Rust resistance genes

Abstract

The present invention relates to rust resistance genes. Specifically, the invention relates to a novel transport polypeptide and a coding gene thereof, which are used for endowing plants with resistance to one or more living nutritional pathogenic fungi.

Description

[0001] The present invention is a divisional application of Chinese patent application 201480052048.5 “Rust resistance gene” filed on August 21, 2014. Technical Field

[0002] The present invention relates to novel transporter polypeptides and genes encoding the same for conferring resistance to one or more biotrophic pathogenic fungi on plants. Background Art

[0003] A variety of genes that confer pathogen resistance have been identified and used in plant breeding. However, single-gene pathogen resistance in plants often becomes ineffective due to the emergence of new virulent strains of pathogens. Instead, long-lasting disease resistance in plants is generally believed to be controlled by multiple genes. Several rust resistance genes have been isolated and cloned from wheat (Feuillet et al., 2003; Huang et al., 2003; Cloutier et al., 2007) and other cereals (Collins et al., 1999; Brueggeman et al., 2002), and these genes are primarily from the nucleotide binding site-leucine-rich repeat (NB-LRR) class of important resistance (R) genes. For example, three wheat R genes (Lr1, Lr10, and Lr21) that confer protection against the wheat leaf rust fungus, Puccinia triticina, have been cloned (Somers et al., 2004; Hayden et al., 2008; Manly et al., 2001). An exception is the barley Rpg1 rust resistance gene, which encodes a protein kinase. These genes confer gene-for-gene resistance to a single pathogen and generally result in a strong hypersensitive response of plant tissues to infection.

[0004] In contrast, wheat (Triticum aestivum L.) rust resistance genes such as Lr34, located on chromosome wall 7DS, confer broad and durable resistance in mature plants to several obligate biotrophic pathogens, including Ascomycetes and Basidiomycetes. These include leaf rust, stripe rust, stem rust, and powdery mildew, and as a result, the Lr34 gene has been widely used in wheat breeding, despite its weak, non-hypersensitive phenotype (Dyck, 1977 and 1987; German and Kolmer, 1992; Bossolini et al., 2006; Spielmeyer et al., 2008). Cultivars with the resistance locus Lr34, such as Frontana, have been shown to confer effective and durable resistance to the leaf rust fungus Puccinia triticina Eriks (Dyck et al., 1966; Singh and Rajaram, 1994). To date, no isolates of wheat leaf rust (P. triticina) have been detected with the full virulence of Lr34 (Kolmer et al., 2003). The Lr34 gene was recently cloned and shown to encode a protein in the ABC transporter family (Krattinger et al., 2009), although its function as a transporter has not been demonstrated. Lr34 resistance remains genetically inseparable from the gene designated Yr18 that confers resistance to stripe rust in P. striiformis (Singh, 1992; McIntosh, 1992). Lr34 / Yr18 has been documented to co-segregate with other traits, such as leaf tip necrosis in mature plants (Ltn1), powdery mildew (recently designated Pm38), and resistance to barley yellow dwarf virus (Bdv1) and leaf spot (Bipolaris sorokiniana) (Singh, 1992a, b; McIntosh, 1992; Joshi et al., 2004; Spielmeyer et al., 2005; Liang et al., 2006), and these phenotypes are all thought to be conferred by the Lr34 resistance polypeptide.

[0005] A second gene, Lr67, that confers broad-spectrum, mature plant resistance to several obligate biotrophic pathogens, is located on wheat chromosome 4DL and has been found in several newly introduced wheat varieties, such as RL6077 (Herrera-Foessel et al., 2011). In contrast to Lr34, the Lr67 gene has not yet been widely used to generate resistant cultivars for commercial wheat production. Although initial reports based on plant phenotypes (Dyck et al., 1994) suggested that the resistance gene in RL6077 might be a translocated Lr34, this was subsequently shown not to be the case (Herrera-Foessel et al., 2011). After mapping the gene in two segregating populations, Hiebert et al. (2010) named the gene in RL6077 Lr67. Although Lr67, like Lr34, also causes leaf tip necrosis and confers partial, broad-spectrum, mature plant resistance to leaf and stripe rust, these are distinct genes.

[0006] There is a need to determine the molecular basis of genes such as Lr67 that confer quantitatively non-cultivar-specific, full-blown phytopathogen resistance or partial resistance to a broad spectrum of pathogens. Summary of the Invention

[0007] The present inventors have identified novel transporter polypeptides and genes encoding same, which are useful for conferring resistance to one or more biotrophic fungal pathogens on plants.

[0008] In one aspect, the present invention provides a recombinant cell comprising an exogenous polynucleotide encoding a polypeptide characterized by one or more or all of the following:

[0009] i) when expressed in a plant, the polypeptide confers resistance to one or more biotrophic fungal pathogens, preferably to one or more or all of leaf rust, stripe rust, stem rust and powdery mildew,

[0010] ii) when expressed in a cell, the polypeptide is less efficient at transporting glucose across the cell membrane than a polypeptide comprising the amino acids of the sequence provided in SEQ ID NO:4,

[0011] iii) when expressed in a cell, the polypeptide functions effectively as a sugar transporter,

[0012] iv) a polypeptide comprising the amino acids of the sequence provided in SEQ ID NO: 1 or an amino acid sequence at least 40% identical to SEQ ID NO: 1 or a biologically active fragment thereof, and

[0013] v) the polypeptide does not comprise glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1, preferably the polypeptide comprises an amino acid other than glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1,

[0014] The polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in the cell.

[0015] In a preferred embodiment, the polypeptide has at least features i) and iv), i), ii) and iv), ii) and iv), or iv) and v), more preferably features i), iv) and v), i), ii), iv) and v), or i), iv) and v).

[0016] In one embodiment, the one or more biotrophic fungal pathogens are rust or mildew or rust and mildew. Examples of biotrophic fungi include, but are not limited to, Blumeria graminis f. sp. tritici, Fusarium graminearum, Bipolaris sorokiniana, Erysiphe graminis f. sp. tritici, Puccinia graminis f. sp. tritici, Puccinia striiformis, Puccinia hordei, and Puccinia recondita f. sp. tritici.

[0017] In one embodiment, the cell is a plant cell or a yeast cell. More preferably, the cell is a plant cell. In one embodiment, the plant cell is a cereal plant cell such as a wheat plant cell. In another embodiment, the plant cell is a grape cell.

[0018] In one embodiment, the promoter directs gene expression in leaves and / or stem cells.

[0019] Preferably, if the polypeptide does not comprise glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1, the polypeptide comprises an amino acid sequence that is at least 40% identical to one or more or all of SEQ ID NO: 1, 4 or 7 to 9 or one or more biologically active fragments thereof.

[0020] In a preferred embodiment, the polypeptide comprises an amino acid at the position corresponding to amino acid number 144 of SEQ ID NO: 1, wherein the amino acid is selected from the group consisting of arginine, lysine, and histidine.

[0021] In another preferred embodiment, the polypeptide does not comprise valine at the position corresponding to amino acid number 387 of SEQ ID NO: 1. Preferably, the polypeptide comprises an amino acid other than valine at the position corresponding to amino acid number 387 of SEQ ID NO: 1. More preferably, the polypeptide comprises an amino acid at the position corresponding to amino acid number 387 of SEQ ID NO: 1, wherein the amino acid is selected from the group consisting of leucine, isoleucine, methionine alanine, and phenylalanine.

[0022] In one embodiment, the exogenous polynucleotide is integrated into the genome of the cell.

[0023] In yet another embodiment, the polypeptide comprises amino acids having a sequence provided in SEQ ID NO: 1, or an amino acid sequence at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1, or a biologically active fragment thereof.

[0024] In one embodiment, the polypeptide comprises 12 transmembrane domains.

[0025] In another aspect, the present invention provides a transgenic plant comprising the cell of the present invention, wherein the transgenic plant is transgenic for an exogenous polynucleotide.

[0026] In a preferred embodiment, each somatic cell of the plant comprises the exogenous polynucleotide.

[0027] In yet another preferred embodiment, the plant has increased resistance to one or more biotrophic fungal pathogens, preferably to rust, mildew, or both rust and mildew, more preferably to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, compared to an isogenic plant lacking the exogenous polynucleotide.

[0028] In yet another embodiment, the plant has enhanced resistance to one or more biotrophic fungal pathogens during the seedling stage of growth.

[0029] In another embodiment, the plant comprises one or more exogenous polynucleotides encoding a plant pathogen resistance polypeptide other than an Lr67 polypeptide, preferably an Lr34 polypeptide, an Sr33 polypeptide, or an Sr35 polypeptide. Other plant pathogen resistance polypeptides include, but are not limited to, Lr1, Lr3, Lr2a, Lr3ka, Lr11, Lr13, Lr16, Lr17, Lr18, Lr21, and LrB.

[0030] Preferably, the plant is a cereal plant. Examples of transgenic cereal plants of the present invention include, but are not limited to, wheat, barley, corn, rice, oats, and rye. In a particularly preferred embodiment, the plant is wheat. In another embodiment, the plant is a grapevine.

[0031] In one embodiment, the promoter directs gene expression in aerial parts of a plant, such as leaves and / or stems.

[0032] Preferably, the plant is homozygous for the exogenous polynucleotide.

[0033] In yet another embodiment, the plants are grown in a field.

[0034] Also provided is a population of at least 100 plants of the invention grown in a field.

[0035] In another aspect, the invention provides a method of determining whether a polypeptide confers resistance or susceptibility to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, more preferably conferring resistance or susceptibility to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, comprising:

[0036] i) obtaining a polynucleotide operably linked to a promoter, the polynucleotide encoding a polypeptide comprising amino acids having the sequence provided in SEQ ID NO: 1 or an amino acid sequence at least 40% identical to SEQ ID NO: 1, or a biologically active fragment thereof,

[0037] ii) introducing the polynucleotide into the plant,

[0038] iii) determining the degree of resistance or susceptibility to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, more preferably determining whether the degree of resistance or susceptibility to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew is increased or decreased relative to an isogenic plant lacking the polynucleotide, and

[0039] iv) Optionally, if the level of resistance or susceptibility is increased, selecting a polynucleotide encoding a polypeptide which, when expressed, confers resistance or susceptibility to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, more preferably conferring resistance or susceptibility to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew.

[0040] In one embodiment, one or more of the following applies to the method,

[0041] a) The polynucleotide comprises nucleotides having a sequence as provided in SEQ ID NO: 2 or SEQ ID NO: 3, a sequence at least 40% identical to one or both of SEQ ID NO: 2 and SEQ ID NO: 3, or a sequence that hybridizes to one or both of SEQ ID NO: 2 and SEQ ID NO: 3.

[0042] b) the plant is a cereal plant such as a wheat plant or a grapevine plant,

[0043] c) the polypeptide is a plant polypeptide or a mutant thereof, and

[0044] d) step ii) further comprises stably integrating the polynucleotide operably linked to the promoter into the genome of the plant

[0045] e) The polypeptide is characterized by one or more of the characteristics defined above in relation to the cell of the invention.

[0046] In another aspect, the present invention provides a substantially purified and / or recombinant polypeptide characterized by one or more or all of the following:

[0047] i) when expressed in a plant, the polypeptide confers resistance to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, more preferably confers resistance to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew,

[0048] ii) when expressed in a cell, the polypeptide is less efficient at transporting glucose across the cell membrane than a polypeptide comprising amino acids having the sequence provided in SEQ ID NO: 4,

[0049] iii) when expressed in a cell, the polypeptide functions effectively as a sugar transporter,

[0050] iv) the polypeptide comprises amino acids having the sequence provided in SEQ ID NO: 1 or an amino acid sequence at least 40% identical to SEQ ID NO: 1, or a biologically active fragment thereof, and

[0051] v) the polypeptide does not comprise glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1, preferably the polypeptide comprises an amino acid other than glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1.

[0052] In a preferred embodiment, the polypeptide is characterized by one or more of the characteristics defined above in relation to the cells of the invention.

[0053] In another aspect, the polypeptide comprises amino acids having the sequence provided in SEQ ID NO: 1, or an amino acid sequence at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1, or a biologically active fragment thereof.

[0054] In one embodiment, the polypeptide of the present invention is a fusion protein further comprising at least one other polypeptide sequence. The at least one other polypeptide may be, for example, a polypeptide that enhances the stability of the polypeptide of the present invention, or a polypeptide that aids in the purification or detection of the fusion protein.

[0055] In yet another aspect, the present invention provides an isolated and / or exogenous polynucleotide comprising nucleotides having a sequence as provided in SEQ ID NO:2 or SEQ ID NO:3, a sequence at least 40% identical to either or both of SEQ ID NO:2 and SEQ ID NO:3, or a sequence that hybridizes to either or both of SEQ ID NO:2 and SEQ ID NO:3.

[0056] In another aspect, the present invention provides a chimeric vector comprising the polynucleotide of the present invention.

[0057] Preferably, the polynucleotide is operably linked to a promoter.

[0058] In yet another aspect, the present invention provides a recombinant cell comprising the exogenous polynucleotide of the present invention and / or the vector of the present invention.

[0059] The cell can be any cell type such as, but not limited to, a plant cell, a bacterial cell, an animal cell, or a yeast cell.

[0060] Preferably, the cell is a plant cell. More preferably, the plant cell is a cereal plant cell. Even more preferably, the cereal plant cell is a wheat cell. In another embodiment, the plant cell is a grape cell.

[0061] In yet another aspect, the present invention provides a method for producing the polypeptide of the present invention, which comprises expressing the polynucleotide of the present invention in a cell expression system or a cell-free expression system.

[0062] Preferably, the method further comprises isolating the polypeptide.

[0063] In another aspect, the present invention provides a method for producing the cell of the present invention, comprising the step of introducing the polynucleotide of the present invention or the vector of the present invention into the cell.

[0064] Preferably, the cell is a plant cell.

[0065] In another aspect, the present invention provides a method for producing the transgenic plant of the present invention, the method comprising the steps of:

[0066] i) introducing the polynucleotide of the present invention and / or the vector of the present invention into cells of a plant,

[0067] ii) regenerating a transgenic plant from the cell, and

[0068] iii) optionally harvesting seeds from the plant, and / or

[0069] iv) optionally producing one or more progeny plants from the transgenic plant, thereby producing a transgenic plant.

[0070] In another aspect, the present invention provides a method for producing a plant having integrated into its genome a polynucleotide encoding a polypeptide of the present invention, the method comprising the steps of

[0071] i) crossing two parental plants, wherein at least one of the plants comprises a polynucleotide encoding a polypeptide,

[0072] ii) screening one or more progeny plants from the cross for the presence or absence of the polynucleotide, and

[0073] iii) selecting progeny plants comprising the polynucleotide, thereby producing plants.

[0074] In one embodiment, the polypeptide comprises an amino acid having a sequence as provided in SEQ ID NO: 1, or an amino acid sequence at least 40% identical to SEQ ID NO: 1, or a biologically active fragment thereof, and wherein when expressed in a plant, the polypeptide confers resistance to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, preferably conferring resistance to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew.

[0075] In one embodiment, at least one of the parent plants is a tetraploid or hexaploid wheat plant. In another embodiment, the parent plant is a grapevine.

[0076] In another embodiment, step ii) comprises analyzing the polynucleotides in a sample containing DNA from a plant.

[0077] In yet another embodiment, step iii) comprises

[0078] i) selecting progeny plants that are homozygous for the polynucleotide, and / or

[0079] ii) analyzing the resistance of the plant or one or more progeny plants thereof to one or more biotrophic fungal pathogens, preferably rust, mildew or both rust and mildew, more preferably analyzing resistance to one or more or all of leaf rust, stripe rust, stem rust and powdery mildew.

[0080] In another embodiment, the method further comprises

[0081] iv) backcrossing the progeny of the hybrid of step i) with a plant having the same genotype as the first parent plant lacking the polynucleotide encoding the polypeptide a sufficient number of times to produce a plant having most of the genotype of the first parent but including the polynucleotide, and

[0082] iv) selecting progeny plants that have become resistant to one or more biotrophic fungal pathogens, preferably rust, mildew or both rust and mildew, more preferably to one or more or all of leaf rust, stripe rust, stem rust and powdery mildew.

[0083] In one embodiment, the method further comprises the step of analyzing the plant for at least one additional genetic marker.

[0084] Also provided is a plant produced using the method of the present invention.

[0085] In another aspect, the present invention provides use of the polynucleotide of the present invention or the vector of the present invention for producing recombinant cells and / or transgenic plants.

[0086] In one embodiment, the transgenic plant has enhanced resistance to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, more preferably to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, compared to an isogenic plant lacking the exogenous polynucleotide and / or vector.

[0087] In another aspect, the present invention provides a method for identifying a plant comprising a polynucleotide encoding a polypeptide of the present invention, the method comprising the steps of:

[0088] i) obtaining a nucleic acid sample from a plant, and

[0089] ii) Screening the sample for the presence or absence of the polynucleotide.

[0090] In one embodiment, the presence of the polynucleotide indicates that the plant has enhanced resistance to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, more preferably enhanced resistance to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, compared to an isogenic plant lacking the exogenous polynucleotide.

[0091] In one embodiment, the genomic region encompassing the polynucleotide is amplified and the amplified product is sequenced to determine whether it encodes a polypeptide. Primers for amplification and for sequencing can be easily designed by a skilled artisan.

[0092] In yet another embodiment, the method identifies the transgenic plants of the invention.

[0093] In embodiments, the method further comprises producing a plant from the seed prior to step i).

[0094] Also provided is a plant part of the plant of the present invention.

[0095] In one embodiment, the plant part is a seed comprising an exogenous polynucleotide encoding a polypeptide of the present invention.

[0096] In yet another aspect, the present invention provides a method for producing a plant part, the method comprising,

[0097] a) growing the plants of the present invention, and

[0098] b) Harvesting the plant parts.

[0099] In another aspect, the present invention provides a method for producing flour, wholemeal, starch or other product obtained from seeds, the method comprising;

[0100] a) obtaining the seeds of the present invention, and

[0101] b) Extraction of flour, wholemeal, starch or other products.

[0102] In a further aspect, the present invention provides a product produced from the plant of the present invention and / or the plant part of the present invention.

[0103] In one embodiment, the portion is a seed.

[0104] In one embodiment, the product is a food product or a beverage product. Examples include, but are not limited to;

[0105] i) a food product selected from the group consisting of flour, starch, leavened or unleavened bread, pasta, noodles, animal feed, breakfast cereals, snack foods, cakes, ale, beer, pastries and foods containing flour-based sauces, or

[0106] ii) Beer or malt liquor beverage products.

[0107] In an alternative embodiment, the product is a non-food product. Examples include, but are not limited to, films, coatings, adhesives, building materials, and packaging materials.

[0108] In a further aspect, the present invention provides a method of preparing a food product of the present invention, the method comprising mixing the seed or flour, wholemeal or starch therefrom with another food ingredient.

[0109] In another aspect, the present invention provides a method of preparing malt liquor, comprising the step of germinating the seeds of the present invention.

[0110] There is also provided the use of a plant of the invention or a part thereof as animal feed or in the production of feed for animal consumption or food for human consumption.

[0111] In yet another aspect, the present invention provides a composition comprising one or more polypeptides of the present invention, polynucleotides of the present invention, vectors of the present invention, or recombinant cells of the present invention and one or more acceptable carriers.

[0112] In yet another aspect, the present invention provides a method for identifying a compound that binds to a polypeptide comprising amino acids having the sequence provided in SEQ ID NO: 1, or an amino acid sequence at least 40% identical to SEQ ID NO: 1, and biologically active fragments thereof, the method comprising:

[0113] i) contacting the polypeptide with a candidate compound, and

[0114] ii) determining whether the compound binds to the polypeptide.

[0115] In one embodiment, the polypeptide is embedded in a cell membrane, preferably a plant cell membrane.

[0116] In yet another aspect, the present invention provides a method for identifying a compound that is transported across a cell membrane by a polypeptide comprising amino acids having a sequence as provided in SEQ ID NO: 1 or SEQ ID NO: 4, or an amino acid sequence at least 40% identical to one or both of SEQ ID NO: 1 or SEQ ID NO: 4, or a biologically active fragment thereof, the method comprising:

[0117] i) contacting a polypeptide embedded in a cell membrane, preferably a plant cell membrane, with a candidate compound,

[0118] ii) determining whether the compound is transported from one side of the membrane to the other by the polypeptide.

[0119] Unless specifically stated otherwise, any embodiment herein should be applied mutatis mutandis to any other embodiment.

[0120] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended to be illustrative only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention, as described herein.

[0121] In this specification, unless specifically stated otherwise or the context dictates otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter should be taken to encompass both one and multiple (i.e., one or more) of these steps, compositions of matter, groups of steps or groups of compositions of matter.

[0122] The invention is hereinafter described by way of the following non-limiting examples and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 - Comparative genomics and mutation analysis.

[0124] Figure 2 – The deleted Hsp70 gene is completely linked to Lr67.

[0125] Figure 3 – Includes comparative genomics and mutational analysis of SUT and PIP.

[0126] Figure 4 – Nucleotide changes found in the Lr67 variant.

[0127] Figure 5 - Amino acid sequence of the SUT polypeptide encoded by the wheat Lr67 (resistant) allele (514 amino acids, SEQ ID NO: 1). The arginine at position 144 and the leucine at position 387 of the predicted fourth transmembrane domain distinguish between resistant and susceptible Lr67 polypeptides.

[0128] Figure 6 Nucleotide sequence of the cDNA corresponding to the Lr67 resistance allele (SEQ ID NO: 3). Two SNPs distinguishing + / - Lr67 are located at positions 514 and 1243; these result in amino acid substitutions in the encoded polypeptide. Translation start codons are at positions 85-87, and translation stop codons are at positions 1627-1629.

[0129] Figure 7 - Amino acid sequence of Lr67 susceptibility polypeptide (SUT) (SEQ ID NO: 4).

[0130] Figure 8 - Nucleotide sequence of DNA corresponding to the Lr67 susceptibility allele SUT (SEQ ID NO: 6).

[0131] Figure 9 The amino acid sequence of the wheat Lr67 (resistance) polypeptide (SEQ ID NO: 1) is aligned with the homologous Arabidopsis thaliana polypeptide (Arath; from GenBank Accession No. NP_198006, 526 amino acids) (SEQ ID NO: 7). Asterisks indicate identical amino acid residues at that position, while "+" indicates similar amino acids at that position.

[0132] Figure 10- Alignment of the amino acid sequence of the wheat Lr67 (resistance) polypeptide (SEQ ID NO: 1) with the homologous rice (Oryza sativa) polypeptide (GenBank Accession No. AAQ24871, 515 amino acids) (SEQ ID NO: 8). Asterisks indicate identical amino acid residues at that position, and "+" indicates similar amino acids at that position.

[0133] Figure 11 – Glucose uptake in yeast cells expressing Lr67(resistant) and Lr67(susceptible) proteins.

[0134] Figure 12 – Glucose uptake kinetics in yeast expressing Lr67 (susceptible).

[0135] Figure 13 –Effects of different amino acids on glucose transport through Lr67 protein.

[0136] Figure 14 The nucleotide sequence of the genomic fragment corresponding to the protein coding region of the Lr67 (susceptibility) gene. This sequence begins with the translation start codon ATG and ends with the translation stop codon TGA. Within the protein coding region, the two introns are nucleotides 137-876 (intron 1, 740 nt) and 1197-3154 (intron 2, 1958 nt).

[0137] Description of Sequence Listing

[0138] SEQ ID NO: 1—Wheat Lr67 (Resistance) protein.

[0139] SEQ ID NO: 2—Open reading frame encoding wheat Lr67 (resistance) protein.

[0140] SEQ ID NO: 3—cDNA encoding wheat Lr67 (resistance) protein.

[0141] SEQ ID NO:4—Wheat Lr67 (susceptible) protein

[0142] SEQ ID NO: 5—Open reading frame encoding wheat Lr67 (susceptible) protein.

[0143] SEQ ID NO:6—cDNA encoding wheat Lr67 (susceptible) protein.

[0144] SEQ ID NO:7—Arabidopsis thaliana Lr67 protein.

[0145] SEQ ID NO:8—Rice Lr67 protein.

[0146] SEQ ID NO:9—Grapevine (Vitis vinifera) Lr67 protein.

[0147] SEQ ID NO: 10—Gene encoding wheat Lr67 (susceptible) protein. DETAILED DESCRIPTION

[0148] General Techniques and Definitions

[0149] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (eg, in the fields of cell culture, molecular genetics, plant molecular biology, protein chemistry, and biochemistry).

[0150] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures well known to those skilled in the art. Such techniques are described and illustrated in the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988) and JE Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updated material up to the present).

[0151] The term "and / or," for example, "X and / or Y" should be understood to mean either "X and Y" or "X or Y" and should be used to provide clear support for both meanings or one of the meanings.

[0152] In this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0153] peptides

[0154] The present invention relates to polypeptides that, when expressed in a plant, confer resistance to one or more biotrophic fungal pathogens, preferably to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew. The present invention also relates to polypeptides that, when expressed intracellularly, are less efficient at transporting glucose across the cell membrane than a polypeptide comprising the amino acid sequence provided in SEQ ID NO:4. Furthermore, the present invention also relates to polypeptides that, when expressed intracellularly, function as sugar transporters. In a preferred embodiment, the polypeptide is encoded by an allele or variant of the Lr67 gene that confers resistance to one or more biotrophic fungal pathogens in a plant. Examples of such polypeptides include, but are not limited to, those comprising the amino acid sequence provided in SEQ ID NO:1. The polypeptides of the present invention confer enhanced resistance to one or more biotrophic fungal pathogens compared to an isogenic plant lacking the polynucleotide encoding the polypeptide.

[0155] As used herein, the term "Lr67" refers to a family of proteins that share a high degree of primary amino acid sequence identity, for example, at least 40%, at least 80%, at least 90%, or at least 95% identity to one or more of the amino acid sequences provided in SEQ ID NOs: 1, 4, or 7 to 9, preferably SEQ ID NO: 1. The present invention has demonstrated that certain variants of the Lr67 protein family, when expressed in plants, confer resistance to one or more biotrophic fungal pathogens, preferably to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew. Examples of such variants include the amino acid sequence provided in SEQ ID NO: 1. Thus, variants that confer resistance are referred to herein as Lr67 (resistant) polypeptides, while those that do not (such as those including the amino acid sequence provided in SEQ ID NO: 4) are referred to herein as Lr67 (susceptible) polypeptides. In a preferred embodiment, the Lr67 (resistance) protein does not comprise a glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1, and preferably the polypeptide comprises an amino acid other than glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1. The amino acid at position 144 is preferably a charged amino acid such as arginine, lysine, or histidine.

[0156] As used herein, "resistance" is a relative term because the presence of a polypeptide of the invention (i) reduces disease symptoms in plants comprising a gene conferring resistance (R (resistance) gene) relative to plants lacking the R gene, and / or (ii) reduces pathogen propagation or spread in a plant or in a population of plants comprising the R gene. Resistance, as used herein, is relative to a plant's "susceptibility" response to the same pathogen. Typically, the presence of an R gene improves at least one production characteristic, such as grain yield, of a plant comprising the R gene when infected by the pathogen, compared to an isogenic plant infected by the pathogen but lacking the R gene. Isogenic plants may have a certain degree of resistance to the pathogen, or may be classified as susceptible. Therefore, the terms "resistance" and "enhanced resistance" are generally used interchangeably herein. In addition, the polypeptides of the invention may not necessarily confer complete pathogen resistance, for example when certain symptoms still occur in a plant or in a population of plants or when some pathogen propagation is infected but in reduced numbers. Resistance may only occur at certain stages of plant growth, for example, in mature plants (fully grown in size) and less mature, or not mature at all, in the seedling stage, or at all stages of plant growth by using a transgenic strategy to express the Lr67 polypeptide in the plant to provide resistance to the plants of the present invention during their growth and development. Enhanced resistance can be determined by methods known in the art, such as analyzing the amount of plant pathogens and / or analyzing the amount of plant growth or plant loss or disease symptoms in the presence of pathogens, and comparing one or more of these parameters with isogenic plants that lack the exogenous gene encoding the polypeptide of the present invention.

[0157] As used herein, " sugar transporter " is a membrane-bound protein that promotes sugar transmembrane movement, for example, from the extracellular space into the cell, or in the opposite direction from the intracellular space to the extracellular space, or through the membrane of the intracellular subcellular organelle. This promotion may be active, using an energy source such as from an ion gradient through the membrane, or passive. For Lr67 (susceptible) protein, sugar may be glucose. In one embodiment, sugar is a monosaccharide, preferably a hexose monosaccharide or a pentose polysaccharide. In one embodiment, sugar may be modified, such as a sugar alcohol or a phosphorylated sugar.

[0158] As used herein, the phrase "is less efficient at transporting glucose across a cell membrane than a polypeptide comprising amino acids having the sequence provided in SEQ ID NO: 4" means that a polypeptide of the invention has less than 50%, or less than 25%, or less than 10% of the ability of a polypeptide comprising amino acids having the sequence provided in SEQ ID NO: 4 to transport glucose into a cell, such as a yeast cell or a plant cell. This can be readily determined as described herein (see, e.g., Figure 11 and relevant experimental details).

[0159] By "substantially purified polypeptide" or "purified polypeptide," we mean a polypeptide that has been separated from lipids, nucleotides, other peptides, and other contaminating molecules with which it is typically associated in its native state. Preferably, a substantially purified polypeptide is at least 90% free from other components with which it is associated in its native state. In one embodiment, a polypeptide of the invention has an amino acid sequence other than that of the naturally occurring Lr67 polypeptide, i.e., it is an amino acid sequence variant.

[0160] Transgenic plants and host cells of the present invention may contain exogenous polynucleotides encoding polypeptides of the present invention. In these cases, plants and cells produce recombinant polypeptides. In the context of polypeptides, the term "recombinant" refers to a polypeptide encoded by an exogenous polynucleotide when produced by a cell, which has been introduced into the cell or progenitor cell by recombinant DNA or RNA technology (e.g., conversion). Typically, the cell contains a non-endogenous gene that causes an altered amount of the polypeptide to be produced. In one embodiment, a "recombinant polypeptide" is a polypeptide produced by expression of an exogenous (recombinant) polynucleotide in a plant cell.

[0161] The terms "polypeptide" and "protein" are often used interchangeably.

[0162] The % identity of the polypeptides is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) using a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 400 amino acids long, and the GAP analysis aligns the two sequences over a region of at least 400 amino acids. More preferably, the query sequence is at least 500 amino acids long, and the GAP analysis aligns the two sequences over a region of at least 500 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their full length, which for the Lr67 polypeptide is approximately 514 amino acid residues.

[0163] As used herein, a "biologically active fragment" is a portion of a polypeptide of the present invention that retains a defined activity of the full-length polypeptide, such as one or both of the following: i) when expressed in a plant, such as wheat, it confers (enhanced) activity against one or more biotrophic fungal pathogens, preferably one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, and (ii) when expressed in a cell, the polypeptide is effective as a sugar transporter, preferably less effective than a polypeptide comprising amino acids having the sequence provided in SEQ ID NO: 4 at transporting glucose across the cell membrane. Biologically active fragments can be of any size as long as they retain the defined activity, but are preferably at least 400 or at least 500 amino acid residues in length. Preferably, the biologically active fragment retains at least 50%, at least 75%, or at least 90% of the activity of the full-length protein. In one embodiment, the biologically active fragment comprises 12 transmembrane domains.

[0164] With respect to the defined polypeptides, it will be appreciated that higher % identity figures than those provided above will encompass preferred embodiments. Thus, where applicable, in accordance with the lowest value of the % identity figures, preferably, the polypeptide comprises an amino acid sequence that is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, even more preferably at least 99.9% identical to the relevant named SEQ ID NO.

[0165] In one embodiment, the polypeptide of the present invention is not a polypeptide consisting of the amino acid sequence provided by SEQ ID NO: 1 or SEQ ID NO: 4 that occurs in nature.

[0166] As used herein, the phrase "at a position corresponding to an amino acid number" or variants thereof refers to the relative position of an amino acid compared to the surrounding amino acids. In this regard, in some embodiments, a polypeptide of the present invention may have a deletion or substitution mutation that alters the relative positioning of an amino acid when compared to, for example, SEQ ID NO: 1. For example, Figure 9 As shown, amino acid number 178 of wheat Lr67(resistant) corresponds to amino acid number 179 of the homologous Arabidopsis Lr67 protein.

[0167] Amino acid sequence mutants of the polypeptides of the present invention can be prepared by introducing appropriate nucleotide variations into the nucleic acids of the present invention, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence. A combination of deletions, insertions, and substitutions may be present in the final construct, provided that the final peptide product possesses the desired characteristics. Preferably, amino acid sequence mutants have only one, two, three, four, or fewer than ten amino acid changes relative to the reference wild-type polypeptide.

[0168] Mutated (altered) polypeptides can be prepared using any technique known in the art, for example, using directed or rational design strategies (see below). Products derived from mutated / altered DNA can be readily screened using the techniques described herein to determine whether they have one or more of the following characteristics: i) when expressed in a plant, such as wheat, they confer (enhanced) resistance to one or more biotrophic fungal pathogens, preferably to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, and (ii) when expressed in a cell, the encoded polypeptide is less efficient than a polypeptide comprising amino acids having the sequence provided in SEQ ID NO: 4 at transporting glucose across the cell membrane, and (iii) when expressed in a cell, the polypeptide acts as an efficient sugar transporter. For example, with respect to i), the method may comprise producing a transgenic plant expressing the mutated / altered DNA and determining the effect of the pathogen on plant growth.

[0169] In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the characteristic to be modified. Mutation sites can be modified individually or sequentially, for example, by (1) first replacing with conservative amino acid selections and then with more radical selections depending on the desired outcome, (2) deleting the target residue, or (3) inserting other residues at adjacently located sites.

[0170] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues, and usually about 1 to 5 contiguous residues.

[0171] Substitution mutants remove at least one amino acid residue in a polypeptide and insert a different residue in its place. It is desirable that some activity is retained, preferably without substitution or with conservative substitutions at amino acid positions that are highly conserved in a family of related proteins. Examples of conservative substitutions are shown in Table 1 under the heading "Exemplary Substitutions."

[0172] In a preferred embodiment, the mutant / variant polypeptide has one, two, three, or four conservative amino acid changes compared to the naturally occurring polypeptide. Table 1 provides details of the conservative amino acid changes. In a preferred embodiment, the changes are not within one or more motifs that are highly conserved between the different polypeptides provided herein, and / or are not within the 12 transmembrane helices of the Lr67 polypeptide. As is known to those skilled in the art, it is reasonable to predict that such minor changes will not alter the activity of the polypeptide when expressed in recombinant cells.

[0173] The primary amino acid sequence of the polypeptide of the present invention can be used to design variants / mutants based on comparison with closely related sugar transporter polypeptides (e.g., Figure 9 and Figure 10 As will be appreciated by the skilled artisan, residues that are highly conserved within closely related proteins are less likely to be altered, particularly with non-conservative substitutions and retention of activity (see above), than residues that are less conserved.

[0174] Also included within the scope of the present invention are polypeptides of the present invention that are differentially modified during or after synthesis, such as by biotinylation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolysis, attachment to antibody molecules or other cellular ligands, etc. The polypeptides may undergo post-translational modifications in cells, such as by phosphorylation, which modulate their activity. These modifications can be used to increase the stability and / or biological activity of the polypeptides of the present invention.

[0175] Table 1. Exemplary substitutions.

[0176]

[0177] Directed evolution

[0178] In directed evolution, random mutagenesis is applied to a protein, and a selection scheme is used to select mutants with the desired property, for example, increased activity. The next round of mutagenesis and selection is then applied. A typical directed evolution strategy involves three steps:

[0179] 1) Diversification: Genes encoding proteins of interest are mutated and / or randomly recombined to create large libraries of gene variants. Variant gene libraries can be prepared by error-prone PCR (see, e.g., Leung, 1989; Cadwell and Joyce, 1992), from a pool of DNase I-digested fragments prepared from the parental template (Stemmer, 1994a; Stemmer, 1994b; Crameri et al., 1998; Coco et al., 2001), from denatured oligonucleotides (Ness et al., 2002, Coco, 2002), or from a mixture of both, or even from a pool of DNase I-digested fragments prepared from the parental template (Zhao et al., 2001). et al., 1998; Eggert et al., 2005; Jézéquek et al., 2008), typically assembled by PCR. Libraries can also be created in vivo or in vitro from recombined parental sequences by homologous or nonhomologous recombination (Ostermeier et al., 1999; Volkov et al., 1999; Sieber et al., 2001). Transgenic gene libraries can also be created by subcloning the gene of interest into a suitable vector, transforming the vector into a "mutator" system such as E. coli XL-1 red (Stratagene), and propagating the transformed bacteria for an appropriate number of generations. Transgenic gene libraries can also be created by DNA shuffling (i.e., homologous recombination of a selected mutant gene pool by random fragmentation and reassembly in vitro) of the gene of interest, as extensively described by Harayama (1998).

[0180] 2) Selection: Screening or selection is used to test the library for the presence of mutants (variants) possessing desired properties. Screening allows for the manual identification and isolation of high-performing mutants, while selection automatically eliminates all non-functional mutants. Screening may involve screening for the presence of known conserved amino acid motifs. Alternatively, or in addition, screening may involve expressing the mutant polynucleotide in a host organism or a portion thereof and measuring activity levels.

[0181] 3) Amplification: Variants identified in a selection or screen are replicated many times, allowing researchers to sequence their DNA to understand what mutations have occurred.

[0182] These three steps together are called a "round" of directed evolution. Most experiments will require more than one round. In these experiments, the "winners" from the previous round are diversified in the next round to create a new library. At the end of the experiment, all evolved protein or polynucleotide mutants are characterized using biochemical methods.

[0183] Reasonable design

[0184] On the basis of known information of protein structure and folding, protein is rationally designed. This can be done by design from scratch (de novo design) or by redesign based on natural scaffold (see, for example, Hellinga, 1997; and Lu and Berry, Protein Structure Design and Engineering, Handbook of Proteins 2, 1153-1157 (2007)). Protein design generally includes identifying the sequence that folds into a given or target structure, which can be completed using computer models. Computer protein design algorithms search for sequence-conformation space of sequences with low energy when folded into the target structure. Computer protein design algorithms use protein thermodynamic models to evaluate how mutations affect the structure and function of proteins. The functions of these energies generally include molecular mechanics, statistics (i.e., knowledge-based), and a combination of other empirical aspects. Suitable available software includes IPRO (iterative protein redesign and optimization), EGAD (genetic algorithm for the design of proteins), Rosetta Design, Sharpen and Abalone.

[0185] Polynucleotides and genes

[0186] The present invention relates to various polynucleotides. As used herein, "polynucleotide" or "nucleic acid" or "nucleic acid molecule" refers to a polymer of nucleotides, which may be DNA or RNA or a combination thereof, and includes genomic DNA, mRNA, cRNA and cDNA. Non-preferred polynucleotides include tRNA, siRNA, shRNA and hpRNA. It may be DNA or RNA of cellular origin, genomic origin or synthetic origin, for example, formed by an automated synthesizer, which may be bound to carbohydrates, lipids, proteins or other substances, labeled with fluorescein or other groups, or attached to a solid support to perform the characteristic activities defined herein, or include one or more naturally occurring, modified nucleotides, all of which are well known to those skilled in the art. The polymer may be single-stranded, double-stranded or partially double-stranded in nature. Base pairing as used herein refers to standard base pairing between nucleotides, including G:U base pairing. "Complementary" means that two polynucleotides can base pair (hybridize) along a portion of their length or the full length of one or both of them. "Hybridized polynucleotide" means that a polynucleotide and its complement are indeed base paired. The terms "polynucleotide" and "nucleic acid" as used herein are used interchangeably. Preferred polynucleotides of the present invention encode polypeptides of the present invention.

[0187] By "isolated polynucleotide," we mean a polynucleotide that has been separated from polynucleotide sequences with which it is typically associated or linked in its natural state, if the polynucleotide is found in nature. Preferably, an isolated polynucleotide that is found in nature is at least 90% free from other components with which it is associated in its natural state. Preferred polynucleotides do not occur in nature, for example, by covalently linking two shorter polynucleotide sequences in a manner not found in nature (chimeric polynucleotides).

[0188] The present invention relates to the modification of gene activity and the construction and use of chimeric genes. As used herein, the term "gene" includes any deoxyribonucleotide sequence comprising a protein coding region or a region that is transcribed but not translated in a cell, as well as associated non-coding regions and regulatory regions. These associated regions are typically located adjacent to the 5' and 3' ends of the coding region or the transcription region at a distance of approximately 2 kb on either side. In this regard, a gene may include natural control signals such as promoters, enhancers, terminations and / or polyadenylation signals, or heterologous control signals associated with a given gene, in which case the gene is referred to as a "chimeric gene." The sequence at the 5' end of the coding region and the sequence present on the mRNA are referred to as 5' non-translated sequences. The sequence at the 3' end or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" includes genes in both cDNA and genomic forms.

[0189] As used herein, "Lr67 gene" refers to a nucleotide sequence homologous to an isolated Lr67 cDNA (as provided in SEQ ID NO: 3 and SEQ ID NO: 6). As described herein, certain alleles and variants of the Lr67 gene family encode proteins that confer resistance to one or more biotrophic fungal pathogens, preferentially conferring resistance to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew (see, e.g., SEQ ID NO: 3). Lr67 genes include naturally occurring alleles or variants found in cereals such as wheat, as well as artificially produced variants.

[0190] A genomic form or clone of a gene comprising a transcribed region may be interrupted by non-coding sequences called "introns" or "insertion regions" or "insertion sequences" which may be homologous or heterologous to the "exons" of the gene. As used herein, an "intron" is a gene segment that is transcribed as part of the primary RNA transcript but is not present in the mature mRNA molecule. Introns are removed or "spliced ​​out" from the nuclear or primary transcript; therefore, introns are not present in the messenger RNA (mRNA). Introns may contain regulatory elements such as enhancers. As described herein, the wheat Lr67 gene (resistant and susceptible alleles) contains two introns in its protein coding region. As used herein, an "exon" refers to a DNA region that corresponds to a mature mRNA or RNA sequence present in a mature mRNA when the RNA molecule is not translated. mRNA is used during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term "gene" includes synthetic or fusion molecules that encode all or part of a protein of the invention described herein, as well as complementary nucleotide sequences of any of the above. The gene can be introduced into a suitable vector for extrachromosomal maintenance in the cell or, preferably, for integration into the host genome.

[0191] As used herein, a "chimeric gene" refers to any gene comprising covalently linked sequences not found in nature. Typically, a chimeric gene comprises regulatory and transcribed sequences or protein-coding sequences that are not found together in nature. Thus, a chimeric gene may comprise regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source, but arranged in a manner different from that found in nature. In one embodiment, the protein-coding region of the Lr67 gene is operably linked to a promoter or polyadenylation / termination region heterologous to the Lr67 gene, thereby forming a chimeric gene. As used herein, the term "endogenous" is used to refer to a substance that is normally present or produced in an unmodified plant at the same developmental stage as the plant being studied. An "endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, a "recombinant nucleic acid molecule," "recombinant polynucleotide," or variations thereof, refers to a nucleic acid molecule that has been constructed or modified using recombinant DNA technology. The terms "exogenous polynucleotide," "exogenous polynucleotide," or "heterologous polynucleotide," etc., refer to any nucleic acid that has been introduced into the genome of a cell through experimental manipulation.

[0192] Foreign or exogenous genes may be genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The term "genetically modified" includes the introduction of genes into cells by transformation or transduction, the mutation of genes in cells, or the alteration or modulation of gene regulation in cells or organisms or their progeny after such actions have been performed.

[0193] In addition, the term "exogenous" in the context of a polynucleotide (nucleic acid) refers to a polynucleotide when present in a cell that does not naturally contain the polynucleotide. The cell may be a cell that contains a non-endogenous polynucleotide that causes an altered production of the encoded polypeptide, such as an exogenous polypeptide that increases expression of an endogenous polypeptide, or a cell that is not naturally capable of producing the polypeptide. Increased production of the polypeptide of the present invention is also referred to herein as "overexpression." The exogenous polynucleotides of the present invention include polynucleotides that have not been separated from other components of the transgenic (recombinant) cell expression system or cell-free expression system in which they are located, as well as polynucleotides produced in these cells or cell-free systems that have been subsequently purified and separated from at least some other components. The exogenous polynucleotide (nucleic acid) can be a continuous stretch of nucleotides that occurs in nature, or a continuous stretch of two or more nucleotides from different sources (naturally occurring and / or synthetic) that are linked to form a single polynucleotide. Typically, this chimeric polynucleotide comprises at least one open reading frame encoding a polypeptide of the present invention, operably linked to a promoter suitable for driving transcription of the open reading frame in the cell of interest.

[0194] The % identity of polynucleotides is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 1,200 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 1,200 nucleotides. More preferably, the query sequence is at least 1,500 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 1,500 nucleotides. Even more preferably, the GAP analysis aligns the entire length of the two sequences.

[0195] With respect to the defined polynucleotides, it will be appreciated that higher % identity figures than those provided above will encompass preferred embodiments. Thus, where applicable, in terms of the lowest value of the % identity figures, preferably the polynucleotide comprises a polynucleotide sequence that is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, even more preferably at least 99.9% identical to the relevant named SEQ ID NO.

[0196] In yet another embodiment, the present invention relates to polynucleotides that are substantially identical to the polynucleotides specifically described herein. As used herein, the term "substantially identical" with respect to polynucleotides refers to the replacement of one or a few (e.g., 2, 3, or 4) nucleotides while retaining at least one activity of the native protein encoded by the polynucleotide. In addition, the term includes the addition or deletion of one or a few (e.g., 2, 3, or 4) amino acid nucleotides that results in an increase or decrease in the size of the encoded native protein while retaining at least one activity of the native protein encoded by the polynucleotide.

[0197] The present invention also relates to the use of oligonucleotides, for example, in methods for screening polynucleotides of the present invention, or in methods for encoding polypeptides of the present invention. As used herein, an "oligonucleotide" is a polynucleotide of up to 50 nucleotides in length. The minimum size of an oligonucleotide is the size required to form a stable hybrid between the oligonucleotide and the complementary sequence of a nucleic acid molecule of the present invention. They can be RNA, DNA, or a combination or derivative of any of these. Typically, oligonucleotides are relatively short, single-stranded molecules of 10 to 30 nucleotides, typically 15-25 nucleotides in length. When used as a probe or as a primer in an amplification reaction, the minimum size of an oligonucleotide is the size required to form a stable hybrid between the oligonucleotide and the complementary sequence of a target nucleic acid molecule. Preferably, the oligonucleotide is at least 15 nucleotides in length, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, and even more preferably at least 25 nucleotides. Oligonucleotides of the present invention used as probes are typically conjugated to a label, such as a radioisotope, an enzyme, biotin, a fluorescent molecule, or a chemiluminescent molecule.

[0198] The present invention includes oligonucleotides that can be used, for example, as probes for identifying nucleic acid molecules or as primers for producing nucleic acid molecules. Probes and / or primers can be used to clone homologs of the polynucleotides of the present invention from other species. In addition, hybridization techniques known in the art can also be used to screen genomic or cDNA libraries for such homologs.

[0199] Polynucleotides and oligonucleotides of the present invention include those that hybridize under stringent conditions to one or more of the sequences provided in SEQ ID NO: 2, 3, 5, or 6. As used herein, stringent conditions are those that (1) use low ionic strength and high temperature for washing, e.g., 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% NaDodSO4 at 50°C; (2) use a denaturing agent such as formamide in hybridization, e.g., 50% (vol / vol) formamide and 0.1% bovine serum albumin, 0.1% polysucrose, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer, pH 6.5, and 750 mM NaCl, 75 mM sodium citrate; or (3) use 50% formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate ... Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate.

[0200] The polynucleotides of the present invention may have, compared to naturally occurring molecules, one or more mutations, i.e., deletions, insertions or substitutions of nucleotide residues. Mutants may be naturally occurring (i.e., isolated from natural sources) or synthetic (e.g., by site-directed mutagenesis of nucleic acids). Variants of the polynucleotides or oligonucleotides of the present invention include molecules of different sizes from the wheat genome close to the genome of the reference polynucleotides or oligonucleotides defined herein, and / or molecules that are capable of hybridizing therewith. For example, variants may include additional nucleotides (e.g., 1, 2, 3, 4 or more) or fewer nucleotides, as long as they still hybridize to the target region. In addition, some nucleotides may be replaced without affecting the ability of the oligonucleotide to hybridize to the target region. In addition, it is easy to design variants that closely hybridize to regions of the plant genome (e.g., within 50 nucleotides) at the locations where hybridization of the specific oligonucleotides defined herein occurs. In particular, this includes polynucleotides that encode the same polypeptide or amino acid sequence, but whose nucleotide sequence varies due to redundancy in the genetic code. The terms "polynucleotide variants" and "variants" also include naturally occurring allele variants.

[0201] Nucleic acid constructs

[0202] The present invention includes nucleic acid constructs comprising the polynucleotides of the present invention, as well as vectors and host cells containing these, methods of producing and using them, and uses thereof. The present invention refers to elements that are operably linked or connected. "Operably linked" or "operably connected" and the like refer to polynucleotide elements that are connected in a functionally related manner. Generally, operably linked nucleic acid sequences are connected continuously, continuously and in reading frame, and when necessary, two protein coding regions are joined. When RNA polymerase transcribes two coding sequences into a single RNA, the coding sequence is "operably linked to" another coding sequence, which, if translated, is then translated into a single polypeptide having amino acids derived from both coding sequences. A coding sequence does not need to be continuous with another sequence, as long as the expressed sequence can ultimately be processed to produce the desired protein.

[0203] As used herein, the terms "cis-acting sequence," "cis-acting element," or "cis-regulatory region," or "regulatory region," or similar terms shall refer to any nucleotide sequence that, when appropriately positioned and linked to an expressible genetic sequence, is capable of regulating the expression of at least a portion of the genetic sequence. Those skilled in the art will appreciate that cis-regulatory regions are capable of activating, silencing, enhancing, inhibiting, or altering the expression level and / or cell type specificity and / or developmental specificity of a gene sequence at the transcriptional or post-transcriptional level. In a preferred embodiment of the invention, the cis-acting sequence is an activator sequence that enhances or stimulates the expression of an expressible genetic sequence.

[0204] To "operably link" a promoter or enhancer element to a transcribable polynucleotide means to place the transcribable polynucleotide (e.g., a protein-coding polynucleotide or other transcript) under the regulation of the promoter, thereby controlling the transcription of that polynucleotide. In constructing a heterologous promoter / structural gene combination, it is generally preferred to place the promoter or a variant thereof at a distance from the transcription start site of the transcribable polynucleotide that is approximately the same distance as the reference promoter and protein-coding region in nature; that is, from the gene from which the promoter originates. As is known in the art, some variation within this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element (e.g., an operator, enhancer, etc.) relative to the transcribable polynucleotide to be placed under its control is defined by the positioning of the element in nature; that is, the gene from which it originates.

[0205] As used herein, "promoter" or "promoter sequence" refers to the region of a gene, generally upstream (5') of the RNA coding region, which controls the initiation and level of transcription in the cell of interest. "Promoters" include transcriptional regulatory sequences of classic genomic genes, such as TATA box and CCAAT box sequences, as well as other regulatory elements (i.e., upstream activating sequences, enhancers, and silencers) that alter gene expression in response to developmental and / or environmental stimuli or in a tissue-specific or cell-type-specific manner. Promoters are often, but not necessarily (e.g., certain PolIII promoters), located upstream of the structural gene whose expression they regulate. In addition, regulatory elements comprising promoters are often located within 2 kb of the transcription start site of a gene. Promoters may contain other specific regulatory elements, located further distal to the start site to further enhance expression in the cell, and / or to alter the timing or inducibility of expression of the structural gene to which they are operably linked.

[0206] "Constitutive promoter" refers to a promoter that directs expression of an operably linked transcribed sequence in many or all tissues of an organism, such as a plant. The term constitutive as used herein does not necessarily mean that a gene is expressed at the same level in all cell types, but rather that a gene is expressed in a wide range of cell types, although some variation in levels is often detectable. "Selective expression" as used herein refers to expression almost exclusively in a specific organ (such as a plant), such as the endosperm, embryo, leaves, fruit, tuber, or root. In a preferred embodiment, the promoter is selectively or preferentially expressed in the leaves and / or stems of a plant, preferably a cereal plant. Thus, selective expression may be in contrast to constitutive expression, which refers to expression in many or all tissues of a plant under most or all conditions experienced by the plant.

[0207] Selective expression may also result in the compartmentalization of gene expression products in specific plant tissues, organs, or developmental stages. Compartmentalization in specific subcellular locations (e.g., plastids, cytosol, vacuoles, or apoplasts) may be achieved by the structural inclusion of gene products with appropriate signals, such as signal polypeptides, for transport to the desired cellular compartments, or for semiautonomous organelles (plastids and mitochondria) by direct integration of transgenes with appropriate regulatory sequences into the genome of the organelle.

[0208] A "tissue-specific promoter" or "organ-specific promoter" is a promoter that is preferentially expressed in one tissue or organ relative to many other tissues or organs, preferably most (if not all) other tissues or organs in, for example, plants. Typically, a promoter is expressed at a 10-fold higher level in a specific tissue or organ than in other tissues or organs.

[0209] In one embodiment, the promoter is a stem-specific promoter, a leaf-specific promoter, or a promoter that directs gene expression in aerial parts of a plant (at least stems and leaves) (green tissue-specific promoter) such as the ribulose-1,5-bisphosphate carboxylase (RUBISCO) promoter.

[0210] Examples of stem-specific promoters include, but are not limited to, those described in US 5,625,136 and Bam et al. (2008).

[0211] The promoters contemplated by the present invention may be derived from the host plant to be transformed or may originate from alternative sources, in which the region in which they are located is functional. Other sources include Agrobacterium T-DNA genes, such as promoters for the biosynthesis of nopaline, octapine, mannopine or other opine promoters, tissue-specific promoters (e.g., US 5,459,252 and WO 91 / 13992); promoters from viruses (including host-specific viruses), or partially or fully synthetic promoters. A large number of functional promoters in monocotyledonous and dicotyledonous plants are well known in the art (e.g., Greve, 1983; Salomon et al., 1984; Garfinkel et al., 1983; Barker et al., 1983); including various promoters isolated from plants and viruses such as the cauliflower mosaic virus promoter (CaMV 35S, 19S). Non-limiting methods for assessing promoter activity are disclosed by Medberry et al. (1992, 1993), Sambrook et al. (1989, supra), and US Pat. No. 5,164,316.

[0212] In some embodiments, the promoter may be an inducible promoter or a developmental regulated promoter that can be introduced into the expression of polynucleotides at the appropriate developmental stage of plants. Other cis-acting sequences that may be adopted include transcription and / or translation enhancers. The enhancer region is well known to those skilled in the art and includes an ATG translation initiation codon and adjacent sequences. When included, the initiator codon may be in phase with the reading frame of the coding sequence relevant to an external source or exogenous polynucleotide to ensure translation of the entire sequence (if it will be translated). The translation initiation region may be provided by the source of the transcription initiation region, or provided by an external source or exogenous polynucleotide. The sequence may also originate from the source of the promoter selected for driving transcription, and is specifically modified to increase the translation of mRNA.

[0213] Nucleic acid constructs of the present invention may comprise a 3' non-translated sequence of approximately 50 to 1000 nucleotide base pairs, which includes a transcription termination sequence. The 3' non-translated sequence may contain a transcription termination signal, which may or may not include a polyadenylation signal and any other regulatory signals that may affect mRNA processing. The polyadenylation signal acts to add polyadenylic acid tracts to the 3' end of the mRNA precursor. Although variations are uncommon, polyadenylation signals are generally recognized by the presence of homology to the typical form of 5'AATAAA-3'. Transcription termination sequences that do not include a polyadenylation signal include terminators for Pol I or Pol III RNA polymerases that include a stretch of 4 or more thymidine deoxyribonucleotides. Examples of suitable 3' non-translated sequences are the 3' transcribed non-translated regions containing the polyadenylation signal of the octopine synthase (ocs) gene or the nopaline synthase (nos) gene from Agrobacterium tumefaciens (Bevan et al., 1983). Examples of suitable 3' non-translated sequences may also be derived from plant genes such as the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene, although other 3' elements known to those skilled in the art may also be employed.

[0214] Since the DNA sequence inserted at the start of the transcription initiation site and the beginning of the coding sequence, i.e., the untranslated 5' leader sequence (5'UTR), can affect gene expression if it is translated and transcribed, one can also use a specific leader sequence. Suitable leader sequences include those containing sequences selected to guide optimal expression of the foreign or exogenous DNA sequence. For example, with reference to the examples described by Joshi (1987), these leader sequences include preferred consensus sequences that can increase or maintain mRNA stability and prevent inappropriate initiation of translation.

[0215] carrier

[0216] The present invention includes the use of vectors for the manipulation and transfer of genetic structures. "Chimeric vectors" refer to nucleic acid molecules, preferably DNA molecules derived from plasmids, phages, or plant viruses, into which nucleic acid sequences may be inserted or cloned. Preferred vectors are double-stranded DNA and contain one or more unique restriction sites, and may be capable of autonomous replication within a defined host cell, including a target cell or tissue or progenitor cell or its tissue, or may be integrated into the genome of a defined host so that the cloned sequence can replicate. Therefore, a vector may be an autonomously replicating vector, i.e., a vector existing as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Or, when introduced into a cell, the vector may be integrated into the genome of the recipient cell and replicate together with the chromosome into which it has been integrated. The vector system may include a single vector or plasmid, two or more vectors or plasmids comprising the total DNA to be introduced into the host cell genome, or a transposon. Selecting a vector will generally depend on the compatibility of the vector with the cell to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene, a herbicide resistance gene or other gene for selecting suitable transformants. Examples of these genes are well known to those skilled in the art.

[0217] Nucleic acid construct of the present invention can be introduced into a vector, such as a plastid. Plasmid vectors generally include additional nucleic acid sequences that can provide convenient selection, amplification, and transformation of expression cassettes in eukaryotic and prokaryotic cells, such as pUC-origin vectors, pSK-origin vectors, pGEM-origin vectors, pSP-origin vectors, pBS-origin vectors, or binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include a replication origin that provides autonomous replication of the vector, a selectable marker gene, preferably encoding antibiotic or herbicide resistance, a unique multiple cloning site for inserting the nucleic acid sequence or gene encoded in the nucleic acid construct, and sequences that enhance transformation of eukaryotic and prokaryotic (particularly plant) cells.

[0218] " marker gene " refers to the gene that different phenotypes are passed on to the cell expressing this marker gene, and therefore this transformed cell can be distinguished from the cell that does not have this mark. Selectable marker gene gives characteristic, and it can select (as, weed killer herbicide, antibiotic, radiation, heat or other damage to non-transformed cell is handled) based on the resistance to selection agent. Screenable marker gene (or reporter gene) gives the characteristic that can be identified by observation or test, that is, by " screening " (as beta-glucuronidase, luciferase, GFP or other in non-transformed cell do not have active enzyme). Marker gene and paid close attention to nucleotide sequence need not be connected.

[0219] For the ease of identifying transformants, ideal nucleic acid constructs comprise selectable or screenable marker genes, such as, or in addition, exogenous or exogenous polynucleotides. The actual selection of mark is not strict, as long as it has function (that is, has selectivity) when associated with the plant cell of selection. It is not necessary to connect marker genes and exogenous or exogenous polynucleotides of interest, because co-transformation of unconnected genes is also an effective method in plant transformation, for example, as described in US4,399,216.

[0220] Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, erythromycin, chloramphenicol or tetracycline resistance, preferably kanamycin resistance. Exemplary selectable markers for selecting plant transformants include, but are not limited to, the hyg gene encoding resistance to hygromycin B; the neomycin phosphotransferase (nptII) gene that confers resistance to kanamycin, paromomycin, G418; the glutathione-S-transferase gene from rat liver that confers resistance to glutathione-derived herbicides, as described in EP 256223; the glutamine synthetase gene that, when overexpressed, confers resistance to glutamine synthetase inhibitors such as phosphinothricin, for example, the gene from Streptomyces viridans that confers resistance to the selective agent phosphinothricin, as described in WO 87 / 05327. viridochromogenes), for example, as described in EP 275 957, encoding a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) conferring resistance to N-phosphonomethylglycine, for example, as described by Hinchee et al. (1988), a bar gene conferring resistance to bialamylin, for example, as described in WO 91 / 02071; a gene from Klebsiella odorifera conferring resistance to bromoxynil ...; a gene from Klebsiella odorifera conferring resistance to bromoxynil, for example, as described in WO 91 / 02071; a gene from Klebsiella odorifera conferring resistance to bromoxynil, for example, as described in EP 275 957; a gene from Klebsiella odorifera conferring resistance to bromoxynil, for example, as described in EP 275 957; a gene from Klebsiella odorifera conferring resistance to bromoxynil, for example, ozaenae) nitrilase genes such as bxn (Stalker et al., 1988); dihydrofolate reductase (DHFR) genes that confer resistance to methotrexate (Thillet et al., 1988); mutant acetolactate synthase genes (ALS) that confer resistance to imidazolinones, sulfonylureas, or other ALS-inhibiting compounds (EP 154,204); mutant anthranilate synthase genes that confer resistance to 5-methyltryptophan; or dalapon dehalogenase genes that confer resistance to the herbicide.

[0221] Preferred screenable markers include, but are not limited to, the uidA gene encoding β-glucuronidase (GUS) (various chromogenic enzymes are known), the β-galactosidase gene encoding an enzyme with a known chromogenic substrate, the aequorin gene for calcium-sensitive bioluminescent detection (Prasher et al., 1985); the green fluorescent protein gene (Niedz et al., 1995) or its derivatives; the luciferase (luc) gene for bioluminescent detection (Ow et al., 1986), and others known in the art. As used herein, a "reporter gene" refers to a molecule that provides an analytically recognizable signal that facilitates identification of promoter activity through protein product, based on its chemical properties.

[0222] Preferably, the nucleic acid construct is stably incorporated into the genome of, for example, a plant. Thus, the nucleic acid comprises appropriate elements that allow the molecule to be incorporated into the genome, or the construct is placed in an appropriate vector that can be incorporated into the chromosome of a plant cell.

[0223] One embodiment of the present invention includes a recombinant vector comprising at least one polynucleotide molecule of the present invention inserted into any vector capable of delivering the nucleic acid molecule to a host cell. Such vectors contain heterologous nucleic acid sequences, i.e., nucleic acid sequences not naturally found adjacent to the nucleic acid molecule of the present invention and preferably derived from a species other than the species from which the nucleic acid molecule is derived. The vector can be RNA or DNA, eukaryotic or prokaryotic, and is typically viral or plasmidic.

[0224] Many vectors suitable for stable transfection of plant cells or for establishing transgenic plants have been described in, for example, Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Supplement, 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, a plant expression vector comprises, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. The plant expression vector also contains a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally regulated or developmentally regulated, or cell-specific or tissue-specific expression), a transcription initiation site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

[0225] Protein level of the present invention can be regulated by increasing the expression level of the nucleotide sequence of this protein in the coding plant cell or by reducing the expression level of the gene of this protein in the coding plant, thereby causing the pathogen resistance of modification.The expression level of gene can be regulated by changing the copy number of each cell, for example, by introducing a synthetic gene construct that comprises the transcription control element that comprises coding sequence and operability connection, that has function in cell.A plurality of transformants may be selected and screen out those transformants with favorable level and / or transgenic expression specificity that result from the influence of endogenous sequence around the transgenic integration site.The favorable level and form of transgenic expression are the level and form that cause pathogen resistance or other phenotypical substantive modification.Or, may screen the seed population of induced mutation or from the plant population of breeding program, have the pathogen resistance of change or the single line of other phenotypes relevant with pathogen resistance.

[0226] recombinant cells

[0227] Another embodiment of the present invention includes a recombinant cell or its daughter cell comprising a host cell transformed by one or more recombinant molecules of the present invention. The nucleic acid molecule is transformed into a cell and can be completed by any method by which the nucleic acid molecule is inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. The recombinant cell can remain a single cell or can grow into a tissue, organ, or multicellular organism. The converted nucleic acid molecule of the present invention can remain extrachromosomal or can be integrated into one or more sites in the chromosome of the transformed (i.e., recombinant) cell in a manner that retains their expression ability. Preferred host cells are plant cells, more preferably cereal plant cells, more preferably barley or wheat cells, even more preferably wheat cells.

[0228] genetically modified plants

[0229] As used herein, the noun term "plant" refers to the entire plant, and any part of the plant kingdom, but as an adjective, refers to any material present in, obtained from, derived from, or related to a plant, such as plant organs (e.g., leaves, stems, roots, flowers), individual cells (e.g., pollen), seeds, plant cells, etc. Plants and germinated seeds from which roots and shoots originate are also included in the meaning of "plant." As used herein, "plant part" refers to one or more plant tissues or organs obtained from a plant that contain the genomic DNA of the plant. Plant parts include vegetative structures (e.g., leaves, stems), roots, floral organs / structures, seeds (including embryos, cotyledons, and seed coats), plant tissues (e.g., vascular tissue, ground tissue, etc.), cells, and their progeny. As used herein, the term "plant cell" refers to a cell obtained from or in a plant, and includes protoplasts or other cells derived from a plant, gamete-producing cells, and cells that regenerate into a complete plant. Plant cells may be cells in culture. "Plant tissue" refers to differentiated tissue in plants or obtained from plants ("grafts") or undifferentiated tissue derived from immature or mature embryos, seeds, roots, seedlings, fruits, tubers, pollen, tumor tissue (crown galls), and various aggregates of plant cells in culture, such as callus. Exemplary plant tissues in or derived from seeds are cotyledons, embryos, and hypocotyls. Thus, the present invention includes plants and plant parts and products comprising these.

[0230] As used herein, the term "seed" refers to a "mature seed" of a plant that is either ready to be harvested or has been harvested from a plant, typically in a field for commercial harvesting, or a "developing seed" that is present in a plant after fertilization and prior to establishment of seed dormancy and harvest.

[0231] " transgenic plant " used in the present invention refers to the plant containing the nucleic acid construct not found in the wild-type plant of the same species, variant or cultivar.That is to say, transgenic plant (transformed plant) contains genetic material (transgene) that did not contain before transformation. Transgenic can include the genetic sequence that obtains or derives from these cells from plant cells, other plant cells, or non-plant sources, or synthetic sequences. Usually, by artificial manipulation such as, for example, by transformation, transgenic is introduced into plant source, but any method can be used as one of the techniques identified in this area. Genetic material is preferably stably integrated into the genome of the plant. The genetic material introduced may be included in the same species and exists in a natural state but in a rearranged order or a sequence of different elements, such as antisense sequences. " transgenic plant " in this article includes plants containing these sequences.

[0232] "Non-transgenic plants" are plants that have been genetically modified by the introduction of genetic material through recombinant DNA technology. In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny will not segregate for the desired phenotype.

[0233] As used herein, the term "compared to an isogenic plant," or similar phrases, refers to an isogenic plant relative to a transgenic plant, but which does not possess the transgene of interest. Preferably, the corresponding non-transgenic plant is of the same cultivar or variety as the precursor of the transgenic plant of interest, or a sibling plant line lacking the construct, often referred to as a "segregant," or a plant of the same cultivar or variety transformed with an "empty vector" construct, which may be a non-transgenic plant. "Wild-type," as used herein, refers to a cell, tissue, or plant that has not been modified according to the present invention. A wild-type cell, tissue, or plant may be used as a control to compare the level of expression of an exogenous nucleic acid or the extent and nature of a property modification with a modified cell, tissue, or plant, as described herein.

[0234] Transgenic plants, as defined in the context of the present invention, include progeny of plants that have been genetically modified using recombinant technology, wherein the progeny contain the transgene of interest. Such progeny may be obtained by self-pollination of the original transgenic plant or by crossing such a plant with other plants of the same species. This generally modulates the production of at least one protein defined herein in the desired plant or plant organ. Transgenic plant parts include all parts and cells of the plant containing the transgene, such as cultured tissue, callus, and protoplasts.

[0235] Plants contemplated for use in the practice of the present invention include monocots and dicots. Target plants include, but are not limited to, the following: cereals (e.g., wheat, barley, rye, oats, rice, corn, sorghum, and related crops); grapes; beets (sugar beets and fodder beets); pome, stone, and soft fruits (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries); legumes (beans, lentils, peas, soybeans); oilseed crops (rape or other Brassicas, mustard, olives, sunflowers, safflower, flax, coconuts, castor oil plants, cocoa beans, peanuts); ); Cucumber plants (cucurbits, cucumbers, melons); Fiber plants (cotton, flax, jute); Citrus fruits (oranges, lemons, grapefruits, mandarins); Vegetables (spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, peppers); Lauraceae (avocado, cinnamon, camphor) or plants such as corn, tobacco, nuts, coffee, sugarcane, tea, vines, hops, turf, bananas and natural rubber plants, as well as ornamental plants (flowers, shrubs, broad-leaved trees and evergreens such as conifers). Preferably, the plant is a cereal plant, more preferably wheat, rice, corn, triticale, oats or barley, even more preferably wheat.

[0236] As used herein, the term "wheat" refers to any species of the genus Triticum, including its precursors, and its progeny produced by hybridization with other species. Wheat includes "hexaploid wheat", which has a genome constitution of AABBDD and consists of 42 chromosomes; and "tetraploid wheat", which has a genome constitution of AABB and consists of 28 chromosomes. Hexaploid wheat includes common wheat (T.aestivum), Spelta (T.spelta), Macha (T.macha), dense spike wheat (T.compactum), Indian round grain wheat (T.sphaerococcum), Vavilovi (T.vavilovii) and interspecific hybrids thereof. The preferred species of hexaploid wheat is T.aestivum ssp aestivum (also known as "bread wheat"). Tetraploid wheat includes T. durum (also referred to herein as durum wheat or Triticum turgidum ssp. durum), wild emmer wheat (T. dicoccoides), emmer wheat (T. dicoccum), Polish wheat (T. polonicum), and interspecific hybrids thereof. In addition, the term "wheat" includes potential precursors of the hexaploid or tetraploid Triticum genus, such as T. uartu, T. monococcum, or wild emmer wheat (T. boeoticum) for the A genome, Aegilops speltoides for the B genome, and T. tauschii (also known as Aegilops squarrosa or Aegilops tauschii) for the D genome. Particularly preferred precursors are precursors of the A genome, and even more preferably, the precursor of the A genome is emmer wheat. The wheat cultivars used in the present invention may belong to, but are not limited to, any of the species listed above. Also contemplated are plants produced by conventional techniques using Triticum as a parent in sexual crosses with non-Triticum species (e.g., Secale cereale), including but not limited to triticale.

[0237] As used herein, the term "barley" refers to any species of the genus Hordeum, including its precursors and progeny produced by hybridization with other species. Preferably, the plant is a commercially grown barley plant such as Hordeum vulgare or a strain, cultivar or variant suitable for commercial food production.

[0238] "Transgenic plants", as defined in the context of the present invention, include plants (and plant parts and cells) that have been genetically altered using recombinant technology so that at least one polypeptide of the present invention is produced in the desired plant or plant organ, and their progeny. Transgenic plants are produced using techniques known in the art, such as those described in A. Slater et al., Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003) and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

[0239] In a preferred embodiment, the transgenic plants are homozygous for each gene (transgene) that has been introduced so that their progeny do not segregate for the desired phenotype. The transgenic plants can also be heterozygous for the introduced transgene, as, for example, in the F1 progeny, which are grown from hybrid seeds. Such plants can provide advantages known in the art such as hybrid vigor.

[0240] As used herein, "other genetic markers" may be any molecule that is linked to a desired trait in a plant. Such markers are well known to those skilled in the art and include molecular markers linked to genes that determine traits such as disease resistance, yield, plant morphology, kernel quality, dormancy characteristics, kernel color, gibberellin acidity in seeds, plant height, powder color, and the like. Examples of such genes are the stripe rust resistance genes Yr10 or Yr17, nematode resistance genes such as Cre1 and Cre3, alleles of the gluten locus that determine dough strength, such as the Ax, Bx, Dx, Ay, By, and Dy alleles, and the Rht gene that determines semi-dwarf growth habit and, thus, lodging resistance.

[0241] Four general methods for delivering genes directly into cells have been described: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, e.g., WO 87 / 06614, US 5,472,869, 5,384,253, WO 92 / 09696 and WO 93 / 21335) and the gene gun (see, e.g., US 4,945,050 and US 5,141,131); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992).

[0242] Acceleration methods that can be used include, for example, microparticle bombardment. An example of a method for delivering transformed nucleic acid molecules to plant cells is microparticle bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microparticles) are coated with nucleic acids and delivered to cells by a propulsive force. Exemplary particles include those composed of tungsten, gold, platinum, etc. In addition to being an effective method for reproducible transformation of monocotyledons, the specific advantage of microparticle bombardment is that it does not require the isolation of protoplasts nor the susceptibility to Agrobacterium infection. A microparticle delivery system suitable for use in the present invention is the helium-accelerated PDS-1000 / He gun, which is available from Bio-Rad Laboratories. For bombardment, immature embryos or target cells derived from immature embryos, such as scutellum or callus, can be arranged on a solid culture medium.

[0243] In another alternative embodiment, plastids can be stably transformed. Methods disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA into the plastid genome by homologous recombination (US 5,451,513, US 5,545,818, US 5,877,402, US 5,932479 and WO 99 / 05265).

[0244] Agrobacterium-mediated transfer is the system that is widely applicable to gene introduction into vegetable cell, because DNA can be introduced in whole plant tissue, thereby bypasses the needs from protoplast regeneration complete plant.This area is well known that Agrobacterium-mediated plant integration vector is used for DNA introduction into vegetable cell (see, for example, US 5,177,010, US5,104,310, US 5,004,863, US 5,159,135).In addition, the integration of T-DNA is a relatively accurate method, hardly causes rearrangement.The DNA district to be transferred is limited by border sequence, and intervening DNA is usually inserted in the plant genome.

[0245] Agrobacterium transformation vectors are capable of replicating in Escherichia coli (E. coli) as well as Agrobacterium, as described in the convenient procedures (Klee et al., Plant DNA Infectious Agents, Hohn and Schell, (eds.), Springer-Verlag, New York, (1985): 179-203). In addition, technological advances in vectors for Agrobacterium-mediated gene transfer have improved gene arrangement and restriction sites in the vectors, so that vectors capable of expressing a variety of polypeptide encoding genes can be constructed. The described vectors have convenient polylinker regions flanked by promoters and polyadenylation sites for direct expression of inserted polypeptide encoding genes, and the vectors are suitable for the purposes herein. In addition, Agrobacterium containing either Ti genes or not can be used for transformation. In those plant varieties where Agrobacterium-mediated transformation is effective, this is the method of choice due to the convenience and limited nature of gene transfer.

[0246] Transgenic plants created using Agrobacterium transformation typically contain a single genetic locus on a chromosome. Such transgenic plants can be referred to as hemizygous for the introduced gene. More preferred are transgenic plants that are homozygous for the introduced structural gene; that is, transgenic plants containing two introduced genes, one gene at the same locus on each chromosome of a chromosome pair. Homozygous transgenic plants can be obtained by sexually mating (selfing) independent segregating transgenic plants containing a single introduced gene, germinating some of the produced seeds, and analyzing the resulting plants for the gene of interest.

[0247] It should also be understood that two different transgenic plants can be mated to produce offspring containing two independently isolated exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for the two exogenous genes. Backcrossing to the parent plant and hybridization with non-transgenic plants, such as asexual propagation, should also be considered. Descriptions of other propagation methods commonly used for different traits and crops can be found in Fehr, Breeding Methods for Cultivar Development, J. Wilcox (editor) American Society of Agronomy, Madison Wis. (1987).

[0248] Use the method based on the combination of calcium phosphate precipitation, polyethylene glycol treatment, electroporation and these treatments to realize the transformation of plant protoplasts.These systems are applied to different plant varieties, depend on the ability of specific plant varieties from protoplast regeneration.Illustrative methods from protoplast regeneration cereals are described (Fujimura etc., 1985; Toriyama etc., 1986; Abdullah etc., 1986).

[0249] Other methods of cell transformation may also be used and include, but are not limited to, introducing DNA into plants by directed DNA transfer into pollen, directed DNA injection into reproductive organs of the plant, or by directed DNA injection into immature embryonic cells followed by rehydration of dried embryos.

[0250] Regeneration, development and planting of plants from individual plant protoplast transformants or from various transformed explants are well known in the art (Weissbach et al., Methods for Plant Molecular Biology, Academic Press, San Diego, (1988)). The regeneration and growth process generally comprises the following steps: selecting transformed cells, culturing these individualized cells through the common stages of embryonic development to the rooted seedling stage. Transgenic embryos and seeds are regenerated in a similar manner. The resulting transgenic rooted seedlings are then planted in a suitable plant growth medium, such as soil.

[0251] The development or regeneration of plants containing foreign, exogenous genes is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated plants is hybridized with plants grown from seeds of agriculturally important varieties. Conversely, pollen from plants of these important varieties is used to pollinate the regenerated plants. Transgenic plants of the present invention containing the desired exogenous nucleic acids are cultivated using methods well known to those skilled in the art.

[0252] Methods for transforming dicotyledons, primarily by using Agrobacterium tumefaciens, and obtaining transgenic plants have been published for cotton (US 5,004,863, US 5,159,135, US 5,518,908); soybean (US 5,569,834, US 5,416,011); Brassica (US 5,463,174); peanut (Cheng et al., 1996) and pea (Grant et al., 1995).

[0253] For cereal plants (such as wheat and barley) conversion into by introducing exogenous nucleic acid genetic deformation is introduced into the method for plant, and the method for plant regeneration from protoplast or plant immature embryo is well known in the art, for example, see CA2,092,588, AU 61781 / 94, AU 667939, US 6,100,447, WO97 / 048814, US 5,589,617, US 6,541,257, and other methods are set forth in WO 99 / 14314. Preferably, transgenic wheat or barley plants are produced by transformation operation mediated by Agrobacterium tumefaciens. The vector carrying the required nucleic acid construct may be introduced into the regenerable wheat cell of tissue culture plant or explant or suitable plant system, for example protoplast. Regenerable wheat cell is preferably from the scutellum of immature embryo, mature embryo and callus derived from these, or meristematic tissue.

[0254] In order to confirm that the transgenic plant exists in transgenic cells and plants, methods known to those skilled in the art can be used to perform polymerase chain reaction (PCR) amplification or Southern blot analysis. Depending on the nature of the transgenic expression product, any one of a variety of methods can be used to detect the product, including Western blot analysis and enzyme assays. A particularly effective method for quantitative protein expression and detection replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants are obtained, plants can be cultivated to produce plant tissues or parts with the desired phenotype. Plant tissues or plant parts can be harvested, and / or seeds can be collected. Seeds can be used as the source for cultivating other plants, and plants comprise tissues or parts with the desired characteristics.

[0255] Marker-assisted selection

[0256] Marker-assisted selection is a recognized method for selecting heterozygous plants when backcrossing to the recurrent parent in a classical breeding program. In each backcross generation, the population of plants will be heterozygous for the gene of interest, normally present in a 1:1 ratio in the backcross population, and molecular markers can be used to distinguish between the two alleles of the gene. By extracting DNA from seedlings and testing the implanted desired characteristics with specific markers, plants can be selected for further backcrossing while energy and resources are concentrated on fewer plants. To further accelerate the backcrossing program, embryos from immature seeds (25 days after flowering) can be excised and grown in a nutrient medium under sterile conditions, rather than mature seeds. This process, known as "embryo rescue," is used in conjunction with DNA extraction at the three-leaf stage and analysis of at least one Lr67 allele or variant that confers resistance to one or more biotrophic fungal pathogens, preferably to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, allowing for the rapid selection of plants carrying the desired characteristics, which can be grown to maturity in a greenhouse or field for subsequent further backcrossing to the recurrent parent.

[0257] Any molecular biology technique known in the art can be used in the methods of the present invention. Such methods include, but are not limited to, the use of nucleic acid amplification, nucleic acid sequencing, nucleic acid hybridization with appropriately labeled probes, single-strand conformation analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalytic nucleic acid cleavage, or a combination thereof (see, for example, Lemieux, 2000; Langridge et al., 2001). The present invention also includes the use of molecular marker technology to detect polymorphisms with respect to alleles of, for example, the Lr67 gene, which confer resistance to one or more biotrophic fungal pathogens, preferably resistance to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew. Such methods include detection or analysis of restriction fragment length polymorphisms (RFLPs), RAPDs, amplified fragment length polymorphisms (AFLPs), and satellite (simple sequence repeat, SSR) polymorphisms. Tightly linked markers can be readily obtained by methods well known in the art, such as Bulked Segregant Analysis, as reviewed by Langridge et al. (2001).

[0258] In one embodiment, the linked site for marker-assisted selection is at least within 1 cM, 0.5 cM, 0.1 cM, or 0.01 cM of the gene encoding the polypeptide of the present invention.

[0259] "Polymerase chain reaction" ("PCR") is a reaction in which replicate copies are formed of a target polynucleotide using a "primer pair" or "primer set" consisting of an "upstream" and a "downstream" primer, a polymerization catalyst (such as a DNA polymerase), and typically a thermostable polymerase. Methods of PCR, known in the art, are taught, for example, in "PCR" (MJ McPherson and S.G. Moller (eds.), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCR can be performed on cDNA obtained from reverse transcribed mRNA isolated from plant cells expressing the Lr67 gene or allele that confers resistance to one or more biotrophic fungal pathogens, preferably to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew. However, it will generally be easier if PCR is performed on genomic DNA isolated from the plant.

[0260] Primer is an oligonucleotide sequence that can hybridize with the target sequence in a sequence-specific manner and is extended during the PCR process. Amplicon or PCR product or PCR fragment or amplification product is an extension product comprising a newly synthesized copy of the primer and the target sequence. The multiplex PCR system contains multiple sets of primers, which result in the simultaneous production of more than one amplicon. Primers can completely match the target sequence or they may contain internal mismatched bases, which can lead to the introduction of restriction enzymes or catalytic nucleic acid recognition / cleavage sites in the specific target sequence. Primers can also contain additional sequences and / or contain modified or labeled nucleotides to facilitate capture or detection of amplicon. Repeated cycles of thermal denaturation of DNA, annealing of primers to their complementary sequences and extension of the annealed primers under the action of a polymerase result in exponential amplification of the target sequence. The term target or target sequence or template refers to the nucleic acid sequence that is amplified.

[0261] Methods for direct sequencing of nucleotide sequences are well known to those skilled in the art, and examples thereof can be found in Ausubel et al., (supra) and Sambrook et al., (supra). Sequencing can be performed by any suitable method, for example, dideoxy sequencing, chemical sequencing, or variations thereof. Direct sequencing has the advantage of determining variations in any base pair of a particular sequence.

[0262] TILLING

[0263] The plants of the present invention can be produced using a process known as TILLING (targeted induced localized lesions in the genome). In a first step, introduced mutations (e.g., new single base changes) are induced in a plant population by treating seeds (or pollen) with a chemical mutagen, and the plants are then propagated for one generation, where the mutations are stably inherited. DNA is extracted and seeds from all members of the population are stored to create a resource that can be repeatedly accessed over time.

[0264] For TILLING assays, PCR primers are designed to specifically amplify a single target gene of interest. Specificity is particularly important if the target is a member of a gene family or part of a polyploid genome. Secondly, dye-labeled primers can be used to amplify PCR products from DNA pooled from multiple individuals. These PCR products are denatured and annealed to allow mismatched base pairs to form. Mismatches, or heteroduplexes, represent naturally occurring single nucleotide polymorphisms (SNPs) (i.e., several plants from a population may carry the same polymorphism) and induced SNPs (i.e., only rare plant individuals may show this mutation). After heteroduplex formation, the use of endonucleases that can recognize and cleave mismatched DNA (such as Cel I) is crucial for discovering new SNPs in the TILLING population.

[0265] Using this method, many plants are screened to identify any individual with single base changes and small insertions or deletions (1-30 bp) in any gene or genome region. The genomic fragment size measured is in the range of 0.3 to 1.6 kb. In 8-fold pools, each assay has 96 lanes and 1.4 kb fragments (ignoring fragment ends, which have problems with noise SNP detection). This combination allows screening of up to 1,000,000 base pairs in genomic DNA in each assay, making TILLING a high-throughput technology.

[0266] TILLING is further described in Slade and Knauf (2005) and Henikoff et al. (2004).

[0267] In addition to allowing effective detection of mutations, high-throughput TILLING technology is also ideal for detecting natural polymorphisms. Therefore, the number and position of polymorphic sites are revealed by interrogating unknown homologous DNA through heteroduplex formation with known sequences. Both nucleotide changes and small insertions and deletions, including polypeptides with at least some repeat counts, can be identified. This has been referred to as Ecotilling (Comai et al., 2004).

[0268] Each SNP is recorded by its approximate position within a number of nucleotides. Thus, each haplotype is archived based on its mobility. Sequence data are acquired using aliquots of the same amplified DNA used for the mismatch-cleavage assay, with relatively small incremental efforts. The left or right sequencing primers for individual reactions are selected using sequencing primers. Sequencher software performs multiple comparisons and finds base changes, which are confirmed in each case by gel banding.

[0269] Ecotilling is cheaper than full sequencing, the current method used to discover most SNPs. Instead of a pool of DNA from mutagenized plants, a plate containing an array of ecological DNA is screened. Because detection is performed on a gel with near-base-pair resolution and a uniform background pattern across lanes, bands of the same size can be matched, allowing SNPs to be discovered and typed in a single step. In this way, final sequencing of the SNP is simple and efficient, and DNA sequencing can be performed directly using aliquots of the same PCR product used for screening, making it even simpler and more efficient.

[0270] Plant / grain processing

[0271] The grains / seeds of the invention, preferably grains and more preferably wheat grains, or other plant parts of the invention, may be processed to produce food ingredients, food or non-food products using techniques known in the art.

[0272] In one embodiment, the product is a whole grain flour such as ultra-fine milled whole grain flour, or flour made from 100% grains. Whole grain flour includes a refined flour component (refined flour or refined flour) and a semolina content (ultra-fine milled semolina content).

[0273] Refined flour is flour that has been prepared, for example, by milling and sifting clean grains such as wheat or barley. Refined flour is described as flour with a particle size in which no less than 98% of the particles pass through a wire mesh with openings no larger than 212 microns (US 70 wire) as specified. The coarse fraction includes at least one of the bran and the germ. For example, the germ is the embryonic plant found within the kernel of a grain. The germ contains lipids, fiber, vitamins, protein, minerals, and phytonutrients such as flavonoids. The bran consists of several cell layers and contains significant amounts of lipids, fiber, vitamins, protein, minerals, and phytonutrients such as flavonoids. Additionally, the coarse fraction may include the aleurone layer, which also contains lipids, fiber, vitamins, protein, minerals, and phytonutrients such as flavonoids. Although technically considered part of the endosperm, the aleurone layer exhibits many of the same characteristics as the bran and is therefore typically removed along with the bran and germ during the milling process. The aleurone layer contains protein, vitamins, and phytonutrients such as ferulic acid.

[0274] In addition, the coarse grain portion may be mixed with a refined flour component. The coarse grain portion may be mixed with a refined flour component to form a whole grain flour, thereby providing a whole grain flour having a higher nutritional value, a higher fiber content, and a higher antioxidant capacity than refined flour. For example, the coarse grain content or whole grain flour may be used in varying amounts to replace refined flour or whole grain flour in baked goods, snack products, and food products. The whole grain flour of the present invention (i.e., ultrafinely milled whole grain flour) may be sold directly to consumers for use in their homemade baked products. In an exemplary embodiment, the granulation of the whole grain flour is such that 98% of the particles are less than 212 microns, calculated on a weight basis.

[0275] In yet another embodiment, enzymes found in the bran and germ of the whole grain flour and / or semolina content are inactivated to stabilize the whole grain flour and / or semolina content. Stabilization is a process that uses steam, heat, radiation, or other treatments to inactivate enzymes found in the bran and germ layers. Stabilized flour retains its culinary characteristics and has a longer shelf life.

[0276] In additional embodiments, whole grain flour, whole grain content, or refined flour may be an ingredient (composition) of a physical product and may be used in a food product. For example, the food product may be a bagel, a cracker, a bread, a bun, a croissant, a dumpling, an English muffin, a muffin, a pita bread, a quickbread, a refrigerated / frozen dough, dough, baked beans, a burrito, chili, a tortilla, a taco, a tortilla, a pot pie, a ready-to-eat cereal, a ready-to-eat meal, a stuffing, a microwavable meal, a brownie, a cake, a cheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet bread, a candy, a pie crust, a pie filling, a baby food, a baking mix, a batter , mixes, sauces, meat enhancers, meat substitutes, seasonings, soup mixes, gravies, rouxes, salad dressings, soups, yogurt, noodles, pasta, ramen, chow mein noodles, lo mein noodles, ice cream inclusions, popsicles, ice cream cones, filled ice cream, crackers, croutons, donuts, egg rolls, puffed foods, fruit and cereal bars, microwavable snack products, nutrition bars, flapjacks, budget-friendly baked products, pretzels, puddings, oat-based products, snack chips, snack foods, snack mixes, waffles, pizza crusts, animal food, or pet food.

[0277] In an alternative embodiment, the whole grain flour, refined flour, or semolina content is a component of a nutritional supplement. For example, a nutritional supplement can be a product added to a daily diet containing one or more additional ingredients, typically including: vitamins, minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices, probiotics, extracts, prebiotics, and fiber. The whole grain flour, refined flour, or semolina content of the present invention includes vitamins, minerals, amino acids, enzymes, and fiber. For example, the semolina portion contains concentrated amounts of dietary fiber as well as other essential nutrients such as B vitamins, selenium, chromium, manganese, magnesium, and antioxidants, which are essential for a healthy diet. For example, 22 grams of the semolina content of the present invention provides 33% of an individual's recommended daily fiber intake. Nutritional supplements can include any known nutritional ingredients that contribute to an individual's overall health, examples of which include, but are not limited to, vitamins, minerals, other fiber components, fatty acids, antioxidants, amino acids, polyols, protein, lutein, ribose, omega-3 fatty acids, and / or other nutrients. The supplement may be provided in the following forms, but is not limited to: ready-to-drink drink mixes, ready-to-drink beverages, nutritional bars, wafers, cookies, crackers, gel pellets, capsules, chews, chewable tablets, and pills. One embodiment provides the fiber supplement in the form of a flavored drink mix or malt liquor-type beverage, which may be particularly attractive as a fiber supplement for children.

[0278] In an additional embodiment, the milling process can be used to make multi-grain flour or multi-grain coarse grain content. For example, the bran and germ from one type of cereal can be ground and mixed with the ground endosperm or whole grain cereal flour of another type of cereal. Or, the bran and germ from one type of cereal can be ground and mixed with the ground endosperm or whole grain cereal flour of another type of cereal. It is contemplated that the present invention encompasses mixing any combination of one or more brans, germ, endosperms with the whole grain flour of one or more cereals. This multi-grain method can be used to make custom flours and utilize the quality and nutritional content of multiple types of cereal grains to make flour.

[0279] It is contemplated that whole grain flour, coarse grain content and / or cereal products of the present invention can be produced by milling methods known in the art. One exemplary embodiment relates to grinding the cereal in a single stream without separating the endosperm, bran and germ of the cereal into separate streams. The cleaned and conditioned cereal is conveyed to a first channel grinder, such as a hammer mill, a roller mill, a pin mill, an impact mill, a disc mill, an air mill, a notch mill, or the like. The cereal is ground and then flowed out and conveyed to a sieve. In addition, it is contemplated that whole grain flour, coarse grain content and / or cereal products of the present invention can be modified or enhanced by many other methods, such as fermentation, instantization, extrusion, packaging, baking, roasting, or the like.

[0280] Wheat germination

[0281] The malt liquor-type beverages provided by the present invention relate to alcoholic beverages (including distilled beverages) and non-alcoholic beverages produced by using malt liquor as part or all of its starting material. Examples include beer, sparkling wine (low-malt liquor beer beverages), whiskey, low-alcohol malt liquor-type beverages (e.g., malt liquor-type beverages containing less than 1% alcohol), and non-alcoholic beverages.

[0282] Malting is a process of controlled soaking and germination followed by drying of grains such as barley and wheat. This sequence of events is important for the synthesis of many enzymes that cause grain modification, a process that primarily depolymerizes dead endosperm cell walls and mobilizes grain nutrients. In the subsequent drying process, aroma and color are produced by chemical browning reactions. Although malt liquor is primarily used in beverage production, it can also be used in other industrial processes, for example as a source of enzymes in the baking industry, or as a flavoring and coloring agent in the food industry, such as malt liquor or malt liquor powder, or directly as malt liquor syrup, etc.

[0283] In one embodiment, the present invention relates to a method for producing a malt liquor composition. The method preferably comprises the following steps:

[0284] (i) providing a cereal, such as barley or wheat cereal according to the present invention,

[0285] (ii) soaking the grains,

[0286] (iii) germinating the soaked grains under predetermined conditions and

[0287] (iv) Drying of germinated grains.

[0288] For example, malt liquor can be produced by any of the methods described by Hoseney (Principles of Cereal Science and Technology, 2nd ed., 1994: American Association of Cereal Chemists, St. Paul, Minn.) However, the present invention also contemplates any other suitable method for producing malt liquor, such as methods for producing specialty malt liquors, including, but not limited to, methods for roasting malt liquor.

[0289] Malt liquor is primarily used to brew beer, but it is also used to produce distilled spirits. Brewing involves producing the wort, primary and secondary fermentation, and post-processing. Malt is first ground, stirred into water, and heated. During this "mashing" process, enzymes activated in the germinating grains break down the starch in the grains into fermentable sugars. The resulting wort is clarified, yeast is added, and the mixture is fermented and post-processed.

[0290] Example

[0291] Example 1. The Lr67 / Yr46 gene is a mature plant resistance gene different from Lr34 / Yr18 and Lr46 / Yr29

[0292] introduction: Rust resistance genes in wheat are divided into two major categories, termed seedling and mature plant resistance (APR) genes. Seedling resistance genes can be detected phenotypically after exposure to the rust pathogen and are observed during the seedling and mature stages; thus, they confer a resistance phenotype across all stages of plant growth. In contrast, APRs are generally not detectable at the seedling stage, but can be detected during later growth stages and often confer resistance to the pathogen in field-grown plants. Conversely, some APR genes are induced to express at the seedling stage by altering growth temperature and light conditions, but are not expressed at the seedling stage under all growth conditions. Furthermore, seedling resistance genes typically exhibit a major effect phenotype and distinct infection types, whereas most APR genes partially influence varying levels of disease severity. Race specificity is more common in seedling resistance genes. Of the few APR genes that have been studied in wheat, most appear to be variety-nonspecific, with a limited number showing clear variety specificity. Those with a non-variety-specific class of partial resistance are related to the slow rust phenotype first described by Caldwell (1968). Generally, slow rust resistant plants show a longer incubation period, fewer and smaller uredia within two weeks of inoculation compared to susceptible plants.

[0293] One of the well-characterized non-variety-specific genes is the mature plant leaf rust resistance gene, Lr34 (WO 2010 / 022443). Previously known as LrT2 (Dyck 1977, 1987), it is present in more mature South American wheat cultivars such as Frontana and its derivatives, certain early hybrid wheat cultivars from the early 20th century, and certain wheat landraces (non-cultivated species isolated from wild wheat), particularly those of Chinese origin (Borghi 2001; Kolmer et al., 2008). A key feature of Lr34 is that the virulence of wheat leaf rust pathogens has not yet been reported, and the enhanced rust resistance effect when the Lr34 gene is combined with other variety-specific leaf rust resistance genes in wheat cultivars has contributed to the persistence of wheat cultivars with Lr34 gene combinations (Kolmer, 1996). However, variability in the degree of rust colony development among leaf rust isolates of the Lr34 gene has been reported (Bender and Pretorius, 2000). The co-segregation of Lr34 with the mature plant stripe rust resistance gene Yr18 in showing double rust resistance in many wheat backgrounds (McIntosh 1992; Singh 1992) may have contributed to the continued widespread use of Lr34 / Yr18 germplasm in wheat breeding. Subsequent observations that the Lr34 / Yr18 locus also confers partial resistance to mature plant powdery mildew (Pm38) highlight the multipathogenic nature of the Lr34 / Yr18 / Pm38 locus on the short arm of wheat chromosome 7D (Spielmeyer et al., 2005; Lillemo et al., 2008).

[0294] Chemical and physical mutagenesis were used to study the multipathogen resistance locus containing Lr34 on wheat chromosome 7DS (Spielmeyer et al., 2008). Susceptible mutants were recovered without loss of DNA markers in the QTL interval on 7DS. These mutants were subsequently shown to carry point mutations in which the chemical mutagen had created a single base substitution. Additional mutants generated by gamma-ray irradiation had a single base deletion in the gene encoding the ATP-binding cassette (ABC) transporter at the multipathogen resistance locus (Krattinger et al., 2009). In addition to the ABC transporter, six other genes co-segregated with the resistance locus and were genetically and physically closely linked. However, none of these mutants (eight separate mutants) had changes in the additional genes. Therefore, mutagenic changes to the ABC transporter alone were sufficient to confer a complete loss of resistance to leaf rust, stripe rust, and powdery mildew encoded by Lr34 / Yr18 / Pm38. A combination of haplotype analysis and high-resolution mapping demonstrated that a single gene, an ABC transporter, conferred all three resistances (Krattinger et al., 2009).

[0295] Lr67 / Yr46 is different from Lr34 / Yr18 and Lr46 / Yr29

[0296] While developing leaf rust-resistant near-isogenic lines within the Thatcher cultivar, Dyck (1987) observed a phenotypic spectrum in line RL6077 (Thatcher*6 / PI250413) that resembled RL6058, a near-isogenic line carrying Lr34 / Yr18. In subsequent studies, the APR in RL6077 segregated independently from Lr34 / Yr18, and there was evidence of translocation differences between the lines. Therefore, Dyck et al. (1994) concluded that RL6077 was a carrier of the Lr34 / Yr18 gene, but on a chromosome other than the 7DS. With the development of closely linked genetic markers and the eventual cloning of Lr34 / Yr18, it became increasingly clear that RL6077 lacked the Lr34 / Yr18a resistance haplotype present in RL6058 and, therefore, contained a different APR gene (Kolmer et al., 2008; Lagudah et al., 2009).

[0297] Studies using isolates of Puccinia tritici and Puccinia spp. from multiple locations in Canada, Mexico, and Australia on a mapping population from a cross containing RL6077 confirmed the co-segregation of individual APRs on chromosome 4DL (normally closed chromosome 4D, the D genome in bread wheat) (Herrera-Foessel et al., 2011; Hiebert et al., 2010). These APR genes, located at the resistance locus on 4DL, have been designated Lr67 / Yr46.

[0298] Example 2 Cloning of the Lr67.(syn=Yr46=Sr55=Pm46) gene

[0299] In addition to the wheat genotype RL6077, two other wheat genotypes, NP876 and Sujata, have been hypothesized to carry Lr67. This is based on the leaf tip necrosis phenotype (Ltn), slow leaf rust infection, and the presence of a simple sequence repeat (SSR) marker allele linked to Lr67 in RL6077 (Hererra et al., 2011). As part of a strategy to identify co-segregating markers and clone the Lr67 gene, the present inventors developed recombinant inbred (RI) families derived from crosses involving Avocet, a parent susceptible to leaf and stripe rust in mature plants. These RI families were derived from crosses of Avocet x RL6077, Avocet x Sujata, and Avocet x NP876. F2 families were also generated from the Thatcher x Thatcher + Lr67 (RL6077) cross. These RI families were then used in genetic mapping studies and mutation experiments as follows.

[0300] First, a dose of 20 krad from 60Gamma irradiation with a Co source was used to induce population mutagenesis of derivative lines from AvocetxRL6077 fixed with Lr67. Secondly, chemical mutagenesis using ethyl p-methanesulfonate (EMS) was used to generate mutant populations using the wheat genotype RL6077 under standard mutagenesis conditions. M2 progeny lines were produced from the mutagenized populations, and each M2 progeny line was tested for loss of resistance phenotype. Field evaluation of the M2 mutant progeny was performed to identify leaf rust resistance and stripe rust resistance caused by inactivation of the Lr67 gene. Putative mutants were tested at the M3 and M4 stages to select homologous susceptible lines. Five gamma irradiated line plants (designated γ318, γ676, γ1183, γ1239, and γ1656) and two EMS lines (designated emsSu1 and emsSu2) were identified as susceptible mutants. They all showed loss of the leaf and stripe rust resistance phenotypes, as well as the leaf tip necrosis phenotype, indicating that a single gene was responsible for all three phenotypes. The wheat genotype background of the EMS mutants facilitated evaluation for stem rust and powdery mildew, and each mutant showed susceptibility to both diseases, in contrast to the resistance observed in its progeny and the original parent.

[0301] To identify DNA sequences at or closely linked to the Lr67 locus, 15 homologous recombinant inbred lines (RILs), each from a resistant (Lr67R) and susceptible (Lr67S) phenotype, were selected and subjected to extensive AFLP and SSR analysis. SNP-based markers from the genome complexity reduction library were evaluated based on a genomic library of pooled DNA from the 15 resistant homologous lines using the PstI restriction endonuclease. Comparative genomic approaches were also used to identify homologous genes from the rice and Brachypodium genomes using sequences from wheat BAC clones containing SSRs closely linked to Lr67 (Hererra et al., 2011) as anchor points. Figure 1 The colinear region between Brachypodium chromosome 1 and rice chromosome 3 was then used to develop DNA markers to amplify the corresponding sequences from wheat and Aegilops spp., which are closely linked to the D genome precursor of wheat chromosome 4DL ( Figure 1 ).

[0302] AFLP and SNP-generated DNA markers from a genome complexity reduction library generated from a marker linked to Lr67 did not co-segregate with any of the 15 homozygous resistant and susceptible recombinant inbred lines. However, a marker containing Hsp70 (heat shock protein, see Figure 1 ) domain of the chaperone protein encoding gene were completely linked to the 15 susceptible recombinant inbred lines and were absent in the 15 resistant lines ( Figure 2). The degree of association of the Hsp70 chaperone protein was confirmed in 500 recombinant inbred lines and confirmed to show complete association with the Lr67 locus. Genomic Southern blot analysis provided additional evidence that the gene encoding the Hsp70 chaperone protein was missing in the Lr67-resistant parental lines, RL6077, Sujata, and NP876. Given that loss-of-function susceptible mutants have been isolated from Lr67, the Hsp70 chaperone protein gene missing in resistant plants is not sufficient to produce the Lr67 resistance phenotype. Therefore, all predicted gene sequences were searched within 21 kb of Hsp70, and three additional genes were identified by comparative genomics. These are genes with protein domains annotated as encoding Sec14B cytoplasmic factor family proteins, monosaccharide transporters (MSTs—tightly linked to sugar transporters—SUTs), and protein interaction / binding proteins (PIPs) on the rice or Brachypodium genome maps.

[0303] The nucleotide sequences of the corresponding genes in several wheat varieties and the Lr67 mutant were determined. Sequence analysis of the parental lines Thatcher (Lr67 susceptible), Avocet (Lr67 susceptible), and Thatcher + Lr67 (RL6077) showed no differences in the Sec14B gene, while the MST and PIP genes revealed sequence polymorphisms. Two SNPs (C / G and T / G) were found in the SUT gene that are unique to wheat containing Lr67 ( Figure 4 ), while the insertion / deletion polypeptide of PIP is not diagnostic for Lr67. A SNP in the SUT gene co-segregated with mature plant rust in Lr67 in 520 recombinant inbred lines. To ensure that no additional gene sequences were lost or unknown rearrangements were detected in the wheat chromosome 4DL region of Lr67, which contains synthetic regions corresponding to Brachypodium (chromosome 1) and rice (chromosome 3), the corresponding region from Aegilops spp. (D genome precursor) was analyzed. A 70 kb sequence contig revealed the presence of Hsp70, SUT, and PIP, located within a 21 kb fragment within the contig; no other predicted genes were found in this region. When 1152 plants from the F2 progeny of a Thatcher x Thatcher + Lr67 cross were analyzed, no recombination between these three genes was detected.

[0304] Analysis of mutants in which the Lr67 phenotype is inactivated revealed that three γ mutants lack SUT, PIP, and some of the markers identified from comparative genomics within the Lr67 region ( Figure 3, as indicated by the dotted line). However, four of the mutants (two EMS mutants and two mutants derived from gamma irradiation, i.e., γ318 and γ1239) retained the SUT and PIP genes that co-isolated in Lr67. Sequence analysis of the SUT gene in these mutants revealed only single nucleotide changes and small deletions in the SUT gene, but not in PIP. Each of the four mutants altered an amino acid found within a conserved region of amino acid hexose transporters ( Figure 4 ). Four additional mutants obtained by sodium azide mutagenesis of RL6077 also displayed single nucleotide changes that altered the amino acid composition of the SUT gene. Based on cosegregation and mutational analysis, the inventors concluded that the SUT gene is sufficient to confer the Lr67 resistance phenotype. When tested against a full range of rust and powdery mildew diseases, all wheat EMS mutants showed susceptibility to leaf rust, stripe rust, stem rust, and powdery mildew. Thus, inactivation of a single gene, SUT, has an impact on multiple diseases. Therefore, the SUT gene was definitively identified as the Lr67 gene.

[0305] The Lr67 gene encodes a protein containing 514 amino acids ( Figure 5), and is predicted to contain 12 transmembrane domains. When predicted using the TMHMM Server v2.0 and PSIPRED v3.3 programs, the amino acid transmembrane domains are predicted to be: TMhelix1, 20-42; TMhelix2, 81-100; TMhelix3, 107-126; TMhelix4, 136-158; TMhelix5, 170-192; TMhelix6; 202-221; TMhelix7, 282-304; TMhelix8, 319-341; TMhelix9, 348-370; TMhelix10, 380-402; TMhelix11, 423-445; and TMhelix12, 450-472. The same program predicted that each of the following amino acid regions: the N-terminal 19 amino acids, the amino acids between TM helices 2 and 3, the amino acids between TM helices 4 and 5, the amino acids between TM helices 6 and 7, the amino acids between TMA helices 8 and 9, and the C-terminal 42 amino acids are located on the inner side of the membrane, with the remaining amino acids located on the outer side. The protein is a member of the Major Facilitator Superfamily (MFS), a large, diverse group of secondary transporters that includes uniporters, cotransporters, and antiporters that facilitate the transport of various compounds, including sugars, across the cytoplasm or inner cell membranes. Homologous polypeptides were identified by querying the amino acid sequence of SEQ ID NO: 1 in the NCBI protein database, and multiple homologs were identified. The amino acid sequence of Lr67 shares approximately 89-93% identity with homologous polypeptides in several cereals, including rice, and approximately 80% identity with its homolog, sugar transporter 13 (Accession No. NP_198006), in Arabidopsis thaliana.

[0306] A DNA fragment corresponding to the Lr67 gene was cloned from wheat and its nucleotide sequence was compared with the cDNA sequence (SEQ ID NO: 10). This revealed the presence of two introns within the protein coding region of the gene ( Figure 14 ).

[0307] RT-PCR analysis showed that all three Lr67 genes homologous in the A, B, and D genomes of wheat were expressed in plants. The polypeptide encoded by the A genome homolog of the Lr67 gene had an amino acid sequence that was 507 / 514 (98.6%) identical to SEQ ID NO:1, including a glycine at position 144 and a valine at position 387, which is typical of a susceptible Lr67 polypeptide. This result suggested to the inventors that the resistant Lr67 polypeptide may function as a dominant negative polypeptide, reducing the activity of the susceptible Lr67 polypeptides encoded by the A and B genomes in hexaploid wheat.

[0308] The molecular basis for the differences between the polypeptides encoded by the Lr67 resistant and susceptible alleles is limited to two nucleotide changes that generate the SNPs used in the Lr67 diagnostic marker. These two nucleotide changes result in amino acid changes, with reference to SEQ ID NO: 1, from a conserved glycine to an arginine (position 144) in the predicted fourth transmembrane domain and from a valine to a leucine (position 387) in the tenth transmembrane domain ( Figure 6 、 Figure 7 、 Figure 8 These two nucleotide changes are rare in wheat and are not found in D-genome progenitors or most commercial wheat varieties, except for a few that carry Lr67. Therefore, Lr67 resistance likely originated after the formation of hexaploid wheat through hybridization from its diploid progenitor. When over 1,000 wheat landraces and accessions from a wide range of geographic origins were analyzed for two Lr67 diagnostic markers, the mutation causing Lr67 was very rare and located in a subset of landraces originating from the Indo-Gangetic Plain.

[0309] Homologs in other plant species, such as sugar transporter 13 in Arabidopsis thaliana and cereals, without exception, do not contain an arginine at the position corresponding to amino acid position 144 of SEQ ID NO: 1, or a leucine at the position corresponding to amino acid position 387. Invariably, homologs have a glycine at the position corresponding to amino acid position 144 and almost always a valine at position 387 (see, e.g., in Figure 9 and Figure 10 Thus, these two amino acids are highly conserved in SUT polypeptides, and mutation of either or both indicates that the altered function of the amino acid is responsible for resistance to rust or powdery mildew pathogens.

[0310] Example 3. Glucose Uptake Study of Lr67(SUT) Expressed in Yeast - Variation of Polypeptide Sequence The demonstration that the Lr67 gene encodes a protein showing homology to known sugar transporters led the present inventors to test the sugar transport function of the Lr67 polypeptide in yeast cells.

[0311] Experimental process: The following experiments were performed using the protein coding regions encoding the Lr67 polypeptide (resistance and susceptible alleles) cloned into the yeast expression vector pRS416. This vector contains the constitutive ADH1 promoter and CYC1 terminator for expression of the inserted coding region. The yeast strain used was a hexose transporter-deficient variant of Saccharomyces cerevisiae designated EBY.VW4000. Radiolabeling was performed using [ 14 C] Glucose uptake was determined by measuring incorporated radioactivity by liquid scintillation counting.

[0312] result

[0313] Glucose uptake over time

[0314] Yeast cells transformed with genetic constructs expressing the resistant (Lr67(res)) or susceptible (Lr67(sus)) allele of Lr67 were treated with 100 μM [ 14 C] glucose incubation for 10 minutes. Glucose uptake was measured at 2-minute intervals. Yeast cells expressing the Lr67 susceptible allele showed a faster glucose transport rate than yeast cells expressing the Lr67 resistant allele or the empty vector ( Figure 11 In fact, cells expressing the Lr67 resistance allele showed no detectable glucose transport activity above that of the control, although this assay was performed for only 10 minutes and is not sensitive.

[0315] Glucose uptake kinetics of Lr67(sus)

[0316] Lr67(sus) 14 The concentration-dependent uptake of glucose showed classic Michaelis–Menten saturation kinetics. The Lineweaver–Burk equation was used to transform the data for linear regression analysis. Lr67(sus) showed a high affinity for glucose, with a K of 0.000 for this substrate. m 73μM, V max 3.02 nmol min -1 FW -1 . ( Figure 12 )

[0317] Amino acid turnover analysis

[0318] As mentioned above, there are two SNP differences in the nucleotide sequence between the susceptible and resistant alleles of Lr67. This creates two amino acid substitutions in the protein product, which include a glycine to arginine (G144R) at position 144 and a valine to leucine (V387L) at position 387. To test whether these amino acid substitutions, alone or together, affect the glucose uptake rate of LR67, the amino acids at positions 144 and 387 in the LR67 resistant allele were converted to the equivalent amino acids (i.e., R144G and L387V) present in the LR67 susceptible allele, respectively, by mutagenesis of the cloned gene.

[0319] Yeast cells transformed with Lr67(sus) or Lr67(res) or Lr67(res)R144G and Lr67(res)L387V were treated with 100 μM [ 14C] glucose for 10 minutes. Yeast cells expressing Lr67(res)R144G showed that they could express Lr67(res) or Lr67(res)L387V( Figure 13 ) transports glucose at a higher rate, but not to the full extent of Lr67 (sus). This indicates that position 144 is the more important of the two substituted amino acids for rust resistance function, and that the conversion of glycine to arginine (G144R) or vice versa (R144G) alters the glucose transport rate of the Lr67 polypeptide. However, the addition of a second amino acid substitution (L387V) also aids glucose transport rate.

[0320] Because the deletion mutation in Avocet x RL6077, created by gamma irradiation, which eliminates Lr67, results in a susceptible allele to rust and powdery mildew, the inventors concluded that the Lr67 (resistance) polypeptide must have a positive function in wheat cells, certainly as a transporter of sugars other than glucose, most likely a sugar transporter. That is, conversion of the Lr67 susceptible allele to a resistant allele in wheat plants such as Avocet requires arginine at position 144 and is ameliorated by the presence of leucine at position 387.

[0321] Example 4. Production of transgenic plants

[0322] Experiments are underway with the cloned genes and amino acid variants introduced into transgenic wheat using standard Agrobacterium-mediated techniques to transform wheat plants of the Fielder variety, and other plants such as cereals, to increase resistance to fungal pathogens such as rust and powdery mildew. Experiments are also underway with modifying the genes encoding the Arabidopsis and grapevine homologs of Lr67 to encode mutant polypeptides with arginine at position 144 and leucine at position 387, and converting them into resistance polypeptides in order to provide resistance genes for these and other plant species.

[0323] Materials and methods: The gene encoding Lr67 was used to transform barley plants as follows. A 7133 bp genomic fragment was isolated from the wheat genotype Thatcher+Lr67. This fragment contained the full-length genomic sequence of the Lr67 gene, including a 1318 bp native promoter region and a 1512 bp native terminator sequence comprising a 3' untranslated region. The Lr67 fragment was inserted into the binary transformation vector pWBVec8. The Lr67 binary vector was transformed into Agrobacterium tumefaciens strain AGL-1 and used to produce stably transformed barley plants (cv. Golden Promise) as described by Tingay et al. (1997).

[0324] result:Eight independent transgenic plants transformed with the Lr67 transgene have been identified. All eight plants and wild-type plants that had undergone the tissue culture steps involved in transformation but lacked the Lr67 transgene were infected with barley leaf rust, which can infect plants in the T0 generation. One of the advanced Lr67 transgenic lines was further tested at the T1 stage. Leaf rust infection and subsequent cultivation were performed on mature plants and seedlings of transgenic barley grown in a humidity chamber using Puccinia hordei pathotype 4653P+ (University of Sydney PBIC culture number 990492). This bacterium is nontoxic to plants with resistance genes Rph3, 5, 7, 10, 11, 14, and 15, but is virulent to lines with resistance genes Rph1, 2, 4, 6, 8, 9, 12, 13, and 19.

[0325] Rust spores were observed on the leaves of the control plants but not in the positive Lr67 transgenic plants of the T0 generation. Compared to the control plants, all Lr67 transgenic plants showed premature leaf senescence, a leaf senescence phenotype similar to that described as leaf tip necrosis, which is a characteristic feature of wheat Lr67-mediated resistance. When the T1 generation of the test plants was tested, all plants containing Lr67 showed a leaf rust resistance phenotype when seedlings were tested, while all plants lacking the Lr67 gene showed leaf rust susceptibility. These results confirm that the isolated Lr67 gene function is active and sufficient to confer a barley leaf rust resistance gene. These experiments also extended the range of pathogenic species to which Lr67 conferred its resistance to barley stem rust (P. hordeii), including in addition to wheat stripe rust, wheat leaf rust, wheat stem rust (P. graminis) and common powdery mildew (Blumeria vulgaris).

[0326] It will be appreciated by those skilled in the art that many variations and / or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, the present embodiments should be considered in all respects as illustrative and not restrictive.

[0327] This application claims priority from AU 2013903161, filed on August 21, 2013, the contents of which are incorporated herein by reference in their entirety.

[0328] All publications discussed and / or cited herein are incorporated herein in their entirety.

[0329] Any discussion of documents, acts, materials, devices, policies, clauses or the like included in this specification is solely for the purpose of providing a context for the present invention and is not an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Claims

1. A method for determining whether a polypeptide confers resistance to one or more biotrophic fungal pathogens, comprising: i) obtaining a polynucleotide operably linked to a promoter, said polynucleotide encoding an Lr67 polypeptide, wherein said polypeptide lacks a glycine at the position corresponding to amino acid 144 of SEQ ID NO: 1, ii) introducing the polynucleotide into a plant, and iii) determining whether the level of resistance to said one or more biotrophic fungal pathogens is increased relative to an isogenic plant lacking said polynucleotide.

2. The method of claim 1, wherein the polynucleotide encoding the polypeptide that confers resistance to the one or more biotrophic fungal pathogens when expressed is selected if the resistance level is increased.

3. The method of claim 1, wherein the biotrophic fungal pathogen is rust, mildew, or both rust and mildew.

4. The method of claim 3, wherein the rust disease is leaf rust, stripe rust, or stem rust. The method according to claim 3 , wherein the mildew is powdery mildew. The method of claim 1 , wherein the plant is a cereal plant.

7. The method of claim 6, wherein the cereal plant is rice, sorghum, barley or wheat.

8. A method for identifying a plant comprising a polynucleotide encoding an Lr67 polypeptide, wherein the polypeptide lacks glycine at the position corresponding to amino acid 144 of SEQ ID NO: 1, the method comprising the steps of: i) obtaining a nucleic acid sample from the plant, and ii) screening said sample for the presence or absence of said polynucleotide.

9. A method for producing a transgenic plant, the method comprising: i) genetically modifying a plant cell to contain a polynucleotide encoding an Lr67 polypeptide, wherein the polypeptide lacks glycine at the position corresponding to amino acid 144 of SEQ ID NO: 1, ii) regenerating a transgenic plant from said cells.

10. A method of producing a polypeptide, the method comprising expressing a polynucleotide encoding an Lr67 polypeptide in a transgenic modified cell, wherein the polypeptide lacks a glycine at a position corresponding to amino acid 144 of SEQ ID NO:

1.

11. An isolated and / or exogenous polynucleotide encoding an Lr67 polypeptide lacking a glycine at the position corresponding to amino acid 144 of SEQ ID NO:

1.

12. A chimeric vector or DNA construct comprising the polynucleotide of claim 11.

13. The vector of claim 12, wherein the polynucleotide is operably linked to a promoter.

14. The vector according to claim 13, wherein the promoter directs gene expression in leaf and / or stem cells.

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