazol-2yl)benzamides (preferably 2-methyl-N-(5-methyl-1,3,4- Oxadiazol-2-yl)-3-(methylsulfonyl)-4(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), N-(tetrazol-5-yl)- or N(triazol-5-yl)arylcarboxamides ( preferably 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1Htetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-N-(1 -methyl-1H-tetrazol-5yl)benzamide; 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-Stetrazol-5-yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, N(triazol-2-yl)arylcarboxamides, triazinones and pyrazolones, are applied to plants (for example, harmful plants such as monocotyledonous or dicotyledonous weeds or unwanted crops), to seeds (for example, grains, seeds or vegetative shoots such as tubers or sprouts with buds) or in the growing area plants (for example, cultivation area). In this context, an effective concentration of one or more GPPD-inhibiting herbicides, for example, one or more GPPD-inhibiting herbicides selected from the group consisting of GPPD-inhibiting herbicides from the class of triketones (preferably benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione), diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxifene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate), N-(1,2,5-oxadiazol-3yl)benzamides, N-(1 ,3,4-oxadiazol-2-yl)benzamides (preferably 2-methyl-N-(5-methyl-1,3,4oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), N-(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamides (preferably 2-chloro-3ethoxy-4-(methylsulfonyl)-N-(1-methyl -1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide, 2-chloro-3-( methylsulfanyl)-M-(1-methyl-1Htetrazol-5-yl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, N-(triazol-2-yl)arylcarboxamides , triazinones and pyrazolones, can be applied, for example, before planting (if necessary by introducing into the soil), pre-emergence or post-emergence, and can be applied in combination with other herbicides to which the crop has an innate resistance or is resistant through the expression of one or more herbicide resistance transgenes. See, for example, U.S. App. Pub. No. 2004/0058427 and PCT application no. pub. WO

- 31 045758

98/20144. By effective concentration is meant the concentration that controls the growth or spread of weeds or other transformed plants without significantly affecting the GPPD inhibitor-resistant plant or seed. Those skilled in the art will appreciate that herbicide application can take many different forms and can occur at various times before and/or during seed planting and the growing process. Pre-emergence application is the application of the herbicide to the appropriate location (e.g., field or growing area) before the plant is visible to emerge from the soil. Post-emergence application is the application of a herbicide to the appropriate location after the plant has visibly emerged from the soil. In some cases, the terms pre-emergence and post-emergence are applied to the weed in the appropriate location, and in some cases the terms are applied to the crop plant in the appropriate location. When applied to a weed, these terms may refer to a specific type or species of weed found or suspected to be present in the relevant location. Preplant herbicide application involves introducing compounds into the soil prior to planting.

Thus, the present invention includes a method of controlling weeds in a field, which consists in planting a plant or its seeds containing the GPPD polypeptide proposed in the present invention in the field, and applying an effective the concentration of one or more GPPD-inhibiting herbicides.

In one embodiment of the present invention, a field to be planted with plants (such as soybeans, cotton, corn, or for example wheat plants) containing the HFPD nucleotide sequence according to the invention may be treated with a HFPD inhibitory herbicide such as isoxaflutole ( IFT), before planting or sowing seeds, which clears the field of weeds destroyed by the GPPD-inhibiting herbicide, allowing the use of zero-till practices, followed by planting or sowing of plants on the same pre-treated field (contact application of the GPPD-inhibiting herbicide) . Residual IFT activity will also protect emerging and growing plants from weed competition in the early stages of growth. When plants reach a certain size and the weeds tend to reappear, glufosinate or glyphosate, or a GPPD-inhibiting herbicide or a mixture of a GPPD-inhibiting herbicide with another herbicide such as glyphosate, may be applied to the plants if such plants are resistant to these. herbicides.

In another embodiment of the present invention, a field sown with seeds containing a GPPD nucleotide sequence according to the invention may be treated with a GPPD inhibitory herbicide, such as IFT, before the plants emerge but after the seeds are sown (the field may be previously cleared of weeds other means, usually standard tillage practices such as plowing, deep plowing or seed preparation), and the residual activity will keep the field clear of weeds killed by the herbicide, so that emerging and growing plants have no competition with weeds (pre-emergence application of a herbicide that inhibits GPPD). When plants reach a certain size and weeds are likely to re-emerge, glufosinate or glyphosate, or a GPPD-inhibiting herbicide or a mixture of a GPPD-inhibiting herbicide with another herbicide, such as glyphosate, can be applied to the plants if such plants are resistant to these herbicides.

In another embodiment of the present invention, plants containing a GPPD nucleotide sequence in accordance with the invention can be treated with a GPPD inhibitory herbicide on top of plants that have already emerged from the sown seeds, thereby clearing the field of weeds killed by the GPPD inhibitory herbicide, application which may be combined with (e.g. in a tank spray), followed or preceded by glyphosate or glufosinate as a post-emergence herbicide applied to plants (post-emergence application of a GPPD inhibitory herbicide (with or without glyphosate)) if such plants are resistant to those herbicides.

Examples of selected monocot and dicot weeds that can be controlled with a GPPD inhibitory herbicide include:

Monocot pests from the genera: Aegilops, Agropyron, Agrostis, Alopecurus, Apera, Avena, Brachiaria, Bromus, Cenchrus, Commelina, Cynodon, Cyperus, Dactyloctenium, Digitaria, Echinochloa, Eleocharis, Eleusine, Eragrostis, Eriochloa, Festuca, Fimbristylis, Heteranthera, Imperata, Ischaemum, Leptochloa, Lolium, Monochoria, Panicum, Paspalum, Phalaris, Phleum, Poa, Rottboellia, Sagittaria, Scirpus, Setaria, Sorghum.

Dicotyledonous harmful plants from the genera: Abutilon, Amaranthus, Ambrosia, Anoda, Anthemis, Aphanes, Artemisia, Atriplex, Bellis, Bidens, Capsella, Carduus, Cassia, Centaurea, Chenopodium, Cirsium, Convolvulus, Datura, Desmodium, Emex, Erysimum, Euphorbia, Galeopsis, Galinsoga, Galium, Hibiscus, Ipomoea, Kochia, Lamium, Lepidium, Lindernia, Matricaria, Mentha, Mercurialis, Mullugo, Myosotis, Papaver, Pharbitis, Plantago, Polygonum, Portulaca, Ranunculus, Raphanus, Rorippa, Rotala, Rumex, Salsola, Senecio, Sesbania, Sida, Sinapis, Solanum, Sonchus, Sphenoclea, Stellaria, Taraxacum, Thlaspi, Trifolium, Urtica, Veronica, Viola, Xan

- 32 045758 thium.

GPPD inhibitory herbicides useful in the present invention, including, but not limited to, GPPD inhibitory herbicides from the class of triketones (preferably benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinothrone), diketonitriles, isoxazoles (preferably isoxaflutole), hydroxypyrazoles (preferably pyrazoxifene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate), N-(1,2,5-oxadiazol-3yl)benzamides, N-(1,3,4-oxadiazol-2-yl)benzamides (preferably 2 -methyl-N-(5-methyl-1,3,4oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 2), N-(tetrazole-5 -yl)- or N(triazol-5-yl)arylcarboxamides (preferably 2-chloro-3ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl) -2-methoxy-3(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide; 2-chloro-3-(methylsulfanyl)-M-(1-methyl-1Htetrazol-5-yl) -4-(trifluoromethyl)benzamide (also referred to hereinafter as Comp. 1); 2(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, pyridazinone derivatives, oxoprazine derivatives, N(triazol-2-yl)arylcarboxamides, triazinones and pyrazolones can be formulated in a variety of ways, depending on the prevailing biological and/or physicochemical parameters. Examples of possible formulations are: wettable powders (WP), water-soluble powders (SP), water-soluble concentrates, emulsifiable concentrates (EC), emulsions (EW) such as oil-in-water and water-in-oil emulsions, spray solutions, suspension concentrates (SC), oil or water based dispersions, oil miscible solutions, capsule suspensions (CS), fine powders (DP), seed treatment products, granules for broadcast and soil application, granules (GR) in the form of microgranules , spray granules, coated granules and adsorption granules, water-dispersible granules (WG), water-soluble granules (SG), ultra-low volume ULV formulations, microcapsules and waxes.

These individual types of compositions are in principle known and are described, for example, in: Winnacker-Kuchler, Chemische Technologie [Chemical Technology], Volume 7, C. Hanser Verlag Munich, 4th ed. 1986; Wade van Valkenburg, Pesticide Formulations, Marcel Dekker, N.Y., 1973; K. Martens, Spray Drying Handbook, 3rd ed. 1979, G. Goodwin Ltd. London.

Necessary formulation auxiliaries, such as inerts, surfactants, solvents and other additives, are also known and described, for example, in: Watkins, Handbook of Insecticide Dust Diluents and Carriers, 2nd ed., Darland Books, Caldwell N.J., H.v. Olphen, Introduction to Clay Colloid Chemistry; 2nd ed., J. Wiley & Sons, N.Y.; C. Marsden, Solvents Guide; 2nd ed., Interscience, N.Y. 1963; McCutcheon's Detergents and Emulsifiers Annual, MC Publ. Corp., Ridgewood N.J.; Sisley and Wood, Encyclopedia of Surface Active Agents, Chem. Publ. Co. Inc., N.Y. 1964; Schonfeldt, Grenzflachenaktive Athylenoxideddukte [Surface-active ethylene oxide adducts], Wiss. Verlagsgesell., Stuttgart 1976; Winnacker-Kuchler, Chemische Technologie [Chemical Technology], Volume 7, S. Hanser Verlag Munich, 4th ed. 1986.

Based on these compositions, it is also possible to prepare combinations with other pesticidal active substances, such as, for example, insecticides, acaricides, herbicides, fungicides, as well as with safeners, fertilizers and/or growth regulators, for example, in the form of ready-to-use formulations or in the form mixture in the tank.

M. Methods of introducing a gene in accordance with the invention into another plant.

The present invention also provides methods for introducing a HFPD nucleotide sequence according to the invention into another plant. The nucleotide sequence of GPPD according to the invention, or a fragment thereof, can be introduced into another plant by recurrent selection, backcrossing, selective breeding, line selection, mass selection, mutation breeding and/or genetic marker enhanced selection.

Thus, in one embodiment, the methods of the invention include crossing a first plant containing a GPPD nucleotide sequence in accordance with the invention with a second plant to produce F1 progeny plants, selecting F1 progeny plants that are resistant to a herbicide that inhibits GPPD or contain the nucleotide sequence HFPD in accordance with the invention. The methods may also include crossing selected progeny plants with a first plant containing a nucleotide sequence in accordance with the invention to produce backcross progeny plants and selecting backcross progeny plants that are resistant to a GPPD inhibitory herbicide or containing a GPPD nucleotide sequence in accordance with the invention. Methods for assessing resistance to herbicides that inhibit GPPD are provided elsewhere in this application. In addition, the methods may also include repeating these steps one or more times sequentially to produce selected second or subsequent generation backcross progeny plants that are resistant to the GPPD inhibitory herbicide or contain the GPPD nucleotide sequence in accordance with the invention.

The method according to the present invention can use any breeding method including selecting plants for a desired phenotype. In some embodiments

- 33 045758 F1 plants can self-pollinate to produce a generation after F2 splitting. Individual plants can then be selected to represent the desired phenotype (eg resistance to GPPD-inhibiting herbicides) in each generation (F3, F4, F5, etc.) until the traits become homozygous or fixed within the population for breeding.

The second plant may be a plant having a desired trait such as herbicide tolerance, insect resistance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency, improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality , resistance to stress, improved reproduction, etc. The second plant may be an elite event, as described elsewhere in the present invention.

In various embodiments, plant parts (whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, shoots, embryos, etc.) can be collected from the resulting single cross and or distributed, or collected for later use for further propagation or processing (such as production of food, feed, biofuel, oil, flour, consumption, etc.).

N. Methods for obtaining plant products.

The present invention also relates to a method for producing a marketable product, comprising harvesting and/or grinding grains of a crop containing the nucleotide sequence of GPPD in accordance with the invention, to obtain a marketable product. Agronomically and commercially important products and/or compositions, including, but not limited to, animal feed, articles, and plant products and by-products, are intended for use as foodstuffs for human consumption or for use in compositions and goods intended for human consumption, in particular devoid of viability seeds/cereals, including (semi-)processed products derived from such grains/seeds, said product being or containing whole or processed seeds or grains, animal feed, coarse corn or soybean meal, corn or soybean meal, corn, corn starch, coarse soybean meal, soybean meal, cereal, soy protein concentrate, soy protein isolates, textured soy protein concentrate, cosmetics, hair care products, soy nut butter, natto, tempeh, hydrolyzed soy protein, whipping cream, shortening, lecithin, edible whole soybeans (raw, roasted or as edamame), soy yogurt, soy cheese, tofu, yubu, and cooked, ground, steamed, baked or parboiled grains, etc., are intended to be included in the present invention if these products and compositions contain detectable amounts of nucleotide and/or amino acid sequence set forth in the present invention as diagnostic for any plant containing such nucleotide sequences.

The following examples are provided for illustrative purposes and not for limitation.

experimental part

Review.

Example 1. Creation of mutated GPPD polypeptides by site-directed mutagenesis.

Example 2: Cloning, expression and purification of recombinant wild-type and mutant GPPD polypeptides.

Example 3: GPPD Enzyme Assay for Analysis of GPPD Mutant Polypeptides with Improved Resistance to GPPD Inhibiting Herbicides.

Example 4: Improved Herbicide Resistance Mediated by Residue Exchanges in HPPD Polypeptides.

Example 5 Brown Assay for Testing Mutant GPPD Polypeptides Resistant to GPPD Inhibiting Herbicides.

Example 6: Transformation of soybeans and resistance of T0 soybean plants.

Example 7. Creation and selection of cotton plant T0.

Example 8 Transformation of maize plant cells by Agrobacterium-mediated transformation.

Example 9: T1 Soybean Plant Resistance to GPPD Inhibiting Herbicides/Field Test.

Example 1. Creation of mutated GPPD polypeptides by site-directed mutagenesis.

The HPPD nucleotide sequence of Pseudomonas fluorescens (SEQ ID NO: 109) as described in WO 2009/144079 encoding the HPPD polypeptide corresponding to SEQ ID NO: 1 was cloned according to well known methods in the art and as described in WO 2014/043435 . Subsequent site-saturated mutagenesis, site-directed mutagenesis and combinatorial variants with one or more mutations of the nucleic acid encoding the wild-type PfHPPD polypeptide sequence encoding the recombinant HPPD polypeptide corresponding to SEQ ID NO: 1 were carried out using a standard method based on PCR technologies, well known in the art (and as described in WO2014/043435). All developed and synthesized mutants

- 34 045758 clones were confirmed by DNA sequencing using plasmid specific oligonucleotides. In table 2 below shows exemplary mutant GPPD polypeptides (SEQ ID NO: 2 - SEQ ID NO: 108).

table 2

Overview of exemplary amino acid exchanges corresponding to amino acid position in SEQ ID NO: 1

The position of amino acids relative to the GPPD polypeptide SEQ Ш NO:1 about st Mr. St. 3 tbsp OO about st O st o 1—H ME 1—N O 1—Н meh 40 7 OO O O meh A R m R T Q T D D E G N F TO A S I R W A Q R S R S R D R D S R D S V R D S V R D S V V

- 35 045758

AND H P D S 12 H P D S V 13 H P D s V 14 H P D s V V 15 S S P D s V V 16 s S P D s V To 17 G E P D s V V 18 R H P D s V 19 R G E H P D s V V 20 P H 21 P H s 22 P H s V 23 P H s V 24 P H s V V 25 H P H s 26 H P H s V 27 H P H s V 28 H P H s V V 29 S S P H s V V thirty s S P H s V To 31 G E P H s V V 32 R E P H s V V 33 G E P D s R V 34 G E H P D s R To 35 G E P D s R To 36 R G E H P D s V M 37 G R H P D s V To 38 G E V P D s V V 39 L G E P D s V V 40 M G E P D s V V 41 s G E P D s V V 42 τ G E P D s V V 43 To G E P D s V V 44 L G E P D s V V 45 G E R P D s V V 46 G E M P D s V V 47 G E H P D s V V 48 G E H P D s V V 49 G E I P D s V V 50 G E P P D s V V 51 G E L P D s V V

-36045758

52 G E S R D S V V 53 G E R D S V V V 54 G E R D S A V V 55 G E R D S E V V 56 G E R D S R V V 57 G E R D S T V V 58 G E R D S V R V 59 G E R D S V R V 60 R G E n R D S V Q V 61 R G E n R D S V R V 62 R G E n R D S V R V 63 G R D S V V 64 G E N R D S V V 65 G E TO R D S V V 66 G E S R D S V V 67 TO G E R D S V V 68 L G E R D S V V 69 Q G E R D S V V 70 R G E R D S V V 71 E R D S V V 72 R E R D S V V 73 S E R D S V V 74 G L R D S V V 75 G R R D S V V 76 G R R D S V V 77 G S R D S V V 78 G E A R D S V V 79 G E F R D S V V 80 G E G R D S V V 81 G E R D S V 82 G E R D S E V 83 G E R D S G V 84 G E R D S L V 85 G E R D S m V 86 G E R D S Q V 87 G E R D S V A 88 G E R D S V 89 G E R D S V To 90 G E R D S V m 91 G E R D S V R 92 R G E N R D S V A 93 R G E N R D S V 94 R G E N R D S V To 95 R G E N R D S V R 96 TO G E R N S V V 97 G R N S R D S V To 98 G E N S R D S R To 99 G E R D S V R V 100 L G E N R D S V Q m 101 L G E R R D S V Q m 102 L R E R R N S V Q m 103 TO L G E R R N S V Q m 104 TO L G E R N S V Q m 105 TO L G E R N S R Q m 106 L G E N R R D S V Q m 107 TO G E R N S V R V 108 G E M N R D S V m

- 37 045758

For clarity, empty cells at the corresponding amino acid positions in SEQ ID NO: 2 - NO: 108 are defined as identical to the amino acids corresponding to SEQ ID NO: 1, highlighting only the exchanges in the mutant GPPD polypeptides. The mutant GPPD polypeptides presented here are examples by way of illustration and not by way of limitation.

Example 2. Cloning, expression and purification of recombinant wild-type and mutant GPPD polypeptides.

All obtained nucleic acid sequences encoding wild type GPPD and mutant encoding recombinant GPPD polypeptide were cloned, produced and purified using methods well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed. ., CSH Laboratory Press, 2001, Cold Spring Harbor, NY). All resulting nucleic acid coding sequences were cloned into pSE420(RI)NX fused with an N-terminal His tag (encoding the amino acid sequence M1-A2-H3-H4-H5-H6-H7-H8-), as described in WO 2014/043435 , and expressed in Escherichia coli strain BL21 (DE3) (New England Biolabs, Frankfurt, Germany). For clarity, all listed positions with the corresponding amino acid exchanges from the HPPD HPPD mutant polypeptides are in Table. 1-5, and table. 7 corresponding to SEQ ID NO: 2 to SEQ ID NO: 108 in the present invention refer to the amino acid sequence of native wild type GPPD without the N-terminal His tag corresponding to SEQ ID NO: 1.

To generate samples of purified GPPD polypeptide, cells were grown for 3 h at 37°C in 5 ml of LB medium containing 100 μg/ml ampicillin in a 50 ml mixing flask at 140 rpm. One ml of this stock culture was used as the inoculum for the expression culture. Cells were grown for approximately 3 h at 37°C in 100 ml LB medium containing 100 μg/ml ampicillin m 150 mM Hepes (Merck, Darmstadt, Germany) in a 500 ml mixing flask at 120 rpm. At an OD600 of approximately 0.6, IPTG (Roth, Karlsruhe, Germany) was added to a concentration of 0.4 mM. After further growth for 60 min. at 37°C, the temperature was reduced to 28°C and growth continued for another 18 h at 140 rpm. Cells were collected by centrifugation at 4°C, 3200 g for 30 min in 50 ml Falcon tubes, and the cell clump was stored at -80°C. Cells were lysed and his-tagged protein purified according to the manufacturer's protocol of the Ni-NTA Fast Start Kit used (Qiagen, Hilden, Germany) followed by adaptations to increase yield: cells from a 50 ml culture were lysed into 4 ml and the lysate supernatant was obtained by centrifugation for 15 min at 18000 g. The amount of matrix in the columns was increased by adding 1 ml NiNTA Superflow (Qiagen, Hilden, Germany) each and thoroughly rebuffered in 20 mM Tris (pH 7.6) (Merck, Darmstadt, Germany). The lysate supernatant was used, and the His-tagged protein was coupled to the Ni-NTA matrix by incubation for 1 h at 4°C.

The resulting protein samples were rebuffered in 20 mM Tris, 20% glycerol (pH 7.8) (SigmaAldrich, St. Louis, USA) using Zeba™ Spin Desalting columns, 7K MWCO, 10 ml (Thermo Fisher Scientific, Waltham, USA) and analyzed on protein concentration and purity by Abs280 (NANODROP 8000, Thermo Fisher Scientific, Waltham, USA) and SDS-PAGE. Purified protein concentrations typically ranged from 0.6 to 4.6 mg/mL, with an estimated purity of approximately 90%.

To obtain crude extract of GPPD polypeptide in microtiter plates (MTP) to determine residual activity in inhibition assays, cells were grown in 40 or 150 μl of LB medium containing 1% glucose (Merck, Darmstadt, Germany) and 100 μg/ml ampicillin in standard A 96-well plate (Thermo Fisher Scientific, Waltham, USA) was incubated for approximately 18 hours in a 37°C humidified incubator.

µl of this stock culture was added to 600 µl of LB medium containing 100 µg/ml ampicillin and 150 mM Hepes (Merck, Darmstadt, Germany) as an inoculum for expression culture in 96-well plates (2 ml deep wells; HJ Bioanalytik, Erkelenz, Germany) . The plates were sealed with aluminum foil and the cells were incubated for 5 h at 37°C on a plate shaker set to 750 rpm. Expression was induced by adding IPTG at a final concentration of 1 mM, followed by further sealed incubation for approximately 18 h at 30°C on a plate shaker at 750 rpm.

Cells were collected by centrifugation at 4°C, 2500 g for 15 min, discarding the supernatant. The cell clump was stored at -80°C and lysed in 250 μl of 1x BugBuster® (Merck, Darmstadt, Germany) in 140 mM Tris (pH 7.8), with 1:25,000 diluted BNase® (Qiagen, Hilden, Germany) by incubating the resuspended cells for 30 minutes at 4°C and 1000 rpm. Lysates were clarified by centrifugation for 15 min. at 4°C, 2500 g, and 150 μl of the supernatant was transferred to a standard 96-well plate (Thermo Fisher Scientific, Waltham, USA) for subsequent quadruplet testing.

Example 3: GPPD Enzyme Assay for Analysis of GPPD Mutant Polypeptides with Improved Resistance to GPPD Inhibiting Herbicides.

The activity of GPPD polypeptides was determined in the absence or presence of GPPD inhibitors using a coupled GPPD activity assay (Fig. 1).

- 38 045758

To determine the residual activity, the apparent kinetic constant (k _app ) of a given substrate conversion was measured as the kinetic changes in absorbance at 320 nm in a coupled assay, since the homogentisate (HH) formed by HPPD from HFP is directly converted to the highly absorbent molecule maleylacetoacetate (MAA) by the second homogentisate dioxygenase (HGD) enzyme, used in excess consistently in all assays (see Fig. 1). Measurements were performed in 384 microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) using plate readers (Tecan infinite M1000 or M1000PRO, Tecan, Männedorf, Switzerland). The ratio k _cat /k _M of enzyme activity is proportional to the apparent kinetic constant k _app and proportional to k _cat /k _M *[E] ([E] = enzyme concentration). A competitive inhibitor exhibits an apparent increase in kM and hence an inverse decrease in kapp at nonsaturable substrate concentrations. Since both kapp measurements in the presence and absence of inhibitor were made using an identical enzyme sample, crude or purified, and thus at the same enzyme concentration, the enzyme concentration is excluded from the calculation of residual activity and the ratio of both kapps directly indicates the change in kM due to inhibition. Notably, this concept refers to enzyme/inhibitor pairs interacting in a competitive inhibitory manner, likely true for almost all polypeptide and inhibitor variants described herein, but certainly not for a wild-type polypeptide that is inhibited irreversibly (by comparison see WO 2014/043435, figures 2 and 3). Therefore, the residual activity of wild-type GPPD polypeptide related to competitive inhibition and ki values cannot be calculated correctly, however, for illustrative purposes, unreasonable values are given in Table. 3 for wild-type GPPD polypeptide, which are calculated from the initial signal changes before irreversible inhibition occurs.

The assay solution used to determine residual activity in crude GPPD polypeptide samples consisted of 200 mM sodium phosphate (Merck, Darmstadt, Germany, pH 7.0), 5 mM MgCl ₂ (Merck, Darmstadt, Germany), 20 mM ascorbate (Sigma-Aldrich , St. Louis, USA), 1 mM DTT (Sigma-Aldrich, St. Louis, USA), 0.1% Pluronic F-68 (Sigma-Aldrich, St. Louis, USA), 40 µM _FeSO4 (Sigma-Aldrich, St. Louis, USA), approximately 8 mg/ml purified HGD and low or high concentrations of GFP substrate (100 or 400 μM) from a 1 M stock solution in DMSO (Sigma-Aldrich, St. Louis, USA) and equilibrated for 20 min. on ice. For each GPPD polypeptide sample, two quadruplet assays were performed whereby 5 μl of GPPD polypeptide sample was first mixed with 5 μl of buffer (1x BugBuster®; (Merck, Darmstadt, Germany); in 140 mM Tris, pH 7.8, with a 1:25,000 dilution BNase®; Qiagen, Hilden, Germany)) or 5 µl inhibitor diluted in the same buffer from a 0.1 M stock solution in DMSO (30, 100 or 120 µM, resulting in 15, 50 and 60 µM in the GPPD polypeptide/inhibitor sample ) in the control and inhibition assays, respectively, and then with 10 μl of the assay solution. The change in optical density at 320 nm was carried out at intervals of 1 min for 30 min. The k _app values were calculated as the slope of the signal over time during the early phase of the kinetic reaction, typically for the first 5-10 minutes of measurements (cf. Fig. 3). Additionally and in accordance with the calculated residual activity, the complete conversion, i.e. The absolute change in signal over the 30 min time interval was monitored as a measure of turnover, and the residual turnover was calculated by dividing the change in signal in the presence of the inhibitor by the change in signal in the reference sample without the inhibitor.

The assay solutions used to determine ki values were formulated in the same manner and contained six different concentrations of GPP substrate (0 - 1350 μM) for each of the four inhibitor concentrations tested. Inhibitors were diluted in 140 mM Tris, 0.05% Pluronic F-68 (Sigma-Aldrich, St. Louis, USA) and applied at the concentrations adopted for the corresponding GPPD polypeptide/inhibitor pairs to generate dynamic data (Fig. 4 a–d); Typically, their concentrations in the GPPD polypeptide/inhibitor sample ranged from 0 to 0.0012 M.

Example 4: Improved Herbicide Resistance Mediated by Residue Exchanges in HPPD Polypeptides.

When the tolerance of mutant GPPD polypeptides was determined to the various available chemical classes of GPPD inhibitory herbicides (triketones, isoxazoles, N-(1,3,4-oxadiazol-2yl)benzamides, or N-(tetrazol-5-yl)-arylcarboxamides), then It has become apparent that some of the new embodiments of the present invention are not only significantly improved over the reference wild-type HPPD (SEQ ID NO: 1), but also unexpectedly better than prior art mutant HPPD polypeptides (such as those which are described in WO 2014/043435) with SEQ ID NO: 2 in this invention as an example.

As indicated in table. 3, the prior art GPPD mutant polypeptides (WO 2014/043435) corresponding to SEQ ID NO: 2 in this invention contain residue exchanges at positions 335, 336, 339 and 340.

Based on a mutant GPPD polypeptide containing 335 (E=>P), 336 (G=>D/H), 337 (N=>S), the introduction of additional residue exchanges at positions 264, 268, 270, 330, 340 and/ or 345, produced by 39 045758 mutant GPPD polypeptides, demonstrating significantly improved stability (Table

3), in relation to many applied GPPD inhibitors belonging to various chemical classes.

Accordingly, the inventors have created and evaluated new mutant GPPD polypeptides by combinatorial exchanges of residues at positions 335 (glutamic acid => proline), 336 (glycine => aspartic acid/histidine), 337 (asparagine => serine) and optionally further including exchanges at positions 204, 213, 264, 268, 270, 310, 315, 330, 331, 338, 339, 340, 344 and/or 345 (Tables 3, 4 and 5), which show improved residual activities, higher residual turnover, higher ki values and thus significantly higher herbicide resistance. Depending on the GPPD inhibitory herbicide tested, the level of improvement may vary for the GPPD polypeptides used in such an assay, ranging from 1.5 to 110 times that of SEQ ID NO: 2 (Table 4).

Analysis of the period of inhibition against various chemical classes of GPPD-inhibiting herbicides showed that GPPD-inhibiting herbicides appear to be reversible inhibitors against the new mutant GPPD polypeptides, in contrast to the slow and tightly binding inhibitor characteristic of the wild-type GPPD polypeptide, corresponding to SEQ ID NO: 1 (see FIGS. 2 and 3). This behavior provides better and more versatile potential for resistance in crop plants to various GPPD-inhibiting herbicides.

For high residual activity in the presence of herbicides that inhibit GPPD, the disclosed positions and residue changes are highlighted in Table. 1 with respect to the position of the amino acid in the HPPD polypeptide corresponding to SEQ ID NO: 1 in the present invention are presented as important.

Table 3. Resistance of mutant GPPD polypeptides to various herbicides that inhibit GPPD, belonging to different chemical classes.

Table 3a

Residual activity and turnover in the presence of comp. 1 in accordance with example 3 at a high concentration of substrate and 15 μM inhibitor

Position of amino acids relative to the GPPD polypeptide SEQIDNO:1 ABOUT £ 0 Residual Residual O SSSSSSSSSSSSSSSSSS activity turnover sch OS 1 MPTDEGNKAI 6% 6% 2 P W A Q 39% 50% 3 R 9% 9% 4 S 13% 12% 5 P S 20% 24% 7 P D S 27% 38% 8 P D S V 21% 44% 9 P D S V 36% 51% 10 P D S V V 41% 66% AND Η Р D S 24% 30% 12 Η Р D S V 43% 55% 13 Η Р D S V 35% 40% 14 Η Р D S V V 44% 60% 15 S S P D S V V 46% 74% 16 S S P D S V K 45% 69% 17 G E R D S V V 43% 68% 18 R Η P D S V 67% 79% 19 RGEHPDS VV 68% 79% 20 R N 14% 16% 21 R N S 18% 30% 22 R N S V 26% 53% 23 R N S V 26% 36% 24 R N S V V 28% 57% 25 N R N S 33% 37% 26 N R N S V 55% 65%

- 40 045758

27 N R N S V 36% 42% 28 N R N S V V 45% 57% 29 S S Р Н S V V 33% 59% thirty S S Р Н S V к 39% 65% 31 G E R N S V V 40% 67%

Table 3b

Residual activity and turnover in the presence of comp. 2 in accordance with example 3 at a high concentration of substrate and 15 μM inhibitor

Position of amino acids relative to the GPPD polypeptide SEQIDNO:1 SEQ ID NO FROM OO O O MO \O O O sh C| CI CI CO CO CO CO co co co Residual activity Residual turnover 1 2 MPTDEGNKAI P W A Q 8% 51% 8% 72% 3 4 5 7 8 9 10 I 12 13 14 15 16 17 18 19 20 21 22 23 P S P S P D S P D S V P D S V P D S V V Η Р D S Η Р D S V Η Р D S V Η Р D S V V S S P D S V V S S P D S V K G E R D S V V R Η P D S V RGEHPDS VV R N R N S R N S V R N S V 16% 18% 36% 75% 67% 79% 89% 59% 93% 82% 82% 90% 94% 90% But. 92% 31% 73% 90% 76% 22% 17% 53% 83% 74% 89% n.a. 67% no. 88% 92% n.a. 94% 90% n.a. 93% 50% 84% n.a. 83% 24 25 26 27 28 29 thirty 31 R N S V V N R N S N R N S V N R N S V N R N S V V S S Р Н S V V S S Р Н S V к G E R N S V V 90% 82% 92% 80% 83% 85% 95% 95% But. 83% 95% 86% 93% But. But. But.

- 41 045758

Table 3c

Residual activity and turnover in the presence of mesotrione (MST) according to example 3 at high substrate concentration and 60 µM inhibitor

Position of amino acids relative to the GPPD polypeptide SEQIDNO:1 SEQ ID NO Ό OO O O ME YU O O O, CNCNCNOOOOOOO Residual activity Residual turnover 1 2 MPTDEGNKAI P W A Q 6% 71% 8% 85% 3 4 5 7 8 9 10 I 12 13 14 15 16 17 19 20 21 P S P S P D S P D S V P D S V P D S V V Η Р D S Η Р D S V Η Р D S V Η Р D S V V S S P D S V V S S P D S V K G E R D S V V RGEHPDS VV R N R N S 13% 16% 46% 70% 70% 71% 82% 64% 80% 72% 70% 83% 88% 85% 51% 25% 62% 17% 15% 71% 85% 85% 86% But. 85% 94% 88% 89% 94% 92% 90% 78% 40% 89% 22 23 24 25 26 27 28 29 thirty 31 R N S V R N S V R N S V V N R N S N R N S V N R N S V N R N S V V S S Р Н S V V S S Р Н S V к G E R N S V V 77% 63% 51% 70% 81% 64% 66% 79% 87% 83% 99% 79% 68% 87% 92% 82% 84% 95% n.a. But.

- 42 045758

Table 3d

Residual activity and turnover in the presence of diketonitrile (DKN) according to example 3 at high substrate concentration and 60 µM inhibitor

Position of amino acids relative to the GPPD polypeptide SEQIDNO:1 SEQ ID NO -G OO O O MCH -Ό t~~ OO MCH O O t~~ O O O O O T T PPPppppyuyuyuyu Residual activity Residual turnover 1 2 MPTDEGNKAI P W A Q 7% 86% 8% 93% 3 4 5 7 8 9 10 I 12 13 14 15 16 17 18 19 R S P S P D S P D S V P D S V P D S V V Η Р D S Η Р D S V Η Р D S V Η Р D S V V S S P D S V V S S P D S V K G E R D S V V R Η P D S V RGEHPDS VV 17% 17% 41% 79% 91% 90% n.a. 81% no. But. 91% no. But. But. But. But. 17% 14% 47% 92% 95% 92% n.a. 95% no. But. But. But. But. 95% no. But. 20 21 22 23 24 25 26 27 28 29 thirty 31 R N R N S R N S V R N S V R N S V V N R N S N R N S V N R N S V N R N S V V S S Р Н S V V S S Р Н S V к G E R N S V V 37% 81% n.a. 83% 93% n.a. But. 92% 90% 94% n.a. But. 40% n.a. But. 87% no. But. But. But. 95% no. But. But.

Residual activity and residual turnover were determined according to example 3 by measuring kapp and total signal change, respectively, in the presence and absence of (a) comp. 1 (2chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide), (b) comp. 2 (2methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide), (c) mesotrione (MST), and (d) diketonitrile (DKN). For each mutant GPPD polypeptide, k _app and total signal change in the absence of GPPD inhibitory herbicides served to normalize k _app and total signal change in the presence of the herbicide. The summed % values in the corresponding tables 3a), 3b), 3c) and 3d) are the means of two independent experiments with an average standard deviation of 5%. The reaction was carried out at high concentrations of substrate with com. 1 and conn. 2 at a concentration of 15 µM and the other two herbicides (DKN, MST) at a concentration of 60 µM. For clarity, empty cells at the corresponding amino acid position in SEQ ID NO: 2 through SEQ ID NO: 108 are defined as identical to the amino acids corresponding to SEQ ID NO: 1, highlighting only the exchanges in the GPPD polypeptide variant. Abbreviation n.o. means that no inhibition was observed under these conditions, i.e. k _app or total signal change in the presence of the inhibitor did not decrease compared to the corresponding value in the absence of inhibitors.

Mutant GPPD polypeptides corresponding to SEQ ID NO: 10 and SEQ ID NO: 24 with amino acid exchanges at positions 335, 336, 337, 340 and 345 relative to the GPPD polypeptide according to SEQ ID NO: 1, in the presence of various GPPD inhibitors tested, exhibit significant improvement in terms of residual activity and residual turnover (Table 3a-d). The depicted herbicide resistance of SEQ ID NO: 10 shows not only an improvement over the wild type GPPD polypeptide (SEQ ID NO: 1), but also compared to the mutant GPPD polypeptide from

- 43 045758 prior art (WO 2014/043435) corresponding to SEQ ID NO: 2 in the present invention on all four different classes of herbicides depicted.

Further improvements in GPPD inhibitor resistance are evident in variants with residue exchanges at exposed amino acid positions 268 and 270.

Based on SEQ ID NO: 24, SEQ ID NO: 31 differ only in the indicated positions 268 and 270. These changes significantly increase the residual turnover in the presence of the GPPD inhibitor Comp. 1 and MST (Tables 3a and 3c), and maintain the already achieved high residual turnover in the presence of Compound. 2 (Table 3b) and DKN (Table 3d). These results can also be seen due to the significantly improved ki values of SEQ ID NO: 31 (Table 4) compared to the HPPD polypeptide (SEQ ID NO: 1), and the prior art mutant HPPD polypeptide (WO 2014/043435) corresponding to SEQ ID NO: 2 in the present invention.

Based on the mutant GPPD polypeptide corresponding to SEQ ID NO: 8, a further change in the position of amino acid 330 (see SEQ ID NO: 12) demonstrates further improved resistance to the GPPD inhibitor (see Table 3)

Based on the mutant GPPD polypeptide corresponding to SEQ ID NO: 12, a further change in the position of amino acid 264 (see SEQ ID NO: 18) demonstrates further improved resistance to the GPPD inhibitor to the compound. 1 and conn. 2 (see tables 3a, 3b).

When all of the above exchanges were introduced at four positions 264, 268, 270, 330 on top of the mutant GPPD polypeptide corresponding to SEQ ID NO: 10, resulting in SEQ ID NO: 19, the strongest stability of all polypeptides depicted in the table was found. 3 a with residual activity and turnover of 68% and 79% to the connection. 1, demonstrating the importance of combinatorial residue exchanges at exposed amino acid positions. Also, the mutant GPPD polypeptide corresponding to SEQ ID NO: 19 has improved ki values for the compound. 1 and conn. 2 (Table 4) compared to SEQ ID NO: 2.

Table 4

Assessment of the resistance of mutated GPPD polypeptides to different GPPD-inhibiting herbicides belonging to different chemical classes by determining ki values

The position of amino acids relative to the GPPD polypeptide SEQ Ш NO: 1 SEQ ID NO 213 264 268 With) 315 330 331 335 336 337 338 O 5 Conn. 1 ki [µM] Conn. 2 ki [µM] MST ki [µM] 1 R M R T T D D E G N F To A S I 2 R W A Q 1 3 22 8 R D S V 4.8 90 72 17 G E R D S V V 19 190 130 31 G E R N S V V 25 120 75 32 R E R N S V V 41 110 120 33 G E R D S R V 17 100 85 34 G E N R D S R To 21 89 120 35 G E R D S R To 88 160 150 16 S S R D S V To 9.5 180 150 18 R N R D S V 19 190 34 19 R G E N R D S V V 33 180 35 36 R G E N R D S V m 39 120 39 37 G R N R D S V To 12 76 81 38 G E V R D S V V 74 170 490 45 G E R R D S V V 9.7 110 120 48 G E N R D S V V 39 160 210 50 G E R R D S V V 47 130 140 60 R G E n R D S V Q V 24 130 32 96 TO G E R N S V V 19 98 93 97 G R n S R D S V To 17 86 130 98 G E n S R D S R To 33 40 150 99 G E R D S V R V 100 160 150 100 L G E n R D S V Q m 100 120 120 101 L G E R R D S V Q m 45 96 89 102 L R E R R N S V Q m 40 230 290

- 44 045758

103 TO L G E R R n S V Q m 24 260 280 104 TO L G E R n S V Q m 43 370 270 105 TO L G E R n S R Q m 140 270 100 106 L G E n R R D S V Q m 95 240 120 107 TO G E R n S V R V 110 190 100 108 G E M n R D S V m 33 150 98

For clarity, empty cells at the corresponding amino acid position in SEQ ID NO: 2 through SEQ ID NO: 108 are defined as identical to the amino acids corresponding to SEQ ID NO: 1, highlighting only the exchanges in the mutant GPPD polypeptides. The mutant GPPD polypeptides provided in the present invention are examples by way of illustration and not by way of limitation.

The data were obtained by measuring the initial reaction rates with increasing concentrations of the compound. 1 (2-chloro-3-(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide), comp. 2 (2-methyl-H-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide), and mesotrione (MST) according to Example 3 Typically, six different concentrations of GFP substrate (0 - 1350 μM) and four different concentrations of the corresponding inhibitor were used (see Fig. 4). Inhibitor concentrations were assumed for the corresponding HPPD/inhibitor polypeptide pairs to generate dynamic data, i.e. variants with lower resistance were analyzed at lower inhibitor concentrations, and concentrations up to 1200 μM were used for variants with maximum resistance. GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla California USA) was used to analyze the data and fit kinetic constants applying constraints according to the competitive inhibition mode. Where obvious outliers occurred, or activities obtained at very high substrate concentrations were not consistent with the mathematical methods underlying the competitive inhibition mode, the corresponding values were excluded from the fit.

Some results of systematic variants created on the basis of two already significantly improved mutated GPPD polypeptides SEQ ID NO: 17 and SEQ ID NO: 19 are shown in table. 5.

SEQ ID NO: 17 and SEQ ID NO: 19 differ at two amino acid positions, and SEQ ID NO: 17 shows a ~20-fold, SEQ ID NO: 19 -30-fold increase in ki relative to the comp. 1 compared to the mutant GPPD polypeptide corresponding to SEQ ID NO: 2 (see Table 4).

The mutant GPPD polypeptide corresponding to SEQ ID NO: 17 has exchanges of residues at positions 268, 270, 335, 336, 337, 340 and 345 and exhibits 29% residual activity, and substrate turnover is reduced by only 46% in the presence of 50 μM comp. 1. Reversion of residues at positions 268, 270, 340, 345 to the corresponding wild-type residue (according to SEQ ID NO: 1) is accompanied by a drop in stability to 19, 24, 17 and 20% of the residual activity, respectively (Table 5; SEQ ID NO : 71, SEQ ID NO: 63, SEQ ID NO: 81, SEQ ID NO: 88, respectively), emphasizing the beneficial properties of these positions and mutations.

Accordingly, reversion of the Valine residue at position 345 in SEQ ID: 19 to the corresponding wild-type isoleucine residue is accompanied by a drop in residual activity from 67 to 57% (Table 5; SEQ ID NO: 93).

On the other hand, the introduction of additional single residual exchanges in SEQ ID: 17 at position 204, 213, 264, 310, 315, 330, 331, 338, 339 or 344 resulted for each position in at least one variant with a further significantly increased residual activity more than 38%, for example, A204M (SEQ ID NO: 40), R213L (SEQ ID NO: 44), M264K (SEQ ID NO: 67), Q310K (SEQ ID NO: 65), T315R (SEQ ID NO: 45), D330V (SEQ ID NO: 38), D331I (SEQ ID NO: 49), F338V (SEQ ID NO: 53), K339E (SEQ ID NO: 55) and S344P (SEQ ID NO: 58).

As a result, specific additional residue exchanges outside the mutations 335 (glutamic acid (E) => proline (P)), 336 (glycine (G) => aspartic acid (D) / histidine (H)) and 337 (asparagine (N) => serine (S)) in HPPD polypeptides provide improved herbicide resistance and demonstrate the additional importance of the disclosed positions 204, 213, 264, 268, 270, 310, 315, 330, 331, 338, 339, 340, 344, 345, imparting improved resistance to herbicides that inhibit GPPD (Table 5). Finally, the combination of these disclosed provisions results in improvements in ki across various classes of GPPD inhibitors, as shown in Table 1. 4 (for example, SEQ ID NO: 99, 100, 105, and 107) to conn. 1 with more than 100 times, to conn. 2 with up to 90-fold and at the same time up to -7-fold improvements against mesotrione (MST) compared to the wild type GPPD polypeptide (SEQ ID NO: 1) and the mutant GPPD polypeptide from the prior art (WO 2014/043435 ), corresponding to SEQ ID NO: 2 in the present invention.

-45 045758

Table 5

The effect of single residue exchanges in SEQ ID NO: 17 and SEQ ID NO: 19, analyzed in accordance with example 3 at low substrate concentration and 50 μM inhibitor compound 1

The position of amino acids relative to the GPPD polypeptide SEQIDNO:! SEQ Ш NO 204 213 264 268 270 310 315 330 1—Н 335 336 337 338 339 O 344 345 Residual activity Residual turnover 17 G E R D S V V 29% 54% 31 G E R n S V V 50% 81% 39 L G E R D S V V 34% 61% 40 M G E R D S V V 65% 77% 41 S G E R D S V V 54% 77% 42 T G E R D S V V 59% 87% 43 TO G E R D S V V 28% 61% 44 L G E R D S V V 58% 78% 67 TO G E R D S V V 73% 96% 68 L G E R D S V V 42% 70% 69 Q G E R D S V V 60% 90% 70 R G E R D S V V 55% 74% 71 E R D S V V 19% 41% 72 R E R D S V V 25% 55% 73 S E R D S V V 29% 58% 63 G R D S V V 24% 54% 74 G L R D S V V 26% 59% 75 G R R D S V V 36% 67% 76 G R R D S V V 19% 50% 77 G S R D S V V 19% 44% 64 G E n R D S V V 60% 64% 65 G E To R D S V V 90% 96% 66 G E S R D S V V 87% 91%

- 46 045758

45 G E R R D S V V 39% 68% 46 G E M R D S V V 32% 59% 47 G E n R D S V V 31% 59% 78 G E A R D S V V 33% 63% 79 G E F R D S V V 39% 66% 80 G E G R D S V V 49% 74% 38 G E V R D S V V 78% 94% 48 G E n R D S V V 47% 74% 49 G E I R D S V V 58% 78% 50 G E R R D S V V 46% 80% 51 G E L R D S V V 27% 56% 52 G E S R D S V V 31% 57% 53 G E R D S V V V 50% 63% 54 G E R D S A V V 33% 55% 55 G E R D S E V V 46% 81% 56 G E R D S R V V 27% 59% 57 G E R D S T V V 33% 68% 81 G E R D S V 17% 51% 82 G E R D S E V 37% 74% 83 G E R D S G V 53% 87% 84 G E R D S L V 17% 43% 85 G E R D S m V 17% 25% 86 G E R D S Q V 23% 45% 33 G E R D S R V 40% 65% 58 G E R D S V R V 49% 59% 59 G E R D S V R V 19% 46% 87 G E R D S V A 17% 53% 88 G E R D S V 20% 44% 89 G E R D S V To 23% 43% 90 G E R D S V m 16% 43% 91 G E R D S V R 25% 52% 19 R G E n R D S V V 67% 88% 60 R G E n R D S V Q V 64% 84% 61 R G E n R D S V R V 61% 88% 62 R G E n R D S V R V 52% 72% 92 R G E n R D S V A 61% 81% 93 R G E n R D S V 57% 75% 94 R G E n R D S V To 20% 62% 36 R G E n R D S V m 67% 85% 95 R G E n R D S V R 40% 81%

For clarity, empty cells at the corresponding amino acid positions in SEQ ID NO: 2 - NO: 108 are defined as identical to the amino acids corresponding to SEQ ID NO: 1, highlighting only the exchanges in the mutant GPPD polypeptide. Residual activity and residual turnover were determined in accordance with example 3 by measuring kapp and total signal change, respectively, in the presence and absence of the compound. 1 (2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide) (50 µM) at low substrate concentration. For each mutant GPPD polypeptide, k _app and total signal change without GPPD inhibitory herbicides were used to normalize k _app and total signal change in the presence of the herbicide, and the resulting % values were summed.

Example 5 Brown Assay for Testing Mutant GPPD Polypeptides Resistant to GPPD Inhibiting Herbicides.

Mutant GPPD polypeptides were analyzed using the brown color assay (WO 2014/043435). Bacterial cells expressing the mutant GPPD polypeptide according to the invention were assayed in a 96-well format for resistance to the GPPD inhibitor by spot coating on a solid medium containing LB agar, a selection agent for the expression vector pSE420(RI)NX (WO 2014/043435 ), 5 mM tyrosine (Sigma-Aldrich, St. Louis, USA), 42 mM succinate (Sigma-Aldrich, St. Louis, USA) and six different concentrations of GPPD inhibitory herbicide, comp. 1 (0 -500 µM).

In the brown color assay, a culture of E. Coli cells expressing overnight one of the corresponding mutant GPPD polypeptides, diluted to OD600 in 1 and 10 μl of extract, was spotted in triplicate on plates containing 0, 25, 50, 100, 250 or 500 µM comp. 1.

- 47 045758

The plates were covered with breathable tape and incubated at 30 degrees C. After 24 hours, the cells were kept in the dark at room temperature and after 7 days the formation of brown pigment was visually assessed. In the presence of a GPPD inhibitory herbicide, this pigment formation is inhibited and the color of the agar plate will not change if the GPPD polypeptide of the stable GPPD inhibitory herbicide is expressed and active. A rating of +++ indicates a dark brown color, as seen for E. coli cells expressing one of the corresponding mutant GPPD polypeptides without inhibitor on an LB agar plate. ++ and + evaluate medium and light brown pigmentation, respectively, and O indicates that no brown pigmentation was detected on the LB agar plates.

Table 6

Assessment of the resistance of mutant GPPD polypeptides to compounds. 1 (0-500 µM) using brown color assay

Concentration Comp. 1 [µM] E. Coli cells expressing the GPPD polypeptide SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 96 SEQ ID NO: 103 SEQ ID NO: 104 0 + + + + + + + + + + + + + + + + + + + + + 25 0 + + + + + + + + + + + + + + + + 50 0 + + + + + + + + + + + + + + + 100 0 + + + + + + + + + + + + + + + 250 0 + + + + + + + + + + 500 0 0 + + + 0 + +

Exemplary mutant GPPD polypeptides presented in table. 6, showed improved resistance to compounds. 1(2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4(trifluoromethyl)benzamide) as compared to the HPPD polypeptide corresponding to SEQ ID NO: 1 in the present invention. Already at a concentration of 25 µM comp. 1, E. coli cells expressing the GPPD polypeptide (corresponding to SEQ ID NO: 1) do not produce brown pigmentation and some mutant GPPD polypeptides exhibit dark to moderate brown pigmentation.

The prior art mutant GPPD polypeptide (WO 2014/043435) corresponding to SEQ ID NO: 2 in the present invention developed only slight brown pigmentation at 250 μM comp. 1. and lost its brown pigmentation at 500 µM compound 1, showing complete inhibition of expressed GPPD polypeptides in the cell. In comparison, some of the mutant GPPD polypeptides shown in Table 6 with SEQ ID NO: 31, 32, 96 and especially with SEQ ID NO: 104 show a stronger brown color in the presence of 25, 50, 100, 250 and even 500 μM comp. 1.

In an additional experiment, mutant GPPD polypeptides were analyzed using the brown colorimetric assay principle (as, for example, described in US 6,768,044). Bacterial cells expressing GPPD polypeptides according to the present invention were assayed in a 96-well plate format (Nunc® 96 DeepWell™ plate, Sigma-Aldrich, St. Louis, USA) for GPPD polypeptides with improved resistance to GPPD inhibitory herbicides.

E. coli cells were grown in liquid LB medium (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) containing the selection agent for the expression vector pSE420(RI)NX (WO 2014/043435) and 5 mM parahydroxyphenylpyruvate (HPP; Sigma-Aldrich , St. Louis, USA), in the presence or absence of a herbicide that inhibits GPPD, for example, Comm. 1 (1000 µM).

An overnight culture of E. coli expressing one of the corresponding HPPD polypeptides according to the present invention was adjusted to an OD600 of 0.3 to 0.5 in a final volume of 500 μl and incubated at 30 degrees Celsius. Residual browning was determined by measuring browning (BCF) in the presence and absence of comp. 1 (2-chloro-3(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide).

Therefore, after 96 h, the culture was centrifuged and the culture supernatant was used to measure the optical density of the formation of soluble brown pigment at 440 nm (OD _440hm ).

- 48 045758

Table 7

Assessment of the resistance of exemplary mutant GPPD polypeptides to the compound. 1 (1000 µM) using brown color bioassay and brown color residue (BCF) detection at 96 h

The position of amino acids relative to the GPPD polypeptide SEQIDNO:! SEQ ID NO: I 204 | I 213 | I 264 | 268 270 1,310 | I 315 | 330 | zz1 | 335 336 I 337 | 338 339 I 340 | I 344 | I 345 | Reference BCF OD440 nm Residual BCF OD440 nm [%] 1 A R m R T Q T D D E G N F To A S I 2.66 15% 2 R W A Q 2.68 18% 7 R D S 1.83 40% 10 R D S V V 1.09 65% 14 N R D S V V 1.42 63% 19 R G E N R D S V V 1.65 60% 21 R N S 0.99 65% 22 R N S V 1.67 58% 24 R N S V V 1.56 48% 29 S S R N S V V 2.03 42% thirty S S R N S V To 1.70 56% 39 L G E R D S V V 1.63 55% 40 M G E R D S V V 1.89 58% 44 L G E R D S V V 1.60 40% 46 G E m R D S V V 2.47 66% 52 G E S R D S V V 2.08 65% 56 G E R D S R V V 1.98 68% 57 G E R D S T V V 1.39 46% 59 G E R D S V R V 1.29 51% 67 TO G E R D S V V 1.93 71% 72 R E R D S V V 2.77 72% 73 S E R D S V V 2.75 72% 76 G R R D S V V 2.73 58% 77 G S R D S V V 2.57 67% 81 G E R D S V 2.05 39% 83 G E R D S G V 1.83 47% 86 G E R D S Q V 1.94 74% 87 G E R D S V A 1.91 68% 88 G E R D S V 2.21 77% 89 G E R D S V To 2.36 67% 92 R G E N R D S V A 1.38 59% 97 G R N S R D S V To 1.54 49% 99 G E R D S V R V 1.52 87% 100 L G E N R D S V Q m 2.48 33% 102 L R E R R N S V Q m 2.47 26% 106 L G E N R R D S V Q m 1.50 73% 107 TO G E R N S V R V 1.38 84%

Exemplary mutant GPPD polypeptides given in table. 7, showed improved resistance to compounds. 1 (2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide) compared to HPPD polypeptide corresponding to SEQ ID NO: 1 and mutant polypeptide HFPD from the prior art (WO 2014/043435), corresponding to SEQ ID NO: 2 in the present invention. Both GPPD polypeptides corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 did not produce significant brown pigmentation in the presence of GPPD inhibitor. Some mutant GPPD polypeptides exhibited medium to dark brown pigmentation. The summed % values in the corresponding Table 7 are the means of at least two independent experiments with

- 49 045758 average standard deviation 5%.

Example 6. Transformation of soybeans and plant resistance of soybeans T0.

Transformation of soybeans was carried out using methods well known in the art, such as those described using Agrobacterium tumefaciens-mediated transformation of semi-seminal soybean explants, essentially applying the method described by Paz et al. (2006), Plant cell Rep. 25:206. Transformants were identified using the GPPD inhibitory herbicide tembotrione as a selection marker. The emergence of green shoots was observed and documented as an indicator of resistance to the GPPD-inhibiting herbicide tebtrotrione. Resistant transgenic shoots showed a normal green color comparable to wild-type soybean shoots untreated with the GPPD-inhibiting herbicide tembotrione, whereas wild-type soybean shoots treated with the same amount of the GPPD-inhibiting herbicide tembotrione bleached completely. This indicated that the presence of the corresponding GPPD polypeptide triggered resistance to GPPD-inhibiting herbicides such as tembotrione.

Resistant green shoots were transferred to a growing substrate or grafted. The rooted plants were transferred to the greenhouse after an acclimatization period. Then the plants containing the transgene were sprayed with herbicides that inhibit GPPD, such as mesotrione with an application rate of 300 - 600 g AB/ha, or comp. 1 (2-chloro-3-(methylsulfanyl)-N-(1-methyl-1Htetrazol-5-yl)-4-(trifluoromethyl)benzamide) with an application rate of 150 g - 300 g AB/ha supplemented with ammonium sulfate and methyl ether rapeseed oil. Symptoms associated with herbicide application were assessed 5-10 days after application and compared with symptoms observed on wild-type plants under the same conditions.

For example, T0 soybean plants having a plant expression cassette that contains a GPPD polypeptide resistant to a GPPD inhibitor according to the present invention were tested for resistance to Comm. 1.

Prior to greenhouse experiments with transgenic plants, soybean transformants were routinely analyzed for transgene expression and presence using the ELISA protein detection method (see detailed description under D and H). To analyze herbicide resistance, only plants that recovered in the selection environment and had detectable expression of the transgenic GPPD protein were used. The DeVries Tracker sprayer was calibrated before each spray. The chemical composition used for the connection. 1 was added with ammonium sulfate and methylated rapeseed oil. Spray tests were carried out at a concentration equal to 300 g AB per hectare (300 g AB/ha). Resistance was assessed 5 days after spraying. Wild-type soybean plants sprayed with the same herbicide formulation were completely bleached and exhibited more than 95% leaf damage on the top two trefoil leaves. A score of 1 was assigned to plants with minor tolerance, i.e., the top two leaves of the trefoil show significant bleaching and minor signs of recovery with 50 - 95% leaf damage. A score of 2 was assigned to plants with moderate resistance, i.e., between 10-49% of the leaf area of the upper two leaves of the trefoil, indicating a significant amount of resistance to herbicide treatment. A rating of 3 was assigned to plants with good resistance, i.e. less than 10% of the leaf area of the top three trefoil leaves showing chlorosis or only very slight bleaching. The results are shown in table. 8.

Table 8

Assessment of damage to leaf area of transgenic T0 soybeans five days after application of the compound. 1 with a rate of 300 g AB/ha

Events of soybeans expressing GPPD polypeptide Herbicide resistance ratings Total number of events processed Percentage of events rated “2 & 3” 0 1 2 3 SEQ Ш NO: 2 5 10 26 49 90 83.3% SEQ GO NO 32 1 0 10 28 39 97.4% SEQ GO NO 96 0 5 43 34 82 93.9% SEQ GO NO 31 4 1 21 thirty 56 91.1%

The results shown in table. 8 show that a significant proportion of the independent events of T0 soybeans are resistant to the herbicide that inhibits GPPD comp. 1 (2-chloro-3-(methylsulfanyl)-N-(1methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide) with a rate of 300 g AB/ha compared to control wild-type soybean plants, also treated with a herbicide that inhibits GPPD comp. 1.

More than 90% of T0 soybean events with mutant GPPD polypeptides corresponding to SEQ ID NO: 32, SEQ ID NO: 96 and SEQ ID NO: 31 have less than 50% leaf damage and thus also better than prior art GPPD polypeptide (WO 2014/043435) T0 population with a proportion of 83%, corresponding to SEQ ID NO: 2 in the present invention. In addition, ~72% of soybean events

- 50,045,758 T0 beans with a mutant GPPD polypeptide corresponding to SEQ ID NO: 32 show less than

10% leaf damage, again showing an improvement over the T0 soybean event population (54%) overexpressing the prior art GPPD polypeptide (WO 2014/043435) corresponding to SEQ ID NO: 2 in the present invention.

In additional greenhouse experiments, 21 to 94 independent events of T0 soybeans per construct containing an exemplary mutant GPPD polypeptide were sprayed with a GPPD inhibitory herbicide Comp.1 (2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H- tetrazol-5-yl)-4(trifluoromethyl)benzamide) with a rate of 300 g AB/ha, supplemented with ammonium sulfate and rapeseed oil methyl ester. Five days after application, the area of leaves damaged due to the action of the GPPD-inhibiting herbicide is scored on a scale from 0 (no damage) to 100 (complete bleaching). Under these conditions, wild-type plants were completely bleached and their damage scores ranged from 95 to 100.

In table 9 shows the distribution of GPPD-inhibiting herbicide injury score data as a percentile for exemplary GPPD-inhibiting herbicide-resistant mutant polypeptides (SEQ ID NOs). Percentiles normalize damage score categories from an individual plant in a population. The 25th percentile value represents the damage scale, where 25% of soybean events in a given population had lower and 75% higher damage rates. The median represents the 50th percentile. Half of the values had higher damage scores; half had lower damage scores. The 75th and 90th percentile values represent damage scores, where 75% and 90% of soybean events had lower damage scores, respectively. The difference between the 75th and 25th percentile is called the interquartile range and a marker for quantifying dispersion in a population. All constructs had only one cassette insertion into the soybean genome.

In table 9, structures were ranked based on damage scores according to the 75th percentile, from lowest to highest. All exemplary GPPD polypeptide variants are better in all percentile analyzes than the prior art GPPD mutant polypeptide (WO 2014/043435) corresponding to SEQ ID NO: 2 in the present invention. The evaluation of the prior art GPPD mutant polypeptide (WO 2014/043435) corresponding to SEQ ID NO: 2 in the present invention is indicated in the bottom row of the table. 9.

Table 9

Assessment of damage to leaf area of transgenic T0 soybeans five days after application of the compound. 1 with a rate of 300 g AB/ha according to percentile distribution

TO soybean events expressing polypeptide Total number of independent sprayed events 25th Median 75th Interquartile range 90th SEQ ID NO: 9 21 10.0 10.0 15.0 5.0 19.0 SEQ ID NO: 30 57 10.0 15.0 15.0 5.0 25.0 SEQ ID NO: 22 50 10.0 10.0 15.0 5.0 34.5 SEQ ID NO: 101 75 5.0 10.0 15.0 10.0 49.0 SEQ ID NO: 105 58 5.0 10.0 15.0 10.0 70.0 SEQ ID NO: 8 70 10.0 10.0 16.3 6.3 38.5 SEQ ID NO: 24 56 10.0 10.0 20.0 10.0 35.0 SEQ ID NO: 102 92 8.0 10.0 20.0 12.0 45.0 SEQ ID NO: 54 44 10.0 10.0 20.0 10.0 62.5 SEQ ID NO: 56 33 10.0 15.0 20.0 10.0 65.0 SEQ ID NO: 43 27 10.0 15.0 20.0 10.0 67.0 SEQ ID NO: 34 44 10.0 15.0 20.0 10.0 70.0 SEQ ID NO: 5 55 10.0 15.0 20.0 10.0 72.0 SEQ ID NO: 104 94 5.0 15.0 20.0 15.0 72.5 SEQ ID NO: 42 59 10.0 15.0 20.0 10.0 75.0 SEQ ID NO: 44 54 10.0 15.0 21.3 11.3 70.0 SEQ ID NO: 21 59 10.0 15.0 25.0 15.0 35.0 SEQ ID NO: 45 74 5.0 15.0 26.3 21.3 67.5 SEQ ID NO: 2 75 15.0 20.0 45.0 30.0 87.0

Example 7. Creation and selection of cotton plant T0.

Cotton transformation is achieved by using methods well known in the art, a particularly preferred method being described in PCT patent publication WO 00/71733. The regenerated plants are transferred to the greenhouse. After an acclimatization period, sufficiently grown plants are sprayed with herbicides that inhibit GPPD, such as, for example, tembotrione, equivalent to 100 or 200 g AB/ha, with the addition of ammonium sulfate and methyl ester of rapeseed oil. Seven days after spray application, symptoms associated with the GPPD-inhibiting herbicide treatment were assessed and compared with symptoms observed on wild-type cotton plants that were subjected to the same treatment under the same conditions.

Example 8. Transformation of maize plant cells through Agrobacterium-mediated transformation.

Construction of a plant expression cassette for suitable expression in a maize plant and transformation of maize are well known in the art and in this particular example the methods of WO 2014/043435 and WO 2008/100353 were described and used. Polynucleotide sequences encoding mutant GPPD polypeptides in the present invention are accumulated with a polynucleotide sequence encoding a variant EPSPS protein to confer resistance to herbicides that target EPSPS. The EPSPS gene was isolated and mutated from Arthrobacter globiformis (WO 2008/100353) and fused in frame to a transit peptide sequence to direct translocation of the translated protein into the chloroplast. Stable expression is achieved using a ubiquitin promoter (sugarcane Ubiquitin 4 promoter, US Patent 6,638,766), and a 35S termination sequence from cauliflower mosaic virus, which is cloned upstream and downstream of the EPSPS gene, respectively.

The corresponding mutant GPPD polypeptide will be cloned with the same promoter, chloroplast transit peptide, and termination sequence as described for the EPSPS gene expression cassette. The coding sequences for both genes are codon optimized for maize expression. For maize transformation, cobs are best harvested 8-12 days after pollination. Embryos are isolated from the cobs, and it is these 0.8-1.5 mm embryos that are preferred for use in transformation. Embryos were placed with the scutellum side up on a suitable incubation medium and incubated overnight at 25°C in the dark.

However, incubating embryos overnight is essentially not necessary. Embryos are contacted with an Agrobacterium strain containing appropriate vectors having the nucleotide sequence of the present invention for Ti plasmid-mediated transfer for approximately 5-10 minutes. and then overlaid on co-culture medium for approximately 3 days (at 25°C in the dark). After co-culture, the explants are transferred to recovery medium for approximately five days (at 25°C in the dark). Explants are incubated in glyphosate selective media for up to eight weeks, depending on the nature and characteristics of the specific selective media used. After a selection period, the resulting callus is transferred to embryo maturation medium until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light and the regeneration process is initiated, as is known in the art. The resulting shoots are allowed to take root in the growing substrate, and the resulting plants are transferred to seedling pots and propagated as transgenic plants. Plants were routinely analyzed for the expression and presence of transgenes using the ELISA protein detection method. To analyze herbicide resistance, only plants isolated in a selective environment and having detectable expression of the transgenic protein GPPD were used.

Example 9: T1 Soybean Plant Resistance to GPPD Inhibiting Herbicides/Field Test.

Soybean plants expressing the GPPD-inhibiting herbicide-resistant polypeptide of the present invention, alone or together with a gene conferring glyphosate resistance and/or a gene conferring resistance to glufosinate, or having a plant expression cassette that includes only the polypeptide , resistant to the GPPD inhibitory herbicide according to the present invention, was tested for resistance to various chemical classes of inhibitory herbicides. Transgenic plants were regularly analyzed for expression and presence of transgenes using the ELISA protein detection method (see details under D and H). Only plants regenerated in selective media and expressing detectable transgenic GPPD protein were regenerated, transferred to a greenhouse, and at V2-V4 growth stages used to analyze GPPD-inhibiting herbicide resistance in T0 soybean events in the greenhouse (Example 6). The chemical composition with herbicides that inhibit GPPD was supplemented with ammonium sulfate and rapeseed oil methyl ester. Herbicide resistance was assessed 5-21 days after spraying.

The most efficient independent T0 soybean events were selfing to produce T1 soybean seeds.

In field trials, improved T1 soybean seeds were planted and treated with either 210 g/ha isoxaflutole or 150 g/ha comp. 1 (2-chloro-3-(methylsulfanyl)-N-(1-methyl-1Ntetrazol-5-yl)-4-(trifluoromethyl)benzamide) at the V2-V4 growth stage (Table 10) and leaf damage was assessed after eight days after application of a herbicide that inhibits GPPD. All wild-type or segregated T1 soybean plants lacking GPPD inhibitor-resistant polypeptide were sprayed with the same herbicide formulation and were completely bleached and showed 100% leaf damage eight days after application.

In table 10 shows the event frequency of soybeans exhibiting good resistance, i.e. equal to or less than 15% damage to the total leaf area.

- 52 045758

All exemplary GPPD herbicide-inhibiting herbicide resistant polypeptide variants provided herein had a higher frequency in a population with good resistance with equal or less than 15% leaf damage and were therefore superior to the prior art GPPD mutant polypeptide (WO 2014/043435), corresponding to SEQ ID NO: 2 in the present invention.

Table 10. Field trial assessment of leaf area damage from sample transgenic events of V2-V4 growth stage T1 soybeans expressing different GPPD polypeptide variants treated with either 210 g/ha isoxaflutole (Table 10a) or 150 g/ha comp. 1 (Table 10b).

Table 10a

Field trials of exemplary transgenic events in soybeans sprayed with 210 g/g isoxaflutole and assessed eight days after application

T1 soybean events expressing GPPD polypeptide Total number of independent sprayed events Independent events with <15% leaf damage after spray Frequency in population SEQ GO NO:2 39 10 26% SEQ GO NO: 16 20 19 95% SEQ GO NO: 18 20 12 60% SEQ GO NO: 31 14 13 93% SEQ ID NO: 32 24 12 50% SEQ ID NO: 96 20 AND 55%

Table 10b

Field trials of exemplary transgenic events in T1 soybeans sprayed with 150 g/ha comp. 1 and assessed eight days after application

T1 soybean events expressing GPPD polypeptide Total number of independent sprayed events Independent events with <15% leaf damage after spray Frequency in population SEQ GO NO:2 39 16 41% SEQ GO NO: 16 20 19 95% SEQ GO NO: 18 20 12 60% SEQ GO NO: 31 14 12 86% SEQ ID NO: 32 24 10 42% SEQ ID NO: 96 20 14 70%

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention relates. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and separately identified as being incorporated by reference.

Although the present invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

Claims (31)

1. A recombinant nucleic acid molecule encoding a 4hydroxyphenylpyruvate dioxygenase (HPPD) polypeptide consisting of an amino acid sequence comprising: (a) a proline at a position corresponding to position 335 in SEQ ID NO: 1, (b) a histidine or aspartic acid at a position corresponding to 336 in SEQ ID NO: 1, and (c) a serine at a position corresponding to position 337 in SEQ ID NO: 1, wherein said GPPD polypeptide is resistant to at least one GPPD inhibitory herbicide. 2. A recombinant nucleic acid molecule according to claim 1, characterized in that said GPPD polypeptide consists of an amino acid sequence additionally containing: i) methionine, threonine, serine or leucine at a position corresponding to position 204 in SEQ ID NO: 1; and/or ii) lysine or leucine at a position corresponding to position 213 in SEQ ID NO: 1; and/or iii) arginine, lysine, glutamine or leucine at a position corresponding to position 264 in SEQ ID NO: 1; and/or iv) arginine, glycine or serine at a position corresponding to position 268 in SEQ ID NO: 1; - 53 045758 and/or v) arginine, leucine, glutamic acid, proline or serine at a position corresponding to position 270 in SEQ ID NO: 1; and/or vi) a serine, histidine or lysine at a position corresponding to position 310 in SEQ ID NO: 1; and/or vii. arginine, methionine or histidine at a position corresponding to position 315 in SEQ ID NO: 1; and/or viii) histidine, alanine, phenylalanine, valine or glycine at a position corresponding to position 330 in SEQ ID NO: 1; and/or ix) proline, histidine, serine, isoleucine or leucine at a position corresponding to position 331 in SEQ ID NO: 1; and/or x) valine at a position corresponding to position 338 in SEQ ID NO: 1; and/or xi) glutamic acid, arginine, alanine or threonine at a position corresponding to position 339 in SEQ ID NO: 1; and/or xii) arginine, glutamine, methionine, glutamic acid, glycine, leucine or valine at a position corresponding to position 340 in SEQ ID NO: 1; and/or xiii) glutamine, proline or arginine at a position corresponding to position 344 in SEQ ID NO: 1; and/or xiv) lysine, arginine, methionine, alanine or valine at a position corresponding to position 345 in SEQ ID NO: 1. 3. A recombinant nucleic acid molecule according to claim 1, characterized in that said GPPD polypeptide consists of an amino acid sequence additionally containing: i) leucine or lysine at a position corresponding to position 213 in SEQ ID NO: 1; and/or ii) arginine or leucine at a position corresponding to position 264 in SEQ ID NO: 1; and/or iii) arginine, glycine or serine at a position corresponding to position 268 in SEQ ID NO: 1; and/or iv) glutamic acid or serine at a position corresponding to position 270 in SEQ ID NO: 1; and/or v) arginine or methionine at a position corresponding to position 315 in SEQ ID NO: 1; and/or vi) a histidine at a position corresponding to position 330 in SEQ ID NO: 1; and/or vii) valine at a position corresponding to position 338 in SEQ ID NO: 1; and/or viii) arginine or valine at a position corresponding to position 340 in SEQ ID NO: 1; and/or ix) glutamine at a position corresponding to position 344 in SEQ ID NO: 1; and/or x) lysine, valine or methionine at a position corresponding to position 345 in SEQ ID NO: 1. 4. A recombinant nucleic acid molecule according to claim 1, characterized in that said GPPD polypeptide consists of an amino acid sequence additionally containing: i) lysine at a position corresponding to position 213 in SEQ ID NO: 1; and/or ii) arginine or leucine at a position corresponding to position 264 in SEQ ID NO: 1; and/or iii) glycine or arginine at a position corresponding to position 268 in SEQ ID NO: 1; and/or iv) glutamic acid at a position corresponding to position 270 in SEQ ID NO: 1; and/or v) arginine at a position corresponding to position 315 in SEQ ID NO: 1; and/or vi) a histidine at a position corresponding to position 330 in SEQ ID NO: 1; and/or vii) valine at a position corresponding to position 338 in SEQ ID NO: 1; and/or viii) arginine or valine at a position corresponding to position 340 in SEQ ID NO: 1; and/or ix) glutamine at a position corresponding to position 344 in SEQ ID NO: 1; and/or x) lysine, valine or methionine at a position corresponding to position 345 in SEQ ID NO: 1. 5. A recombinant nucleic acid molecule according to any one of claims 1 to 4, characterized in that said HPPD polypeptide contains an amino acid sequence that is at least 95% identical to the amino acid sequence presented in SEQ ID NO: 1. 6. Recombinant nucleic acid molecule according to any one of claims 1 to 5, characterized in that it is a synthetic polynucleotide for expression in a plant. 7. Recombinant nucleic acid molecule according to any one of claims 1 to 5, characterized in that it is operably linked to a promoter capable of controlling its expression in a plant cell. 8. Recombinant nucleic acid molecule according to any one of claims 1 to 5, characterized in that said herbicide is triketone, diketonitrile, isoxazole, hydroxypyrazole, N-(1,2,5oxadiazol-3-yl)benzamide, N-(1 ,3,4-oxadiazol-2-yl)benzamide, N-(tetrazol-5-yl)- or N-(triazol-5yl)arylcarboxamide, pyridazinone derivative, oxoprazine derivative, N-(triazol-2yl)arylcarboxamide, triazinone or pyrazolone. 9. The recombinant nucleic acid molecule according to claim 8, characterized in that said herbicide is a benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione, isoxaflutole, diketonitrile, pyrazoxyphene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate, 2 -methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4(trifluoromethyl)benzamide, 2-chloro-3-ethoxy-4-(methylsulfonyl)-N -(1-methyl-1H-tetrazol-5-yl)benzamide, - 54 045758 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-N-tetrazol-5-yl)benzamide, 2-chloro-3(methylsulfanyl)-N-(1-methyl-1H -metrazol-5-yl)-4-(trifluoromethyl)benzamide or 2-(methoxymethyl)-3(methylsulfinyl)-M-(1-methyl-N-tetrazol-5-yl)-4-(trifluoromethyl)benzamide. 10. A host cell containing a recombinant nucleic acid molecule according to any one of claims 1 to 4. 11. The host cell according to claim 10, which is a bacterial host cell. 12. The host cell according to claim 10, which is a plant cell. 13. A transgenic plant containing a recombinant nucleic acid molecule according to any one of claims 1 to 4. 14. The plant of claim 13, wherein said plant is maize, sorghum, wheat, sunflower, tomato, cruciferous plant, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley or oilseed rape. 15. Transgenic plant seed according to claim 13, containing a recombinant nucleic acid molecule according to any one of claims 1-4. 16. A recombinant HPPD polypeptide consisting of an amino acid sequence comprising (a) a proline at a position corresponding to position 335 in SEQ ID NO: 1, (b) a histidine or aspartic acid at a position corresponding to position 336 in SEQ ID NO: 1, and (c) a serine at a position corresponding to position 337 in SEQ ID NO: 1, wherein said GPPD polypeptide is resistant to at least one GPPD-inhibiting herbicide. 17. The recombinant polypeptide according to claim 16, characterized in that it consists of an amino acid sequence that additionally contains: i) methionine, threonine, serine or leucine at a position corresponding to position 204 in SEQ ID NO: 1; and/or ii) lysine or leucine at a position corresponding to position 213 in SEQ ID NO: 1; and/or iii) arginine, lysine, glutamine or leucine at a position corresponding to position 264 in SEQ ID NO: 1; and/or iv) arginine, glycine or serine at a position corresponding to position 268 in SEQ ID NO: 1; and/or v) arginine, leucine, glutamic acid, proline or serine at a position corresponding to position 270 in SEQ ID NO: 1; and/or vi) a serine, histidine or lysine at a position corresponding to position 310 in SEQ ID NO: 1; and/or vii) arginine, methionine or histidine at a position corresponding to position 315 in SEQ ID NO: 1; and/or viii) histidine, alanine, phenylalanine, valine or glycine at a position corresponding to position 330 in SEQ ID NO: 1; and/or ix) proline, histidine, serine, isoleucine or leucine at a position corresponding to position 331 in SEQ ID NO: 1; and/or x) valine at a position corresponding to position 338 in SEQ ID NO: 1; and/or xi) glutamic acid, arginine, alanine or threonine at a position corresponding to position 339 in SEQ ID NO: 1; and/or xii) arginine, glutamine, methionine, glutamic acid, glycine, leucine or valine at a position corresponding to position 340 in SEQ ID NO: 1; and/or xiii) glutamine, proline or arginine at a position corresponding to position 344 in SEQ ID NO: 1; and/or xiv) lysine, arginine, methionine, alanine or valine at a position corresponding to position 345 in SEQ ID NO: 1. 18. The recombinant polypeptide according to claim 16, characterized in that it consists of an amino acid sequence that additionally contains: i) leucine or lysine at a position corresponding to position 213 in SEQ ID NO: 1; and/or ii) arginine or leucine at a position corresponding to position 264 in SEQ ID NO: 1; and/or iii) arginine, glycine or serine at a position corresponding to position 268 in SEQ ID NO: 1; and/or iv) glutamic acid or serine at a position corresponding to position 270 in SEQ ID NO: 1; and/or v) arginine or methionine at a position corresponding to position 315 in SEQ ID NO: 1; and/or vi) a histidine at a position corresponding to position 330 in SEQ ID NO: 1; and/or vii) valine at a position corresponding to position 338 in SEQ ID NO: 1; and/or viii) arginine or valine at a position corresponding to position 340 in SEQ ID NO: 1; and/or ix) glutamine at a position corresponding to position 344 in SEQ ID NO: 1; and/or x) lysine, valine or methionine at a position corresponding to position 345 in SEQ ID NO: 1. 19. Recombinant polypeptide according to claim 16, characterized in that it consists of an amino acid - 55 045758 sequence, which additionally contains: i) lysine at a position corresponding to position 213 in SEQ ID NO: 1; and/or ii) arginine or leucine at a position corresponding to the position in 264 SEQ ID NO: 1; and/or iii) glycine or arginine at a position corresponding to position 268 in SEQ ID NO: 1; and/or iv) glutamic acid at a position corresponding to position 270 in SEQ ID NO: 1; and/or v) arginine at a position corresponding to the position in 315 SEQ ID NO: 1; and/or vi) a histidine at a position corresponding to position 330 in SEQ ID NO: 1; and/or vii) valine at a position corresponding to position 338 in SEQ ID NO: 1; and/or viii) arginine or valine at a position corresponding to position 340 in SEQ ID NO: 1; and/or ix) glutamine at a position corresponding to position 344 in SEQ ID NO: 1; and/or x) lysine, valine or methionine at a position corresponding to position 345 in SEQ ID NO: 1. 20. A recombinant GPPD polypeptide consisting of an amino acid sequence comprising glycine or arginine at a position corresponding to position 268 in SEQ ID NO: 1, glutamic acid at a position corresponding to position 270 in SEQ ID NO: 1, proline at a position corresponding to position 335 in SEQ ID NO: 1, a histidine or aspartic acid at a position corresponding to position 336 in SEQ ID NO: 1, a serine at a position corresponding to position 337 in SEQ ID NO: 1, a valine at a position corresponding to 340 in SEQ ID NO: 1 ; and valine at a position corresponding to position 345 in SEQ ID NO: 1, wherein said GPPD polypeptide is resistant to at least one GPPD-inhibiting herbicide. 21. A recombinant polypeptide according to any one of claims 16-20, characterized in that said GPPD polypeptide contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1. 22. Recombinant polypeptide according to any one of claims 16-20, characterized in that said herbicide is triketone, diketonitrile, isoxazole, hydroxypyrazole, N-(1,2,5-oxadiazol-3yl)benzamide, N-(1,3 ,4-oxadiazol-2-yl)benzamide, N-(tetrazol-5-yl)- or N-(triazol-5yl)arylcarboxamide, pyridazinone derivative, oxoprazine derivative, N-(triazol-2yl)arylcarboxamide, triazinone or pyrazolone. 23. The recombinant polypeptide according to claim 22, characterized in that said herbicide is a benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione, isoxaflutole, diketonitrile pyrazoxifene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate, 2-methyl- N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide, 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-( 1-methyl-1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2methoxy-3-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide, 2-chloro- 3 -(methylsulfanyl)-N-(1 methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide or 2-(methoxymethyl)-3-(methylsulfinyl)-N-(1methyl-1H-tetrazol-5 -yl)-4-(trifluoromethyl)benzamide. 24. A method for producing a GPPD polypeptide that is resistant to herbicides that inhibit GPPD, comprising culturing the host cell of claim 10 under conditions in which a nucleic acid molecule encoding the polypeptide of any one of claims 16 to 20 is expressed. 25. A transgenic plant having a DNA construct stably integrated into its genome containing a promoter operably linked to a recombinant nucleic acid molecule according to any one of claims 1-5. 26. The plant of claim 25, wherein said plant is maize, sorghum, wheat, sunflower, tomato, cruciferous plant, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley or oilseed rape. 27. A method of controlling weeds in a field, comprising planting a plant according to claim 25 or a seed according to claim 15 in a field and treating said field with an effective concentration of a herbicide that inhibits GPPD. 28. The method according to claim 27, characterized in that the herbicide is triketone, diketonitrile, isoxazole, hydroxypyrazole, N-(1,2,5-oxadiazol-3-yl)benzamide, N-(1,3,4-oxadiazole -2-yl)benzamide, N(tetrazol-5-yl)- or N-(triazol-5-yl)arylcarboxamide, pyridazinone derivative, oxoprazine derivative, N-(triazol-2-yl)arylcarboxamide, triazinone or pyrazolone. 29. The method according to claim 27, characterized in that the herbicide is benzobicyclone, sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone, fenquinotrione, isoxaflutole, diketonitrile pyrazoxifene, benzophenap, pyrazolinate, pyrasulfotol, topramesone, tolpyralate, 2-methyl-N-( 5-methyl1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide, 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H -tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3(methylsulfonyl)-N-(1 -methyl-1 H-tetrazol-5-yl)benzamide, 2-chloro-3-(methylsulfanyl) )-N-(1 -methyl-1Нtetrazol-5-yl)-4-(trifluoromethyl)benzamide or 2-(methoxymethyl)-3-(methylsulfinyl)-N-(1-methyl-1Нtetrazol-5-yl)-4 -(trifluoromethyl)benzamide. 30. Use of a nucleic acid according to any one of claims 1 to 4 to impart resistance to at least one herbicide that inhibits GPPD to a plant. 31. A commercial product containing a nucleic acid molecule according to any one of claims 1-4 or re- - 56 045758 combinant polypeptide according to any one of claims 16 to 20, characterized in that said product is whole or processed seeds or grains, animal feed, corn or soy flour, corn starch, soy flakes, soy protein concentrate, soy isolates protein, cosmetics, hair care products, soy nut butter, natto, tempeh, hydrolyzed soy protein, whipping cream, shortening, lecithin, edible whole soybeans, soy yogurt, soy cheese, tofu, yubu, and cooked, ground , steamed, baked or steamed grain.