(en)The present invention relates to a new method for improving glyphosate resistance of a plant. The method encompasses providing one or more specific mutations in a specific nucleotide sequence in said plant. In comparison to a plant not manipulated according to the method, the plant obtained by the method displays (improved) glyphosate resistance. Also provided are a (transgenic) plant, including a seed thereof, and a plant product that can be obtained by the method according to the invention.

1.ApplicationNumber: US-201214114599-A
1.PublishNumber: US-2014206850-A1
2.Date Publish: 20140724
3.Inventor: BUNDOCK PAUL

4.Inventor Harmonized: BUNDOCK PAUL(NL)

5.Country: US
6.Claims:
(en)The present invention relates to a new method for improving glyphosate resistance of a plant. The method encompasses providing one or more specific mutations in a specific nucleotide sequence in said plant. In comparison to a plant not manipulated according to the method, the plant obtained by the method displays (improved) glyphosate resistance. Also provided are a (transgenic) plant, including a seed thereof, and a plant product that can be obtained by the method according to the invention.

7.Description:
(en)FIELD OF THE INVENTION
The present invention relates to a new method for providing a glyphosate resistant plant and/or enhancing glyphosate resistance of a plant. The method encompasses providing to a plant a nucleotide sequence that encodes an EPSPS enzyme comprising (a) specific mutation(s). In comparison to an unmodified plant, the plant obtained by the method displays (improved) glyphosate resistance. Also provided are a (transgenic) plant including a seed thereof and plant products that can be obtained by the method according to the invention.
DESCRIPTION OF THE BACKGROUND ART
Glyphosate (N-phosphonomethyl-glycine) is the active compound in Round Up, the most commonly used herbicide. It is used in agriculture, horticulture, and silviculture. Typically it is sprayed and absorbed through the leaves.
Glyphosate acts broadly against plant species and works via inhibition of the enzyme 5-eno/pyruvylshikimate-3-phosphate synthase (EPSPS) which is a key enzyme in the shikimate pathway, essential for the production of aromatic amino acids. Glyphosate has a chemical structure similar to phosphoenolpyruvate (PEP), the natural EPSPS enzyme substrate, and thus competes with PEP for the enzyme active site. Inhibition of EPSPS disrupts amino acid synthesis and thereby kills the affected plant cells. Glyphosate is non-selective and it kills both weeds and crop plants.
The popularity of glyphosate as a herbicide is partly due to its low toxicity to animals. The shikimate pathway is only found in plants and bacteria. Monsanto originally patented glyphosate in the 1970s.
The observation that certain bacterial types were able to survive in glyphosate led to the discovery that some bacterial EPSPS enzymes are insensitive to glyphosate. This led to the development of Monsanto's transgenic Round Up Ready crops which express these glyphosate resistant bacterial EPSPS enzymes.
Current Roundup Ready crops include soybean, maize (corn), sorghum, canola, alfalfa, cotton and sugar beet. These crops greatly improved farmers' ability to control weeds, since glyphosate can be sprayed on fields without severely affecting the crops. As of 2005, 87% of USA soybean fields were planted with glyphosate-resistant crops (National Agriculture Statistics Service (2005) in Acreage eds. Johanns, M. & Wiyatt, S. D. 6 30, (U.S. Dept. of Agriculture, Washington, D.C.)).
It is however found that Roundup Ready soybean crops, compared with the top conventional varieties, have a 6.7% lower yield (Charles Benbrook. Evidence of the Magnitude and Consequences of the Roundup Ready Soybean Yield Drag from University-Based Varietal Trials in 1998. Ag BioTech InfoNet Technical Paper Number 1). Conferring glyphosate resistance to a plant may involve significant fitness cost of said plant. Such fitness cost may, next to a decreased crop yield, also be expressed by decreased biomass accumulation over time.
An important mutation that has been found to give plants glyphosate resistance under field conditions is a single nucleotide change, altering the proline at position 106 in the EPSPS enzyme into a leucine (P106L). Many weed species around the world have independently developed this mutation but thus far other spontaneous mutations in EPSPS have not been found (Baerson et al. Plant Physiol, July 2002, Vol. 129, pp. 1265-1275 2002). Gassert et al. (J. Biol. Chem Vol 263(9) pp 4280-4289) describe structure, expression, and evolution of the 5-Enolpyruvylshikimate-3-phosphate Synthase Genes of Petunia and Tomato.
There is a need to identify further mutations that are useful in providing EPSPS enzymes that provide (improved) resistance to glyphosate, and plants carrying such mutation(s) and/or expressing such enzymes, that are thereby able to grow in the presence of (increased levels of) glyphosate. Such plants may be able to grow in the presence of higher concentrations of glyphosate in comparison with prior art plants, including glyphosate resistant plants, or show enhanced growth in the presence of similar concentrations of glyphosate in comparison with prior art plants, including glyphosate resistant plants.
Furthermore, there is also a need to identify mutations that further enhance glyphosate resistance of prior art glyphosate resistant plants or mutations that preferably reduce the fitness cost associated with the glyphosate resistance of said prior art glyphosate resistant plants.
It is an object of the present invention to provide for at least one of the above-mentioned needs.
DESCRIPTION OF THE INVENTION
Definitions
In the following description and examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The disclosures of all publications, patent applications, patents and other references are incorporated herein in their entirety by reference.
As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. For example, a method for isolating “a” DNA molecule, as used above, includes isolating a plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).
Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.
“Cultivated plant” and “wild plant” refers, respectively, to plants which have been bred for good agronomic characteristics by humans and plants which are found in nature in the wild. Genes and alleles found in the latter are also referred to as “wild type”. However, with respect to the EPSPS genes, genes present both in “cultivated plant” and “wild type plants” are referred to as “wild-type genes”. Expression of such wild-type genes in the plant to levels normally found in these plants does not provide for “glyphosate-resistant” EPSPS, but for “glyphosate-sensitive” EPSPS (see below).
A “nucleic acid construct” or “vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell. The vector backbone may for example be a binary or superbinary vector (see e.g. U.S. Pat. No. 5,591,616, US 2002138879 and WO95/06722), a co-integrate vector or a T-DNA vector, as known in the art and as described elsewhere herein, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence is already present, only a desired nucleic acid sequence (e.g. a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream of the transcription regulatory sequence. Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like.
As used herein, a “glyphosate-resistant” EPSPS refers to an EPSPS, the expression of which in a plant cell confers glyphosate resistance upon the plant cell. An EPSPS is “glyphosate-sensitive” if it does not confer glyphosate-resistance when being expressed in plant cells at levels normally found in such plants, i.e. when not brought to over-expression.
As used herein, a “glyphosate-resistant” cell or plant refers to a cell or plant that can survive or continue to grow in the presence of certain concentrations of glyphosate that typically kill or inhibit the growth of other cells or plants. Growth includes, for instance, photosynthesis, increase of rooting, increase of height, increase of mass, or development of new leaves.
For example, an EPSPS is glyphosate resistant when it confers glyphosate resistance to a plant. E.g. at the same level of expression of EPSPS, a plant carrying the glyphosate resistant EPSPS continues to survive or grow at levels of glyphosate at least 5%, 10%, 20%, 30%, or 40% higher than the level at which a plant not carrying the glyphosate resistant EPSPS stops growth or dies.
In one embodiment, a glyphosate-resistant (plant) cell can grow and divide on a culture medium containing 50 μM (or 50 mg/l) or more glyphosate, preferably a glyphosate-resistant cell can grow and divide on a culture medium containing 100 μM (or 100 mg/l) or more glyphosate, such as 200 μM (or 200 mg/l), 300 μM (or 300 mg/l) or 400 μM (or 400 mg/l) glyphosate. Even more preferably a glyphosate-resistant cell can grow and divide on a culture medium containing 500 μM (or 500 mg/l) or more glyphosate, such as 600 μM (or 600 mg/l). For purposes of the present invention, the term “glyphosate” includes any herbicidal effective form of N-phosphonomethylglycine (including any salt thereof) and other forms which result in the production of the glyphosate anion in plants.
Glyphosate resistance”, as defined in the current application, is in the field also commonly referred to as glyphosate tolerance.
“Functional”, in relation to proteins (or variants, such as orthologs or mutants, and fragments), refers to the capability of the gene and/or encoded protein to modify the (quantitative and/or qualitative) characteristic by modifying the expression level of the gene (e.g. by overexpression or silencing) in a plant. For example, the functionality of a putative protein obtained from plant species X can be tested by various methods. Preferably, if the protein is functional, silencing of the gene encoding the protein in plant species X, using e.g. gene silencing vectors, will lead to a reduction or suppression of the characteristic while overexpression in a susceptible plant will lead to enhanced resistance. Also, complementation with a functional protein will be capable of restoring or conferring the characteristic. The skilled person will have no difficulties in testing functionality.
The term “gene” means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5′ leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3′non-translated sequence comprising e.g. transcription termination sites. “Expression of a gene” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment) or which is active itself (e.g. in posttranscriptional gene silencing or RNAi). An active protein in certain embodiments refers to a protein being constitutively active. The coding sequence is preferably in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment. In gene silencing approaches, the DNA sequence is preferably present in the form of an antisense DNA or an inverted repeat DNA, comprising a short sequence of the target gene in antisense or in sense and antisense orientation. “Ectopic expression” refers to expression in a tissue in which the gene is normally not expressed.
A nucleic acid as disclosed herein may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry , at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes). The present disclosure contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. The term “isolated” thus means isolated from naturally occurring sources or artificially or synthetically produced. A nucleotide sequence can for example be isolated by cloning it into a host organism such as a BAC clone. Typically, when a nucleotide sequence is isolated, it comprises at most 500, preferably at most 250, more preferably at most 100, more preferably at most 50 or most preferably at most 20 contiguous nucleotides of the nucleotide sequence(s) that naturally directly flanks the nucleotide sequence which is now isolated. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
Plants according to the invention include Cucurbitaceae, Gramineae, Solanaceae or Asteraceae (Compositae), maize/corn ( Zea species), wheat ( Triticum species), barley (e.g. Hordeum vulgare ), oat (e.g. Avena sativa ), sorghum ( Sorghum bicolor ), rye ( Secale cereale ), soybean ( Glycine spp, e.g. G. max ), cotton ( Gossypium species, e.g. G. hirsutum, G. barbadense ), Brassica spp. (e.g. B. napus, B. juncea, B. oleracea, B. rapa , etc), sunflower ( Helianthus annus ), safflower, yam, cassava, alfalfa ( Medicago sativa ), rice ( Oryza species, e.g. O. sativa indica cultivar-group or japonica cultivar-group), forage grasses, pearl millet ( Pennisetum spp. e.g. P. glaucum ), tree species ( Pinus , poplar, fir, plantain, etc), tea, coffea , oil palm, coconut, vegetable species, such as pea, zucchini, beans (e.g. Phaseolus species), hot pepper, cucumber, artichoke, asparagus, eggplant, broccoli, garlic, leek, lettuce, onion, radish, turnip, tomato, potato, Brussels sprouts, carrot, cauliflower, chicory, celery, spinach, endive, fennel, beet, fleshy fruit bearing plants (grapes, peaches, plums, strawberry, mango, apple, plum, cherry, apricot, banana, blackberry, blueberry, citrus, kiwi, figs, lemon, lime, nectarines, raspberry, watermelon, orange, grapefruit, etc.), ornamental species (e.g. Rose, Petunia, Chrysanthemum , Lily, Gerbera species), herbs (mint, parsley, basil, thyme, etc.), woody trees (e.g. species of Populus, Salix, Quercus, Eucalyptus ), fibre species e.g. flax ( Linum usitatissimum ) and hemp ( Cannabis sativa ), and others.
A “recombinant plant” or “recombinant plant part” or “transgenic plant” is a plant or plant part (seed or fruit or leaves, for example) which comprises the chimeric gene in all cells and plant parts, at the same locus, even though the gene may not be expressed in all cells.
The term “sequencing” refers to determining the order of nucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA. Many techniques are available such as Sanger sequencing and Next-Generation sequencing technologies such as offered by 454 or Solexa technologies.
SEQ ID NO:1-8 are based on the relevant regions of EPSPS as shown in FIG. 1 : Maize (SEQ ID NO:1), Rice (SEQ ID NO:2), Wheat (SEQ ID NO:3), Tomato (SEQ ID NO:4), Arabidopsis (SEQ ID NO:5), Onion (SEQ ID NO:6), Salmonella (SEQ ID NO:7), and E. coli (SEQ ID NO:8). With respect to any unintended difference between the sequences of any of SEQ ID NO:1-8 and the corresponding sequence as shown in FIG. 1 , it is hereby noted that the sequences as shown in FIG. 1 are leading and should be considered as the basis for correction of any such unintended difference.
For amino acids the following common abbreviations may be used throughout the text:
Ala A Alanine
Arg R Arginine
Asn N Asparagine
Asp D Aspartic acid (Aspartate)
Cys C Cysteine
Gln Q Glutamine
Glu E Glutamic acid (Glutamate)
Gly G Glycine
His H Histidine
Ile I Isoleucine
Leu L Leucine
Lys K Lysine
Met M Methionine
Phe F Phenylalanine
Pro P Proline
Ser S Serine
Thr T Threonine
Trp W Tryptophan
Tyr Y Tyrosine
Val V Valine
Asx B Aspartic acid or Asparagine

Glx Z Glutamine or Glutamic acid

Xaa X Any amino acid (sometime—is used to refer to any amino acid).

DETAILED DESCRIPTION OF THE INVENTION
The invention relates to a newly identified mutation in an EPSPS coding sequence that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 . In a preferred embodiment this change is in combination with at least one further mutation in said EPSPS coding sequence, preferably with a further mutation that corresponds to a change of an amino acid at a position chosen from the group consisting of 101, 106, and 179, as shown in FIG. 1 . FIG. 1 shows the mature protein lacking the chloroplast targeting signal while the other figures show the complete protein, including such target signal, as will be clearly understood by the person skilled in the art. A skilled person will therefore have no problem determining corresponding positions and mutations according to the invention in either nucleotide sequences coding for EPSPS enzymes or in amino acid sequences of EPSPS enzymes, for example by aligning sequences of various EPSPS genes, in accordance with the data of FIG. 1 .
During experimentation with various plant materials including protoplasts, including tomato protoplasts, plant material, e.g. callus, was obtained that was capable of growing on medium containing glyphosate and the plant material, e.g. the callus, appeared resistant against this herbicide while maintaining growth.
The EPSPS genes from these different glyphosate-resistant materials were sequenced using methods available to the skilled person. A new and unexpected mutation was indentified in the coding region for the EPSPS, which mutation in the genetic materials leads to the change of an amino acid in the encoded EPSPS enzyme at position 44 as indicated in FIG. 1 . It was found that a plant (cell) carrying this mutation shows (enhanced) glyphosate resistance. In fact, and even more surprisingly, it was found that already existing glyphosate resistance of a plant (cell) may be further enhanced by introducing the above-identified mutation at position 44 in the EPSPS coding sequence present in said plant. For example, and in particular, the glyphosate resistance of a plant expressing an EPSPS coding sequence that confers glyphosate resistance by having a mutation leading to change of an amino acid in the original EPSPS enzyme at a position chosen from the group consisting of 101, 106, and 179, as shown in FIG. 1 , may be further enhanced by introduction of a mutation leading to a change of an amino acid at position 44, as shown in FIG. 1 . In other words, the newly indentified mutation may advantageously be used to enhance the glyphosate resistance conferring capabilities of other and known mutations in EPSPS enzymes.
Certain aspects of the present invention thus relate to four different mutations in said EPSPS coding sequence that corresponds to a change of amino acids at specific positions. In general these mutations can be referred to in the remainder of the text as mutations A, B, C, and D:
Mutation A
The first mutation in the EPSPS coding sequence may be referred to as Mutation A and leads to a change of the amino acid at position 44 in the EPSPS enzyme, as shown in FIG. 1 , e.g. leading to glyphosate resistance, in comparison to the same EPSPS coding sequence not having said mutation. For example, and preferably, the amino acid at position 44 that is changed due to a mutation in the EPSPS-coding sequence is an Asparagine (N), as shown in the amino acid sequences of the plants shown in FIG. 1 (in other words the original amino acid at position 44 is to be changed according to the invention and the original amino acid may be an Asparagine). Thus, preferably this mutation results in an amino acid at position 44 in the EPSPS-coding sequence which is not Asparagine (N).
Although the amino acid on position 44, preferably an Asparagine (N), may, according to the invention, be altered into any other amino acid, as long as it is useful in providing glyphosate resistance, preferably the EPSPS-coding sequence comprises a mutation that leads to a change of the amino acid at position 44, preferably Asparagine, into an Aspartic acid (D), again as shown in FIG. 1 .
Thus, in a preferred embodiment, the EPSPS encoding sequence encodes an EPSPS enzyme wherein the amino acid at position 44 is an Aspartic acid (D), independently of the amino acid that was present at said position before.
Mutation B
The second mutation (reference may be made to this mutation as Mutation B) leads to the change of the amino acid at position 101 in the EPSPS enzyme, as shown in FIG. 1 , e.g. leading to glyphosate resistance, in comparison to the same EPSPS coding sequence not having said mutation. For example, and preferably, the amino acid at position 101 that is changed due to a mutation in the EPSPS-coding sequence is a Glycine (G), as shown in the amino acids sequences for the plants shown in FIG. 1 (in other words the original amino acid at position 101 may be altered according to the invention, and the original amino acid may be a Glycine). Thus, preferably this mutation results in an amino acid at position 101 in the EPSPS-coding sequence which is not Glycine (G). In the E. coli EPSPS amino acid sequence of FIG. 17 , Mutation B corresponds to a change of the amino acid at position 96. Although the amino acid on position 101, preferably a Glycine (G), may be changed into any other amino acid, as long as it is useful in providing glyphosate resistance, preferably the EPSPS-coding sequence comprises a mutation that leads to a change of the amino acid at position 101, preferably Glycine, into an Alanine (A), again as shown in FIG. 1 .
Thus, in a preferred embodiment, the EPSPS encoding sequence encodes an EPSPS enzyme wherein the amino acid at position 101 is a Alanine (A), independently of the amino acid that was present at said position before.
The structure of the G101A mutation revealed that an alanine at position 101 sterically hinders the binding of glyphosate to the PEP binding site, which is accompanied by a corresponding increase in the Km of PEP (see Eschenburg, S., Healy, M. L., Priestman, M. A., Lushington, G. H. and Schonbrunn, E. (2002) How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli . Planta 216, 129-135).
Mutation C
The third mutation (Reference may be made to this mutation as Mutation C) leads to the change of the amino acid at position 106 in the EPSPS enzyme, as shown in FIG. 1 , e.g. leading to glyphosate resistance, in comparison to the same EPSPS coding sequence not having said mutation. For example, and preferably, the amino acid at position 106 that is changed due to a mutation in the EPSPS-coding sequence is a Proline (P), as shown in the amino acids sequences for the plants shown in FIG. 1 (in other words the original amino acid at position 106 may be a Proline, and that amino acid may be changed). Thus, preferably this mutation results in an amino acid at position 106 in the EPSPS-coding sequence which is not Proline (P).
Although the amino acid on position 106, preferably a Proline (P), may be changed into any other amino acid, as long as it is useful in providing glyphosate resistance, preferably the EPSPS-coding sequence comprises a mutation that leads to a change of the amino acid at position 106, preferably Proline, into a Leucine (L), again as shown in FIG. 1 .
Thus, in a preferred embodiment, the EPSPS encoding sequence encodes an EPSPS enzyme wherein the amino acid at position 106 is a Leucine (L), independently of the amino acid that was present at said position before.
Mutation D
The fourth mutation (which is newly identified) (Reference may be made to this mutation as Mutation D) leads to the change of the amino acid at position 179 in the EPSPS enzyme, as shown in FIG. 1 , e.g. leading to glyphosate resistance, in comparison to the same EPSPS coding sequence not having said mutation. For example, and preferably, the amino acid at position 179 that is changed due to a mutation in the EPSPS-coding sequence is a Serine (S), as shown in the amino acids sequences for the plants shown in FIG. 1 (in other words the original amino acid at position 179 may be a Serine, and that amino acid may be changed). Thus, preferably this mutation results in an amino acid at position 179 in the EPSPS-coding sequence which is not Serine (S).
Although the amino acid on position 179, preferably a Serine (S), may be changed into any other amino acid, as long as it is useful in providing glyphosate resistance, preferably the EPSPS-coding sequence comprises a mutation that leads to a change of the amino acid at position 179, preferably Serine, into an Asparagine (N), again as shown in FIG. 1 .
Thus, in a preferred embodiment, the EPSPS encoding sequence encodes an EPSPS enzyme wherein the amino acid at position 179 is an Asparagine (N), independently of the amino acid that was present at said position before.
Various (amino acid or nucleic acid encoding such amino acid) sequences of EPSPS enzymes of various organisms are shown in any of the FIGS. 1-22 ; in most of the FIGS. 4-22 the amino acid positions referred to as 44, 101, 106, and 179 and in FIG. 1 are indicated by underlining. It will be understood by the skilled person that the mutations described herein, under reference to FIG. 1 , can also be present/introduced in the corresponding positions in any EPSPS coding sequence and/or EPSPS enzyme of any organism, for example those shown in the Figures. The corresponding position in other sequences is easily established by comparison of the sequences as shown in FIG. 1 . This can be done by taking the sequence of the EPSPS protein from the relevant organism (particularly plant), performing an alignment with the A. thaliana protein and identifying the amino acid positions corresponding to N44, G101, P106 and S179. The sequences shown in FIG. 4 and further include the chloroplast targeting signal; FIG. 1 shows the mature protein lacking the chloroplast targeting signal.
In a first aspect, the invention provides for a (isolated) nucleotide sequence that encodes an EPSPS enzyme, preferably a plant EPSPS enzyme, that confers glyphosate resistance to a plant (cell), characterized in that the nucleotide sequence comprises at least one mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 . Accordingly, the amino acid at position 44 in the EPSPS-coding sequence preferably is not Asparagine (N).
Preferably, the amino acid that is changed at position 44 is Asparagine. Preferably the amino acid at position 44 is changed into a proteinogenic amino acid having a pl (isoelectric point) of 3.50 or below, being Aspartic acid or Glutamic Acid, preferably Aspartic Acid. In other words, the amino acid at position 44 in the EPSPS enzymes according to the invention (providing glyphosate resistance) is preferably an Aspartic acid or a Glutamic Acid, preferably an Aspartic Acid.
Preferably, said change is in comparison to a/the wild-type EPSPS enzyme, for example obtained from a plant in which glyphosate resistance is to be introduced and/or enhanced.
It was found that this particular amino acid change at position 44, as shown in FIG. 1 (change into a proteinogenic amino acid having a pl (isoelectric point) of 3.50 or below, being Aspartic acid or Glutamic Acid, preferably Aspartic Acid) renders advantageous results with respect to glyphosate resistance conferred by the resulting EPSPS enzyme and/or leads to an EPSPS enzyme with an improved activity towards its normal substrate, in comparison with a corresponding wild-type EPSPS, or an EPSPS enzyme wherein the amino acid at position 44 is another amino acid than Aspartic acid or Glutamic Acid.
Thus, a plant (cell) carrying said nucleotide sequence, e.g. by alteration of the original sequence present in said plant, may display (enhanced) glyphosate resistance. In the context of the present invention, glyphosate-resistance of a plant (cell) refers to the ability of said plant (cell) to survive or continue to grow in the presence of certain concentrations of glyphosate that typically kill or inhibit growth of a wild-type plant (cell). Preferably, said plant (cell) maintains normal or only slightly reduced (e.g. up to 20% less mass accumulation) growth under such circumstances.
Enhancing glyphosate resistance of a plant (cell) refers to conferring to said plant (cell) the ability to survive or continue to grow in the presence of a concentration of glyphosate that is higher than the concentration of glyphosate that would normally inhibit survival and/or growth of said plant (cell) e.g. not carrying a glyphosate resistant EPSPS. This provides for a plant (cell) with enhanced glyphosate resistance. In other words, a plant exhibiting a certain level of resistance to glyphosate, for example due to the presence of a mutation, may show resistance to a higher concentration of glyphosate by the presence of the mutation at position 44.
For example, if a reference glyphosate-resistant plant (cell) can only grow on a culture medium containing max. 50 μM (or 50 mg/l) glyphosate, a plant (cell) with enhanced glyphosate resistance may grow on a culture medium containing more than 50 μM (or 50 mg/l) glyphosate, for example 55 μM (or 55 mg/l) glyphosate or more, such as 100 μM (or 100 mg/l), 200 μM (or 200 mg/l) or 300 μM (or 300 mg/l) glyphosate. If a reference glyphosate-resistant plant (cell) can grow on a culture medium containing 100 μM (or 100 mg/l) glyphosate, a plant (cell) with enhanced glyphosate resistance can grow on a culture medium containing more than 100 μM (or 100 mg/l) glyphosate, for example 110 μM (or 110 mg/l) glyphosate or more, such as 200 μM (or 200 mg/l), 300 μM (or 300 mg/l) or 400 μM (or 400 mg/l) glyphosate. The enhanced capability of the plant (cell) to grown under conditions of increased concentration of glyphosate can be provided for by the introduction of a mutation leading to the change of an amino acid at position 44 as shown in FIG. 1 , preferably the amino acid in the improved EPSPS enzyme being an Aspartic acid.
In the context of the present invention, growth includes, for instance, photosynthesis, increased rooting, increased height, increased biomass, developments of new leaves, development of crops, and/or increased crop yield. An example is detailed below.
Enhanced glyphosate-resistance of a plant (cell) may however also refer to increased survival and/or increased growth of a plant (cell) in the presence of a specific concentration of glyphosate, in comparison to the normally displayed survival and/or growth of such plant (cell) in the presence of said concentration of glyphosate. In other words, a plant exhibiting a certain level of resistance to glyphosate, for example due to the presence of a mutation, will show increased survival or growth in the presence of a specific concentration of glyphosate by the presence of the mutation at position 44, as detailed herein.
For example, if a reference glyphosate-resistant plant(s) (cell(s)) growing on a culture medium containing 50 μM (or 50 mg/l) glyphosate show(s) a survival rate of 90% after 50 days and a growth rate of 100 g dry weight per 50 days, plant(s) (cell(s)) with enhanced glyphosate resistance growing on a culture medium containing 50 μM (or 50 mg/l) glyphosate may show increased survival and/or increased growth. Comparably, if reference glyphosate-resistant plant(s) (cell(s)) growing on a culture medium containing 100 μM (or 100 mg/l) glyphosate show(s) a specific survival rate and a specific growth rate, plant(s) (cell(s)) with enhanced glyphosate resistance growing on a culture medium containing 100 μM (or 100 mg/l) glyphosate may show increased survival and/or increased growth.
In a preferred embodiment, the nucleotide sequence that encodes an EPSPS enzyme according to the invention and as disclosed herein, is further characterized in that the nucleotide sequence comprises at least one further mutation that on its own renders said nucleotide sequence to encode an EPSPS enzyme that confers glyphosate resistance to a plant, preferably wherein said at least one further mutation corresponds to a change of an amino acid at a position chosen from the group consisting of 101, 106, or 179, as shown in FIG. 1 . Accordingly, in the EPSPS-coding sequence, the amino acid at position 101 preferably is not Glycine (G), the amino acid at position 106 preferably is not Proline (P), and/or the amino acid at position 179 preferably is not Serine (S).
For example, a nucleotide sequence that encodes an EPSPS enzyme with in its amino acid sequence a mutation (for example corresponding to position 101, 106, or 179 as shown in FIG. 1 ) that confers glyphosate resistance to said EPSPS enzyme may according to the invention be provided with at least one further mutation that corresponds to a change of an amino acid at position 44 as disclosed herein.
In a preferred embodiment, the amino acid that is changed at position 101 is Glycine.
In a preferred embodiment, the amino acid that is changed at position 106 is Proline.
In a preferred embodiment, the amino acid that is changed at position 179 is Serine.
Preferably, the amino acid at position 101 is changed into an Alanine.
Preferably, the amino acid at position 106 is changed into a Leucine.
Preferably, the amino acid at position 179 is changed into an Asparagine.
In other words, according to the invention preferably, the amino acid at position 101 is an Alanine or the amino acid at position 106 is a Proline or the amino acid at position 179 is an Asparagine. It was found that, in combination with preferably an Aspartic acid or a Glutamic Acid, preferably an Aspartic Acid, at position 44, as shown in FIG. 1 in an EPSPS enzyme, these particular amino acid (changes) render advantageous results with respect to glyphosate resistance conferred by the resulting EPSPS enzyme and/or leads to an EPSPS enzyme with an improved activity towards its normal substrate, in comparison with a corresponding wild-type EPSPS, or with an EPSPS that is changed at said positions into other than the preferred amino acids.
In the literature and in practice, different numbers are used to refer to identical amino acid positions in the amino acid sequence of an EPSPS enzyme, particularly if amino acid sequences of different organisms are aligned. To obviate ambiguity with respect to the numbers used in this description, we introduced FIG. 1 wherein the relevant region of EPSPS enzyme amino acid sequences of maize (corn), rice, wheat, tomato, Arabidopsis , onion, Salmonella , and E. coli are aligned and numbered. Furthermore, the important amino acids highlighted. The amino acid positions disclosed and referred herein are relative to FIG. 1 .
Where in this description (or in the claims) reference is made to amino acid positions 44, 101, 106, and/or 179 it is to be construed that also is meant a change of an amino acid at a position analogous to said position 44, 101, 106, and/or 179 in an amino acid sequence that is substantially homologous to EPSPS protein having amino acid sequences as shown in FIG. 1 , as will be understood by the skilled person, for example has a amino acid identity of at least 70%, preferably at least 75%, more preferably at least 80%, even more preferable 84%, 88%, 92%, 95%, 98% or 99% identity over its entire length with a protein having an amino acid sequence shown in FIG. 1 , and/or is functional, in other words has EPSPS enzymatic activity.
For example, although the relevant region of EPSPS of potato, or sunflower are not shown in FIG. 1 , they can be aligned to the sequences disclosed in FIG. 1 and according to methods known to the skilled person (for Example using the CLC Bio Main Workbench package (www.clcbio.com). For example, an amino acid sequence of a potato EPSPS enzyme that has EPSPS enzymatic activity and comprises the sequence Threonine (T)-Valine (V)-Valine (V)-Aspartic acid (D)-Aspartic acid (D)-Leucine (L)-Leucine (L)-Asparagine (N), will correspond to position 40-47 as shown in FIG. 1 . As this sequence comprises a change at a position corresponding to position 44 as shown in FIG. 1 , namely Aspartic acid instead of Asparagine, it is according to the present invention.
The inventors have realized that the limiting factor of mutations in the EPSPS enzyme might be the reduced biochemical activity they confer to the EPSPS enzyme. For instance, a T42M mutation in Salmonella gives a 25 fold lower affinity for its normal substrate (PEP) but a 26 fold higher tolerance for glyphosate. This fitness penalty may mean that such mutant genes are not sufficient for normal growth under field conditions. Comparably, Roundup Ready soybean crops, compared to the top conventional varieties, show a 6.7% lower yield (Charles Benbrook. Evidence of the Magnitude and Consequences of the Roundup Ready Soybean Yield Drag from University-Based Varietal Trials in 1998. Ag BioTech InfoNet Technical Paper Number 1).
It is believed that having an EPSPS mutant with Mutation A, i.e. at position 44, preferably as described above, alone or incombination with another mutation that renders glyphosate resistance, preferably a mutation as described herein, may lead to (improved) resistance to glyphosate. This may be without (further) inhibiting of enzyme activity or even improved enzyme activity. It was particularly unexpected to find that the combination of Mutation A (at position 44) with another mutation that renders glyphosate resistance, e.g. Mutation B (at position 101), Mutation C (at position 106), or Mutation D (at position 179) enhances glyphosate resistance beyond what may be expected on the basis of Mutation A alone and said another mutation alone.
For example, a nucleotide sequence encoding an EPSPS enzyme, that comprises a mutation that renders said EPSPS enzyme to confer glyphosate resistance to a plant (cell), preferably a mutation that corresponds to a change of an amino acid at a position chosen from the group consisting of 101, 106, or 179, as shown in FIG. 1 , may be provided with a further mutation corresponding to a change of an amino acid at a position 44, as disclosed herein, to obtain a nucleotide sequence encoding an EPSPS enzyme that may have increased activity towards its normal substrate or is better resistant to glyphosate, in comparison to when said mutation corresponding to a change of an amino acid at position 44 is not provided in said nucleotide sequence encoding an EPSPS enzyme. For example said increased activity encompasses a 1.5, 2, 4, or 5 fold increase of the affinity of the EPSPS enzyme for its normal substrate. Without being bound to theory, the current inventors believe that said effects may relate to changes in the active site of the EPSPS enzyme that lead to a lowered affinity of the enzyme for glyphosate and/or an increased affinity of the enzyme for its normal substrate.
Other mutated or modified EPSPSs, such as those described in U.S. Pat. Nos. 5,310,667, 5,866,775, 6,225,114, and 6,248,876, or natural EPSPS variants showing glyphosate-resistance, can also be used in combination with the present invention. In addition, bacteria-derived, glyphosate-resistant EPSPS variants, and for example after fusion with a chloroplast transit peptide, can also be used in combination with the present invention.
As will be understood by a skilled person, a mutation may be introduced in a nucleotide sequence encoding EPSPS as defined herein by the application of mutagenic compounds, such as ethyl methanesulfonate (EMS) or other compounds capable of (randomly) introducing mutations in nucleotide sequences. Said mutagenic compounds or said other compound may be used as a means for creating plant(s) cell(s) harboring a mutation in a nucleotide sequence encoding an EPSPS enzyme. Plant(s) cell(s) harboring a mutation according to the invention may then be selected by means of sequencing.
Alternatively, the introduction of a mutation in a nucleotide sequence encoding an EPSPS enzyme according to the invention is effected by the introduction of transfer-DNA (T-DNA) in a plant, for instance T-DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens . A T-DNA element comprising a nucleotide sequence encoding an EPSPS enzyme that comprises an amino acid change at position 44 according to the invention, optionally in combination with another mutation described herein, may be introduced in said plant, leading to a plant (cell) with (enhanced) glyphosate resistance obtained by the method according to the invention (see for example Krysan et al. 1999 The Plant Cell, Vol 11. 2283-2290). Likewise advantage can be taken from the use of transposable element insertion (See for Example Kunze et al (1997) Advances in Botanical Research 27 341-370 or Chandlee (1990) Physiologia Planta 79(1) 105-115).
Preferably, introducing a mutation in a nucleotide sequence encoding a EPSPS enzyme according to the invention is introduced by genome engineering techniques, such as techniques based on homologous recombination, or oligo-directed mutagenesis (ODM), for instance as described in WO2007073170; WO2007073149; WO2009002150; and WO2007073166). By applying ODM, specific nucleotides can be altered in a nucleotide sequence encoding EPSPS, whereby a mutation according to the invention may be introduced.
The invention also provides for an EPSPS enzyme that confers glyphosate resistance to a plant, preferably a plant EPSPS enzyme, encoded by a nucleotide sequence according to the invention as described herein. Such EPSPS enzyme may be derived from a bacterium or a plant. It is however preferred that the EPSPS enzyme, or the nucleotide encoding said EPSPS enzyme, is derived from a crop plant, more preferably cotton, tomato, potato, onion, rice, wheat, maize, or sunflower, because the EPSPS enzyme may then more closely resemble the natural EPSPS enzyme of the plants that are most used in agriculture. Such a more natural EPSPS enzyme may render crop plants to generate higher crop yield in agriculture.
The EPSPS enzyme according to the invention may be expressed in a plant, preferably a crop plant, more preferably cotton, tomato, potato, onion, rice, wheat, maize, sunflower, sugar beet or Brassica species.
Cotton includes Gossypium species, e.g. G. hirsutum, G. barbadense , tomato includes Solanum lycopersicum species, potato includes Solanum tubersosum species, onion includes Allium species, specifically Allium cepa species, rice includes Oryza sativa species e.g. O. sativa indica cultivar-group or japonica cultivar-group and Oryza glaberrima species, wheat includes Triticum species including T. monococcum and T. dicoccoides , maize/corn includes Zea species, sunflower includes Helianthus annus, Brassica species include canola, oilseed rape, cauliflower, broccoli, cabbage, B. napus, B. juncea, B. rapa and B. oleracea , and sugar beet includes Beta vulgaris.
The invention also provides for a vector comprising a nucleotide sequence according to the invention and a host comprising such vector. Said vector may be used to transfer a nucleotide according to the invention into another cell such as a plant cell. Different types of vectors include plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes.
Also provided is a plant or transgenic plant or part thereof comprising a nucleotide sequence according to the invention and/or a fragment thereof and/or an EPSPS enzyme according to the invention. Said fragment is at least of a length that is sufficient to determine that the fragment is derived from a nucleotide sequence encoding an EPSPS enzyme, and further comprises Mutation A according to the invention, preferably in combination with at least one further mutation that would confer, when in a complete nucleotide sequence encoding an EPSPS enzyme, glyphosate resistance upon a plant. Preferably, said at least one further mutation comprises Mutation B, Mutation C, and/or Mutation D, as described herein and according to the invention.
The fragment may for example have a length of at least 10, 30, 100, 500 consecutive nucleotides. A skilled person will have no problem in determining whether such fragment comprises a mutation as described herein. He can do so, for example, by aligning the fragment with the EPSPS coding sequence.
A transgenic crop plant contains a gene or genes which have been artificially inserted, i.e. wherein said gene or genes have not initially been acquired through pollination. A non-transgenic plant thus does not contain a gene or genes which have been artificially inserted. Also provided is a seed derived from a plant or transgenic plant or part thereof as mentioned-above, comprising a nucleotide sequence according to the invention and/or a fragment thereof and/or an EPSPS enzyme according to the invention.
Said fragment is at least of a length that is sufficient to determine that the fragment is derived from a nucleotide sequence encoding an EPSPS enzyme, and further comprises at least the Mutation A (position 44) according to the invention.
The fragment may for example have a length of at least 10, 30, 100, 500 consecutive nucleotides. A skilled person will have no problem in determining whether such fragment comprises a mutation as described herein. He can do so, for example, by aligning the fragment with the EPSPS coding sequence.
In another aspect, the invention relates to a method for providing a glyphosate resistant plant (cell), the method comprising:
a) providing a plant (cell) that comprises a nucleotide sequence that encodes an EPSPS enzyme, preferably a plant EPSPS enzyme, characterized in that the nucleotide sequence comprises at least one mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 .

In the above-mentioned method it is preferred that the amino acid that is changed at position 44 is Asparagine and/or that the amino acid at position 44 is changed into an Aspartic Acid, preferably in comparison to wild-type EPSPS. In other words, the amino acid at position 44, as shown in FIG. 1 , is preferably an Aspartic Acid.
In yet another aspect, the invention relates to a method for providing a plant (cell) with resistance to glyphosate, the method comprising:
a) providing a plant (cell) that comprises a nucleotide sequence that encodes an EPSPS enzyme, preferably a plant EPSPS enzyme, characterized in that the nucleotide sequence comprises at least one mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 , and at least one further mutation that on its own renders said nucleotide sequence to encode an EPSPS enzyme that confers glyphosate resistance to a plant, preferably wherein said at least one further mutation corresponds to a change of an amino acid at a position chosen from the group consisting of 101, 106, or 179, as shown in FIG. 1 .

In yet another aspect, the invention relates to a method for providing a plant (cell) with resistance to glyphosate, the method comprising:
a. providing a plant (cell) that comprises a nucleotide sequence that encodes an EPSPS enzyme, preferably a plant EPSPS enzyme; b. providing a mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 , in said nucleotide sequence of step a), preferably in said plant of step a).

Thus, preferably thereby a mutation is provided that results in an amino acid at position 44 in the EPSPS-coding sequence which is not Asparagine (N), e.g. leading to glyphosate resistance, in comparison to the same EPSPS coding sequence not having the mutation.
In yet another aspect, the invention relates to a method for enhancing glyphosate resistance of a plant (cell), the method comprising:
a) providing a plant (cell) that comprises a nucleotide sequence that encodes an EPSPS enzyme that confers glyphosate resistance to a plant, preferably a plant EPSPS enzyme, and preferably characterized in that the nucleotide sequence comprises at least one mutation that corresponds to a change of an amino acid at a position chosen from the group consisting of 101, 106, or 179, as shown in FIG. 1 ; b) providing a mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 , in said nucleotide sequence of step a), preferably in said plant (cell) of step a).

Thus, preferably thereby a mutation is provided that results in an amino acid at position 44 in the EPSPS-coding sequence which is not Asparagine (N), e.g. leading to glyphosate resistance, in comparison to the same EPSPS coding sequence not having the mutation.
Providing a mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 , in a nucleotide sequence that encodes an EPSPS enzyme, preferably a plant EPSPS enzyme, may be performed by Targeted Nucleotide Exchange (TNE), i.e. by introduction of at least one oligonucleotide capable of hybridizing to the nucleotide sequence of step a) and comprising a mismatch with respect to the nucleotide sequence of step a), wherein the position of the mismatch corresponds to the position of a mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 , in a nucleotide sequence that encodes an EPSPS enzyme, preferably a plant EPSPS enzyme.
Once introduced into the cell, e.g. by electroporation or PEG-mediated oligonucleotide uptake, such oligonucleotide can hybridize (basepair) with the complementary sequence of the nucleotide sequence to be altered (i.e. the target locus in the nucleotide sequence that encodes an EPSPS enzyme). By deliberately designing a mismatch in the oligonucleotide, the mismatch may impart a nucleotide conversion at the corresponding position in the target genomic sequence. This may result in the provision of a mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 . Likewise, and depending on the type of mismatch(es) that is deliberately designed, this may result in the provision of a mutation that corresponds to a change of an amino acid at position 101, 106, and/or 179, as shown in FIG. 1 .
The oligonucleotide may have a length of between 10-500 nucleotides, preferably 15-250 nucleotides, more preferably between 10-200 nucleotides, most preferably between 15-150 nucleotides. The oligonucleotide may contain locked nucleic acids (LNAs), preferably located one nucleotide upstream or downstream from the mismatch. The oligonucleotide may alternatively or additionally contain propynylated bases. The TNE method is described in Applicant's patent publications WO2007073166, WO2007073170, WO2009002150. The oligonucleotide may further contain at least 4, at least 3, 3, 2 or 1 phosphorothioate linkages at the 5′ end and/or the 3′ end, both ends or flanking the mismatch.
It is further preferred that in the above-mentioned method, the amino acid that is changed at position 44 is Asparagine and/or that the amino acid at position 44 is changed into an Aspartic Acid and/or the amino acid that is changed at position 101 is Glycine and/or the amino acid that is changed at position 106 is Proline and/or the amino acid that is changed at position 179 is Serine and/or the amino acid at position 101 is changed into an Alanine and/or the amino acid at position 106 is changed into a Leucine and/or the amino acid at position 179 is changed into an Asparagine (see also specific combinations of mutations as described under the heading “Specific combinations of mutations encompassed by the invention”).
The plant provided by a method according to the invention can be used for the production of further plants and or plant products derived therefrom. The term plant products refers to those materials that can be obtained from the plants grown, and include fruits, leaves, plant organs, plant fats, plant oils, plant starch, plant protein fractions, either crushed, milled or still intact, mixed with other materials, dried, frozen, and so on. In general such plant products can, for example be recognized by the presence of a nucleotide according to the present invention.
The invention also provides for a method for generating a plant product, the method comprising:
a) processing a plant or transgenic plant or part thereof comprising a nucleotide according to the invention and/or a EPSPS enzyme according to the invention, or a plant obtainable by any of the methods according to the invention.

In the above-mentioned method it is preferred that processing is performed by cooking, grinding, drying, milling, baking, cutting, sieving, flaking, peeling, soaking, washing, heating, cooling, crushing, or wetting, to obtain a plant product.
Also provided is a plant product obtainable by the above-mentioned method, preferably a starch-based product or a plant-oil-based product, characterized in the presence of a nucleotide sequence according to the invention and/or a fragment thereof.
Starch-based products may include products such as tomato purée, flour, or any other starch-containing product. Plant-oil-based products may include products such as olive oil, sunflower oil, or any other plant-oil-containing product.
The invention also provides for the use of a nucleotide sequence according to the invention and/or an EPSPS enzyme according to the invention, for enhancing glyphosate resistance of a plant. For example, such nucleotide sequence may be introduced in a plant (cell) thereby effecting (enhanced) glyphosate resistance.
Another aspect of the present invention relates to the use of a mutation that corresponds to a change of an amino acid at position 44, as shown in FIG. 1 , for enhancing glyphosate resistance of a glyphosate resistant plant, preferably wherein said plant comprises a nucleotide sequence that encodes an EPSPS enzyme that confers glyphosate resistance to a plant, preferably a plant EPSPS enzyme, preferably characterized in that the nucleotide sequence comprises at least one mutation that corresponds to a change of an amino acid at a position chosen from the group consisting of 101, 106, or 179, as shown in FIG. 1 .
In other words, use of a mutation that corresponds to a change of an amino acid at position 44 involves changing an amino acid at position 44, preferably so that the amino acid at position 44 is Aspartic Acid, thereby effecting (enhanced) glyphosate resistance. Yet in other words, said use involves the use of the information that a change of an amino acid at position 44, according to the invention, effects (enhanced) glyphosate resistance.
With respect to the above-mentioned use, it is preferred that the amino acid that is changed at position 44 is Asparagine, and/or the amino acid that is changed at position 101 is Glycine, and/or the amino acid that is changed at position 106 is Proline and/or the amino acid that is changed at position 179 is Serine and/or the amino acid at position 44 is changed into an Aspartic Acid and/or the amino acid at position 101 is changed into an Alanine and/or the amino acid at position 106 is changed into a Leucine, and/or the amino acid at position 179 is changed into a Asparagine. Said changes are preferably in comparison to wild-type EPSPS.
Also provided is the use of a mutation as described above, in an EPSPS enzyme resistant to glyphosate, for restoration or improvement of enzyme activity. For example, the mutation leading to the change of the amino acid at position 44, preferably an Asparagine, into another amino acid, preferably Aspartic acid, may be used through introducing it in an enzyme already providing resistance to glyphosate, to further improve activity of the enzyme towards its normal substrate PEP. Preferably, a mutation leading to a change of the amino acid at position 101, preferably a Glycine, into another amino acid, preferably Alanine, and/or a mutation leading to a change of the amino acid at position 106, preferably a Proline, into another amino acid, preferably Leucine, and/or the mutation leading to a change of the amino acid at position 179, preferably Serine, into another amino acid, preferably Asparagine, may (in addition) be introduced in an enzyme already providing resistance to glyphosate, to further improve activity of the enzyme towards its normal substrate PEP.
Below, specific combinations of mutations A, B, C, and D are described that may be used according to the invention.
Specific Combinations of Mutations Encompassed by the Invention:
(Mutations A+B)
In a preferred embodiment, the nucleotide sequence that encodes an EPSPS enzyme comprises a mutation that leads to change of the amino acid at position 44 and a mutation that leads to a change of the amino acid at position 101. In a preferred embodiment, the amino acid that is changed at position 44 is Asparagine. In another preferred embodiment the amino acid that is changed at position 101 is Glycine. In a further preferred embodiment, the amino acid that is changed at position 44 in Asparagine and the amino acid that is changed at position 101 is Glycine. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 44 leads to a change into the amino acid Aspartic acid, i.e. a aspartic acid is present at position 44. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 101 leads to a change into the amino acid Alanine, i.e. a alanine is present at position 101. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 44 leads to a change into the amino acid Aspartic acid, i.e. a aspartic acid is present at position 44 and at the same time the mutation in said nucleotide sequence that leads to the change of the amino acid at position 101 leads to a change into the amino acid Alanine, i.e. a alanine is present at position 101.
(Mutations A+C)
In another preferred embodiment, the nucleotide sequence that encodes an EPSPS enzyme comprises a mutation that leads to change of the amino acid at position 44 and a mutation that leads to a change of the amino acid at position 106. In a preferred embodiment, the amino acid that is changed at position 44 is Asparagine. In another preferred embodiment the amino acid that is changed at position 106 is Proline. In a further preferred embodiment, the amino acid that is changed at position 44 in Asparagine and the amino acid that is changed at position 106 is Proline. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 44 leads to a change into the amino acid Aspartic acid, i.e. a aspartic acid is present at position 44. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 106 leads to a change into the amino acid Leucine, i.e. an Leucine (L) is present at position 106. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 44 leads to a change into the amino acid Aspartic acid, i.e. a aspartic acid is present at position 44 and at the same time the mutation in said nucleotide sequence that leads to the change of the amino acid at position 106 leads to a change into the amino acid Leucine, i.e. an Leucine (L) is present at position 106.
(Mutations A+D)
In another preferred embodiment, the nucleotide sequence that encodes an EPSPS enzyme comprises a mutation that leads to change of the amino acid at position 44 and a mutation that leads to a change of the amino acid at position 179. In a preferred embodiment, the amino acid that is changed at position 44 is Asparagine. In another preferred embodiment the amino acid that is changed at position 179 is Serine. In a further preferred embodiment, the amino acid that is changed at position 44 in Asparagine and the amino acid that is changed at position 179 is Serine. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 44 leads to a change into the amino acid Aspartic acid, i.e. a aspartic acid is present at position 44. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 179 leads to a change into the amino acid Asparagine (D), i.e. a Asparagine is present at position 179. Preferably, the mutation in said nucleotide sequence that leads to the change of the amino acid at position 44 leads to a change into the amino acid Aspartic acid, i.e. a aspartic acid is present at position 44 and at the same time the mutation in said nucleotide sequence that leads to the change of the amino acid at position 179 leads to a change into the amino acid Asparagine, i.e. a Asparagine is present at position 179.



FIGURES
In FIG. 1 , the relevant region of EPSPS from Maize (SEQ ID NO:1), Rice (SEQ ID NO:2), Wheat (SEQ ID NO:3), Tomato (SEQ ID NO:4), Arabidopsis (SEQ ID NO:5), Onion (SEQ ID NO:6), Salmonella (SEQ ID NO:7), and E. coli (SEQ ID NO:8) are aligned and the important amino acids highlighted. The amino acid positions disclosed and referred herein are indicated in FIG. 1 . As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 43, 99, 104, and 177 respectively in SEQ ID NO:1, positions 44, 101, 106, and 179 respectively in SEQ ID NO:2, positions 44, 101, 106, and 179 respectively in SEQ ID NO:3, positions 44, 101, 106, and 179 respectively in SEQ ID NO:4, positions 44, 101, 106, and 179 respectively in SEQ ID NO:5, positions respectively 30, 87, 92, and 165 in SEQ ID NO:6, positions 42, 95, 100, and 169 respectively in SEQ ID NO:7, positions 42, 95, 100, and 169 respectively in SEQ ID NO:8. FIG. 1 shows the mature proteins lacking the chloroplast targeting signal.
FIG. 2 shows the results of a complementation assay in E. coli using A. thaliana EPSPS variants. Legend of FIG. 2 is shown below:
(1) N44D P106L (2) S179N (3) P106L (4) Wild Type (5) N44D (6) P106L S179N (7) Empty

FIG. 3 shows the complete Arabidopsis EPSPS amino acid sequence, including the chloroplast targeting signal (SEQ ID NO:9). The relevant amino acids are underlined. As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 120, 177, 182, and 255 respectively in SEQ ID NO:9.
FIG. 4 shows the complete Arabidopsis thaliana EPSPS ORF (SEQ ID NO:10).
FIG. 5 shows the complete Arabidopsis thaliana EPSPS ORF comprising mutation A358G that corresponds to N44D (SEQ ID NO:11).
FIG. 6 shows the complete Arabidopsis thaliana EPSPS ORF comprising mutation C545T that corresponds to P106L (SEQ ID NO:12).
FIG. 7 shows the complete Arabidopsis thaliana EPSPS ORF comprising mutations A358G and C545T that correspond to N44D and P106L respectively (SEQ ID NO:13).
FIG. 8 shows the complete Arabidopsis thaliana EPSPS ORF comprising mutation G902A that corresponds to S179N (SEQ ID NO:14).
FIG. 9 shows the complete Arabidopsis thaliana EPSPS ORF comprising mutations C545T and G902A that correspond to P106L and S179N respectively (SEQ ID NO:15).
FIG. 10 shows the complete Arabidopsis thaliana EPSPS ORF comprising mutations A358G and G902A that correspond to N44D and S179N respectively (SEQ ID NO:16).
FIG. 11 shows the complete Arabidopsis thaliana EPSPS ORF comprising mutation G530C that corresponds to G101A (SEQ ID NO:17).
FIG. 12 shows the complete Arabidopsis thaliana EPSPS ORF comprising mutations A358G and G530C that correspond to N44D and G101A respectively (SEQ ID NO:18).
FIG. 13 shows the complete Tomato EPSPS nucleotide sequence (SEQ ID NO:19).
FIG. 14 shows the complete Tomato EPSPS2 Nucleotide sequence (SEQ ID NO:20).
FIG. 15 shows the complete Tomato EPSPS1 Amino Acid Sequence. The relevant amino acids are underlined (SEQ ID NO:21). As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 118, 175, 180, and 253 respectively in SEQ ID NO:21.
FIG. 16 shows the complete Tomato EPSPS2 amino acid sequence. The relevant amino acids are underlined (SEQ ID NO:22). As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 120, 177, 182, and 255 respectively in SEQ ID NO:22.
FIG. 17 shows the complete E. coli EPSPS amino acid sequence. The relevant amino acids are underlined (SEQ ID NO:23). As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 43, 96, 101, and 170 respectively in SEQ ID NO:23.
FIG. 18 shows the complete Cotton EPSPS amino acid sequence. The relevant amino acids are underlined (SEQ ID NO:24). As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 121, 178, 183, and 256 respectively in SEQ ID NO:24.
FIG. 19 shows the complete Maize EPSPS amino acid sequence. The relevant amino acids are underlined (SEQ ID NO:25). As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 46, 102, 107, and 180 respectively in SEQ ID NO:25.
FIG. 20 shows the complete Rice EPSPS amino acid sequence. The relevant amino acids are underlined (SEQ ID NO:26). As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 115, 172, 177, and 250 respectively in SEQ ID NO:26.
FIG. 21 shows the complete Wheat EPSPS amino acid sequence. The relevant amino acids are underlined (SEQ ID NO:27). As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, 106, and 179 relate to positions 110, 167, 172, and 245 respectively in SEQ ID NO:27.
FIG. 22 shows the complete Sunflower EPSPS amino acid sequence. The relevant amino acids are underlined (SEQ ID NO:28). As can be seen, the amino acid positions disclosed and referred herein as positions 44, 101, and 106 relate to positions 118, 175, and 180 respectively in SEQ ID NO:28.
FIG. 23 shows glyphosate tolerance in transgenic Arabidopsis lines: ratio of average seedling weight (+Gly/−Gly). Legend of FIG. 23 is shown below:
(1) Wild type EPSPS gene (2) EPSPS with mutation N44D (3) EPSPS with mutation P106L (4) EPSPS with mutations P106L and N44D (1) (5) EPSPS with mutations P106L and N44D (2) (6) EPSPS with mutations P106L and N44D (3) (7) EPSPS with mutations N44D and S179N




EXAMPLE 1
Applying Mutation a in an EPSPS Enzyme Enhances Glyphosate Resistance Conferred by Said EPSPS Enzyme.
We tested whether a mutation that corresponds to a change of Asparagine at position 44 into Aspartic Acid, in an EPSPS enzyme (N44D mutation), would be able to provide the EPSPS enzyme with resistance to glyphosate. This was tested by introducing the N44D mutation into EPSPS alone and in combination with other mutations which confer glyphosate resistance to said EPSPS enzyme. These were a mutation that corresponds to a change of Glycine at position 101 into Alanine, in an EPSPS enzyme (G101A), a mutation that corresponds to a change of Proline at position 106 into Leucine, in an EPSPS enzyme (P106L) and a mutation that corresponds to a change of Serine at position 179 into Asparagine, in an EPSPS enzyme (S179N).
Constructs
All experiments utilized the Arabidopsis thaliana EPSPS (At2g45300) open reading frame (see FIGS. 4-12 ). The Arabidopsis EPSPS amino acid sequence is depicted in FIG. 3 .
First, this sequence was synthesized synthetically (www.geneart.com) and cloned into a vector where it was flanked by BamHI and EcoRI sites. This was then used as a basis construct for the introduction of the N44D (A350G), G101A (G530C), P106L (C545T) and S179N (G902A) mutations which were introduced by site specific mutagenesis (www.geneart.com). The following double mutants were also constructed:
N44D+G101A; N44D+P106L; N44D+S179N; and P106L+S179N.

Each of these EPSPS variants was then cloned as a 1576 bps EcoRI-BamHI fragment into the vector pET302 NT HIS (Invitrogen, product K630203), fusing the EPSPS variants to a 6×His tag and allowing inducible protein expression with IPTG in E. coli . These constructs were introduced into the E. coli expression strain BL21 pLYS (Invitrogen) for complementation assays.
Complementation Assays
E. coli is unable to grow in minimal M9 growth medium supplemented with glyphosate due to inhibition of the activity of the endogenous bacterial EPSP synthase AroA. Bacterial growth can be restored by complementation with a glyphosate resistant plant EPSPS gene. The E. coli strains containing the A. thaliana EPSPS variants were grown overnight in 10 ml LB medium containing 100 μg/ml carbenicillin (Duchefa). Strains were then diluted 4 fold in the same medium and allowed to grow for a further 4 hours. The OD600 of each culture was then measured and 1.5 ml culture was centrifuged at 4000 rpm for 10 minutes. The bacterial pellet was resuspended in 500 μM9 medium (12.8 g/l Na 2 HPO 4 , 3.0 g/l KH 2 PO 4 , 0.5 g/l NaCl, 1.0 g/l NH 4 Cl, 2.0 g/l glucose, 0.4940 g/l MgSO 4 .7H 2 O, 15 mg/l CaCl 2 .2H 2 O, 10 mg/l thiamine and 10 mg/l FeSO 4 .7H 2 O) used to inoculate 10 ml M9 medium containing 1 mM IPTG and 100 μg/ml carbenicillin and 30 mM glyphosate to an OD600 of 0.1. Identical cultures for each strain were also made lacking glyphosate and these were used to assess the growth of each strain without selection. The cultures were then grown at 25° C. and the OD600 was measured over time to assess bacterial growth (see FIG. 2 ).
Results
The results of the complementation assays are shown in FIG. 2 . As expected, the strain containing the empty pET302NT His vector was unable to grow in the presence of glyphosate whereas the strain containing the P106L mutation showed a good level of resistance. Overexpression of the wild type (WT) EPSPS enzyme was able to complement bacterial growth at later time points, presumably due to titration of glyphosate due to high protein expression levels. In other words, presumably, at said later time points the amount of glyphosate was no longer enough to inactivate all expressed EPSPS enzymes.
The results showed that the N44D mutation alone may provide for some resistance to glyphosate, but when combined with the P106L mutation was able to enhance the resistance levels 2.5 fold after 18 hours of growth. This suggests that N44D can be used, preferably as secondary mutation that is able to improve glyphosate resistance. Similar results can be obtained when Mutation A, in this case N44D, was combined with the other mutations described above. Also good resistance was conferred by the S179N mutation alone, but when this was combined with the P106L mutation the EPSPS enzyme was inactive.
EXAMPLE 2
An EPSPS Gene Containing the N44D and P106L Mutations Confers Enhanced Tolerance to Glyphosate when Expressed in Arabidopsis
In the first example using the E. coli assay we have been able to show that the N44D mutation in itself provides tolerance to glyphosate, but when combined with e.g. the P106L mutation glyphosate tolerance is strongly enhanced. The aim of the present example is to demonstrate that the N44D mutation shows the same effect in plants and so the effect of a number of mutant EPSPS genes on glyphosate resistance in Arabidopsis thaliana was tested.
Construction of Binary Vectors Containing Mutant EPSPS Genes
The mutated Arabidopsis EPSPS genes tested in E. coli (see Example 1) were cloned into the binary vector pEARLEYGATE100 (The Plant J. 2006; 45:616-629). Briefly, the EPSPS genes were isolated as BamHI and SacI fragments comprising the complete ORF's and then inserted into the same unique sites present in the binary vector. This resulted in a series of binary vectors carrying a T-DNA on which the EPSPS genes are expressed by the constitutive 35S promoter and also carrying a BAR gene (conferring resistance to glufosinate) that can be used for plant selection. This series of binary vectors were electroporated to the Agrobacterium tumefaciens strain GV2260 and bacterial colonies were selected on LB medium containing 100 μg/ml spectinomycin, using methods known to the skilled person.
Transformation of Arabipdopsis thaliana
Seeds of Arabidopsis thaliana (ecotype Colombia) were sown on soil and once germinated a single seedling was placed in a 5 cm×5 cm pot and allowed to grow until the first influorescence appeared. At this stage the plants were then transformed using the floral dip method. Briefly, the Agrobacterium strains were grown overnight in 50 ml liquid LB medium containing 100 μg/ml spectinomycin at 28° C. and then this culture was centrifuged at 4000 rpm for 10 minutes. The pellets were then resuspended in a 5% sucrose solution containing 0.02% Silwet (Lehle Seeds). The Arabidopsis plants were dipped into this culture and then incubated overnight in the dark at 25° C. The plants were then allowed to grow further in the greenhouse and after 7 days the dipping procedure was repeated. The plants were then allowed to set seed which was then germinated on soil. For selection of transformed Arabidopsis seedlings, a solution of glufosinate was prepared (0.1 mg/ml glufosinate ammonium+0.005% silwet) and sprayed on the seedlings. After 7 days the surviving seedlings were identified, grown to maturity and the seeds were collected.
Copy Number Determination of the Transgenic Arabidopsis Lines
We performed a segregation analysis on the seed of the transgenic lines to estimate the T-DNA copy number. Seed from each of the transgenic lines was sterilized with a 2% hypochlorite solution and washed with sterile water. Approximately 100 seeds per line were then placed on MS20 medium containing 0.1 mg/ml glufosinate ammonium and the number of surviving seedlings was scored after 20 days. The lines with a single T-DNA copy that segregated 3:1 (resistance:sensitivity) to the glufosinate were selected for further phenotyping.
Glyphosate Tolerance of the Transgenic Arabidopsis Lines
Seeds from the transgenic Arabidopsis lines were sterilized and then placed on MS20 medium containing 0.1 mg/ml glufosinate ammonium and +/−400 μM glyphosate and were grown for 4 weeks. After this period the seedlings were removed from the growth plates and the average seedling weight was determined. The degree of glyphosate resistance for each line was determined by dividing the average seedling weight in the presence of glyphosate by the average seedling weight found in the absence of glyphosate. The results of this analysis is shown in FIG. 23 .
The data in plants, i.e. from Arabidopsis confirms the resistance that we found in the E. coli growth assays. Overexpression of the EPSPS genes containing only the N44D mutation lead to glyphosate resistance as compared to the EPSPS unmutated WT form. Overexpression of the EPSPS P106L gene gave reasonable resistance, while this could be enhanced in the 3 independent lines expressing EPSPS with both the P106L and the N44D mutation. The data for the S179N mutation shows that the mutation also confers resistance when combined with N44D.