Rust resistance gene

Abstract

The present invention relates to novel transport polypeptides for conferring resistance to one or more biotrophic pathogenic fungi in plants and genes encoding the same.

Description

Technical Field

[0001] The present invention relates to novel transport polypeptides for conferring resistance to one or more biotrophic pathogenic fungi on plants and their encoding genes. Background Art

[0002] A variety of genes conferring pathogen resistance have been identified and used in plant breeding. However, due to the emergence of new virulent strains of pathogens, single-gene pathogen resistance in plants often becomes ineffective. In contrast, durable disease resistance in plants is generally considered to be controlled by multiple genes. Some 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 mainly belong to 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 can defend 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 encoding a protein kinase. These genes encode gene-for-gene resistance to a single pathogen and generally result in a strong hypersensitive response of plant tissues to infection.

[0003] In contrast, wheat (Triticum aestivum L.) rust resistance genes such as Lr34, located on chromosome arm 7DS, confer broad-spectrum and durable resistance in mature plants to several obligate biotrophic pathogens including ascomycete and basidiomycete fungi. These include leaf rust, stripe rust, stem rust and powdery mildew, and thus the Lr34 gene has been widely used in wheat breeding, although it has a weak, non-hypersensitive response 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 shown effective and durable resistance to the leaf rust fungus Puccinia triticina Eriks (Dyck et al., 1966; Singh and Rajaram, 1994). To date, no isolate of wheat leaf rust (P. triticina) has been detected with full virulence to Lr34 (Kolmer et al., 2003). Recently the Lr34 gene was cloned and shown to encode a protein in the ABC transporter family (Krattinger et al., 2009), although the function of the gene as a transporter has not been demonstrated. Lr34 resistance remains genetically inseparable from the gene designated Yr18 that confers resistance to stripe rust (Puccinia striiformis) in wheat (Singh, 1992; McIntosh, 1992). Co-segregation of Lr34 / Yr18 with other traits such as leaf tip necrosis at the mature plant stage (Ltn1), powdery mildew (recently designated Pm38), tolerance to barley yellow dwarf virus (Bdv1) and spot blotch (Bipolaris sorokiniana) has been recorded ((Singh, 1992a, b; McIntosh, 1992; Joshi et al., 2004; Spielmeyer et al., 2005; Liang et al., 2006)), and these phenotypes are thought to be all conferred by the Lr34 resistance polypeptide.

[0004] The second gene, Lr67, which confers broad-spectrum, adult-plant resistance to several obligate biotrophic pathogens in plants, is located on wheat chromosome 4DL and has been found in some advanced wheat lines such as RL6077 (Herrera-Foessel et al., 2011). In contrast to Lr34, the Lr67 gene has not been widely used to produce resistant cultivars for commercial wheat production. Although initial reports based on plant phenotypes (Dyck et al., (1994)) indicated 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 as Lr67. Although Lr67 also causes leaf tip necrosis like Lr34 and provides partial, broad-spectrum, adult-plant resistance to leaf rust and stripe rust, these are distinct genes.

[0005] It is necessary to determine the molecular basis of genes such as Lr67 that provide quantitative, non-race-specific, adult-plant pathogen resistance or partial resistance to broad-spectrum pathogens. SUMMARY OF THE INVENTION

[0006] The present inventors have identified novel transporter polypeptides and their encoding genes for conferring resistance in plants to one or more biotrophic fungal pathogens.

[0007] In one aspect, the invention provides a recombinant cell comprising an exogenous polynucleotide encoding a polypeptide characterized by one or more or all of the following:

[0008] i) When expressed in a plant, the polypeptide confers resistance in the plant to one or more biotrophic fungal pathogens, preferably confers resistance to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew,

[0009] ii) When expressed in a cell, the polypeptide is less effective in transporting glucose across the cell membrane than a polypeptide comprising the amino acids of the sequence provided in SEQ ID NO:4,

[0010] iii) When expressed in a cell, the polypeptide functions effectively as a sugar transporter,

[0011] iv) The polypeptide comprises the sequence provided in SEQ ID NO:1 or an amino acid sequence having at least 40% identity to SEQ ID NO:1 or an amino acid of a biologically active fragment thereof, and

[0012] v) The polypeptide does not contain glycine at the position corresponding to amino acid number 144 of SEQ ID NO:1, preferably the polypeptide contains an amino acid other than glycine at the position corresponding to amino acid number 144 of SEQ ID NO:1,

[0013] wherein the polynucleotide is operably linked to a promoter capable of directing the expression of the polynucleotide in a cell.

[0014] In a preferred embodiment, the polypeptide has at least characteristics i) and iv), i), ii) and iv), ii) and iv), or iv) and v), more preferably characteristics i), iv) and v), i), ii), iv) and v), or i), iv) and v).

[0015] In one embodiment, 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.

[0016] 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.

[0017] In one embodiment, the promoter directs gene expression in leaves and / or stem cells.

[0018] Preferably, if the polypeptide does not contain glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1, the polypeptide comprises an amino acid sequence having at least 40% identity to one or more or all of SEQ ID NO: 1, 4, or 7 to 9 or a bioactive fragment thereof.

[0019] In a preferred embodiment, the polypeptide contains 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.

[0020] In yet another preferred embodiment, the polypeptide does not contain valine at the position corresponding to amino acid number 387 of SEQ ID NO:1. Preferably, the polypeptide contains an amino acid other than valine at the position corresponding to amino acid number 387 of SEQ ID NO:1. More preferably, the polypeptide contains 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.

[0021] In one embodiment, the exogenous polynucleotide is integrated into the genome of the cell.

[0022] In yet another embodiment, the polypeptide comprises an amino acid sequence having the sequence provided in SEQ ID NO:1 or an amino acid sequence having at least 80% identity, at least 90% identity, or at least 95% identity with SEQ ID NO:1 or a bioactive fragment thereof.

[0023] In one embodiment, the polypeptide comprises 12 transmembrane domains.

[0024] On the other hand, the present invention provides a transgenic plant comprising the cell of the present invention, wherein the transgenic plant is an exogenous polynucleotide transgenic.

[0025] In a preferred embodiment, each somatic cell of the plant comprises the exogenous polynucleotide.

[0026] In yet another preferred embodiment, the plant has enhanced resistance to one or more biotrophic fungal pathogens as compared to an isogenic plant lacking the exogenous polynucleotide, 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.

[0027] In yet another embodiment, the plant has enhanced resistance to one or more biotrophic fungal pathogens during the seedling growth stage.

[0028] In another embodiment, the plant comprises one or more other exogenous polynucleotides encoding plant pathogen resistance polypeptides other than the Lr67 polypeptide, preferably the Lr34 polypeptide, the Sr33 polypeptide, or the 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.

[0029] Preferably, the plant is a cereal plant. Examples of the 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.

[0030] In one embodiment, the promoter directs gene expression in the aerial parts of the plant, such as leaves and / or stems.

[0031] Preferably, the exogenous polynucleotide of the plant is homozygous.

[0032] In yet another embodiment, the plant is grown in a field.

[0033] Also provided is a population of at least 100 plants of the invention grown in a field.

[0034] In another aspect, the invention provides a method for 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 confers resistance or susceptibility to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, comprising:

[0035] i) obtaining a polynucleotide operably linked to a promoter, the polynucleotide encoding a polypeptide comprising an amino acid sequence having the sequence provided in SEQ ID NO:1 or an amino acid sequence having at least 40% identity to a SEQ ID NO:1, or a bioactive fragment thereof,

[0036] ii) introducing the polynucleotide into a plant,

[0037] 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 the degree of resistance or susceptibility to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, relative to an isogenic plant lacking the polynucleotide, and

[0038] iv) optionally, if the level of resistance or susceptibility is increased, selecting the polynucleotide encoding the polypeptide that confers resistance or susceptibility to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, more preferably confers resistance or susceptibility to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew when expressed.

[0039] In one embodiment, one or more of the following apply to the method,

[0040] a) The polynucleotide comprises nucleotides having the sequence provided in SEQ ID NO:2 or SEQ ID NO:3, a sequence having at least 40% identity 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.

[0041] b) The plant is a cereal plant such as a wheat plant or a grapevine plant,

[0042] c) The polypeptide is a plant polypeptide or a mutant thereof, and

[0043] d) Step ii) further comprises stably integrating a polynucleotide operably linked to a promoter into the genome of the plant

[0044] e) The polypeptide is characterized by one or more of the features defined above in relation to the cells of the invention.

[0045] On the other hand, the invention provides a substantially purified and / or recombinant polypeptide characterized by one or more or all of the following:

[0046] i) When expressed in a plant, the polypeptide confers resistance to one or more biotrophic fungal pathogens on the plant, which fungal pathogens are preferably rust, mildew or both rust and mildew, and more preferably confers resistance to one or more or all of leaf rust, stripe rust, stem rust and powdery mildew,

[0047] ii) When expressed intracellularly, the polypeptide is less effective than a polypeptide comprising the amino acids having the sequence provided in SEQ ID NO:4 in transporting glucose across the cell membrane,

[0048] iii) When expressed intracellularly, the polypeptide is effective as a sugar transporter,

[0049] iv) The polypeptide comprises the amino acid sequence having the sequence provided in SEQ ID NO:1 or an amino acid sequence having at least 40% identity with SEQ ID NO:1, or the amino acids of a biologically active fragment thereof, and

[0050] v) The polypeptide does not contain glycine at the position corresponding to amino acid number 144 of SEQ ID NO:1, and preferably the polypeptide contains an amino acid other than glycine at the position corresponding to amino acid number 144 of SEQ ID NO:1.

[0051] In a preferred embodiment, the polypeptide is characterized by one or more of the features defined above in relation to the cells of the invention.

[0052] On the other hand, the polypeptide comprises the amino acid sequence provided in SEQ ID NO:1 or an amino acid sequence having at least 80% identity, at least 90% identity or at least 95% identity with SEQ ID NO:1 or the amino acids of a biologically active fragment thereof.

[0053] 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 facilitates the purification or detection of the fusion protein.

[0054] In another aspect, the present invention provides an isolated and / or exogenous polynucleotide comprising a nucleotide having a sequence provided in SEQ ID NO:2 or SEQ ID NO:3, a sequence having at least 40% identity to either 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.

[0055] In another aspect, the present invention provides a chimeric vector comprising the polynucleotide of the present invention.

[0056] Preferably, the polynucleotide is operably linked to a promoter.

[0057] In 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.

[0058] 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.

[0059] 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.

[0060] In another aspect, the present invention provides a method for producing the polypeptide of the present invention, the method comprising expressing the polynucleotide of the present invention in a cell expression system or a cell-free expression system.

[0061] Preferably, the method further comprises isolating the polypeptide.

[0062] In another aspect, the present invention provides a method for producing the cell of the present invention, the method comprising the step of introducing the polynucleotide of the present invention or the vector of the present invention into a cell.

[0063] Preferably, the cell is a plant cell.

[0064] In another aspect, the present invention provides a method for producing a transgenic plant of the present invention, the method comprising the following steps:

[0065] i) introducing the polynucleotide of the present invention and / or the vector of the present invention into a cell of a plant,

[0066] ii) regenerating a transgenic plant from the cell, and

[0067] iii) Optionally harvesting seeds from the plant, and / or

[0068] iv) Optionally producing one or more progeny plants from the transgenic plant, thereby producing a transgenic plant.

[0069] On the other hand, the present invention provides a method for producing a plant that has integrated a polynucleotide encoding the polypeptide of the present invention into its genome, the method comprising the steps

[0070] i) Crossing two parental plants, wherein at least one of the plants comprises a polynucleotide encoding a polypeptide,

[0071] ii) Screening for the presence or absence of the polynucleotide in one or more progeny plants from the cross, and

[0072] iii) Selecting the progeny plants that comprise the polynucleotide, thereby producing a plant.

[0073] In one embodiment, the polypeptide comprises an amino acid sequence having the sequence provided in SEQ ID NO:1 or an amino acid sequence that is at least 40% identical to SEQ ID NO:1, or a bioactive fragment thereof, and wherein when expressed in a plant, the polypeptide confers on the plant resistance to one or more biotrophic fungal pathogens, which fungal pathogen is preferably rust, mildew or both rust and mildew, and preferably confers resistance to one or more or all of leaf rust, stripe rust, stem rust and powdery mildew.

[0074] In one embodiment, at least one of the parental plants is a tetraploid or hexaploid wheat plant. In another embodiment, the parental plant is a grapevine.

[0075] In another embodiment, step ii) comprises analyzing the polynucleotide in a sample from the plant containing DNA.

[0076] In yet another embodiment, step iii) comprises

[0077] i) Selecting progeny plants that are homozygous for the polynucleotide, and / or

[0078] ii) Analyzing the resistance of the plant or one or more of its progeny plants to one or more biotrophic fungal pathogens, which fungal pathogen is preferably rust, mildew or both rust and mildew, and more preferably analyzing the resistance to one or more or all of leaf rust, stripe rust, stem rust and powdery mildew.

[0079] In another embodiment, the method further comprises

[0080] iv) Backcrossing the progeny of the hybridization of step (i) with a plant having the same genotype as the first parental plant lacking the polynucleotide encoding the polypeptide a sufficient number of times to produce a plant having the genotype of the majority of the first parental plant but including the polynucleotide, and

[0081] iv) Selecting progeny plants that are resistant to one or more biotrophic fungal pathogens, preferably rust, mildew, or both rust and mildew, more preferably resistant to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew.

[0082] In one embodiment, the method further comprises the step of analyzing at least one other genetic marker of the plant.

[0083] Also provided is a plant produced by the method of the present invention.

[0084] On the other hand, the present invention provides the use of the polynucleotide or vector of the present invention for producing recombinant cells and / or transgenic plants.

[0085] In one embodiment, the transgenic plant has enhanced resistance to one or more biotrophic fungal pathogens compared to an isogenic plant lacking the exogenous polynucleotide and / or vector, 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.

[0086] In yet another aspect, the present invention provides a method for identifying a plant comprising a polynucleotide encoding the polypeptide of the present invention, the method comprising the following steps

[0087] i) Obtaining a nucleic acid sample from a plant, and

[0088] ii) Screening the sample for the presence or absence of the polynucleotide.

[0089] In one embodiment, the presence of the polynucleotide indicates that the plant has enhanced resistance to one or more biotrophic fungal pathogens compared to an isogenic plant lacking the exogenous polynucleotide, 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.

[0090] In one embodiment, the genomic region encompassing the polynucleotide is amplified and the amplification product is sequenced to determine whether it encodes a polypeptide. Primers for amplification and for sequencing can be readily designed by a person skilled in the art.

[0091] In yet another embodiment, the method identifies the transgenic plant of the present invention.

[0092] In an embodiment, the method further comprises producing a plant from the seed prior to step i).

[0093] There is also provided a plant part of a plant of the present invention.

[0094] In one embodiment, the plant part is a seed comprising an exogenous polynucleotide encoding a polypeptide of the present invention.

[0095] In another aspect, the present invention provides a method for producing a plant part, the method comprising,[[]]

[0096] a) growing a plant of the present invention, and

[0097] b) harvesting the plant part.

[0098] In another aspect, the present invention provides a method for producing flour, whole wheat flour, starch or other products obtained from seeds, the method comprising;

[0099] a) obtaining a seed of the present invention, and

[0100] b) extracting flour, whole wheat flour, starch or other products.

[0101] In another aspect, the present invention provides a product produced from a plant of the present invention and / or a plant part of the present invention.

[0102] In one embodiment, the part is a seed.

[0103] In one embodiment, the product is a food product or a beverage product. Examples include, but are not limited to;

[0104] i) a food product selected from the group consisting of flour, starch, fermented or unfermented bread, pasta, noodles, animal feed, breakfast cereals, snack foods, pastries, malt beverages, beer, cakes and flour-containing sauces, or

[0105] ii) a beverage product of beer or malt beverage.

[0106] 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.

[0107] In another aspect, the present invention provides a method for preparing a food product of the present invention, the method comprising mixing a seed or flour, whole wheat or starch from the seed with another food ingredient.

[0108] In another aspect, the present invention provides a method for preparing a malt beverage, which comprises the step of germinating a seed of the present invention.

[0109] The use of the plant of the present invention or a part thereof as animal feed or for producing feed for animal consumption or food for human consumption is also provided.

[0110] In another aspect, the present invention provides a composition comprising one or more polypeptides, polynucleotides, vectors, or recombinant cells of the present invention and one or more acceptable carriers.

[0111] In another aspect, the present invention provides a method for identifying a compound that binds to a polypeptide comprising an amino acid sequence provided in SEQ ID NO:1 or having at least 40% identity to the amino acid sequence of SEQ ID NO:1 and its bioactive fragments, the method comprising:

[0112] i) contacting the polypeptide with a candidate compound, and

[0113] ii) determining whether the compound binds to the polypeptide.

[0114] In one embodiment, the polypeptide is embedded in a cell membrane, preferably in a plant cell membrane.

[0115] In another aspect, the present invention provides a method for identifying a compound that is transported across a cell membrane by a polypeptide comprising an amino acid sequence provided in SEQ ID NO:1 or SEQ ID NO:4 or having at least 40% identity to one or both of the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:4, or its bioactive fragments, the method comprising:

[0116] i) contacting the polypeptide embedded in a cell membrane, which is preferably the membrane of a plant cell, with a candidate compound,

[0117] ii) determining whether the compound is transported from one side of the membrane to the other side by the polypeptide.

[0118] Unless otherwise specifically stated, any embodiment herein should be modified as necessary to apply to any other embodiment.

[0119] The present invention is not limited to the scope of the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly also within the scope of the present invention, as described herein.

[0120] In this specification, unless otherwise specifically stated or the context otherwise requires, reference should be made to a single step, composition of matter, group of steps, or group of compositions of matter to cover one and more (i.e., one or more) of these steps, compositions of matter, group of steps, or group of compositions of matter.

[0121] The present invention will be described hereinafter by way of the following non-limiting examples and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 - Comparative genomics and mutation analysis.

[0123] Figure 2 – The deleted Hsp70 gene is completely linked to Lr67.

[0124] Figure 3 – Comparative genomics and mutation analysis including SUT and PIP.

[0125] Figure 4 – Nucleotide changes found in the Lr67 variant.

[0126] Figure 5 – Amino acid sequence of the SUT polypeptide encoded by the wheat Lr67 (resistant) allele (514 amino acids, SEQ ID NO: 1). Arginine at position 144 and leucine at position 387 in the predicted fourth transmembrane domain distinguish the resistant Lr67 and susceptible Lr67 polypeptides.

[0127] Figure 6 – Nucleotide sequence of the cDNA corresponding to the Lr67 resistant allele (SEQ ID NO: 3). The positions of two SNPs at positions 514 and 1243 that distinguish + / - Lr67; these result in amino acid substitutions in the encoded polypeptide. The translation start codon is at positions 85 - 87, and the translation stop codon is at positions 1627 - 1629.

[0128] Figure 7 – Amino acid sequence of the Lr67 susceptibility polypeptide (SUT) (SEQ ID NO: 4).

[0129] Figure 8 – Nucleotide sequence of the DNA corresponding to the Lr67 susceptibility allele SUT (SEQ ID NO: 6).

[0130] Figure 9 – Alignment of the amino acid sequence of the wheat Lr67 (resistant) polypeptide (SEQ ID NO: 1) with a homologous Arabidopsis thaliana polypeptide (Arath; from GenBank accession number NP_198006, 526 amino acids) (SEQ ID NO: 7). Asterisks indicate identical amino acid residues at that position, and “+” indicates similar amino acids at that position.

[0131] Figure 10– Alignment of the amino acid sequence of wheat Lr67 (resistant) polypeptide (SEQ ID NO:1) with the homologous rice (Oryza sativa) polypeptide (GenBank accession number 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.

[0132] Figure 11 – Glucose uptake by yeast cells expressing Lr67 (resistant) and Lr67 (susceptible) proteins.

[0133] Figure 12 – Glucose uptake kinetics in yeast expressing Lr67 (susceptible).

[0134] Figure 13 – Effect of different amino acids on glucose transport by Lr67 protein.

[0135] Figure 14 – Nucleotide sequence of the genomic fragment corresponding to the protein-coding region of the Lr67 (susceptible) gene. The sequence starts at the translation initiation codon ATG and ends at the translation termination TGA. The two introns within the protein-coding region are nucleotides 137 - 876 (intron 1, 740 nt) and 1197 - 3154 (intron 2, 1958 nt).

[0136] Sequence Listing Description

[0137] SEQ ID NO:1 – Wheat Lr67 (resistant) protein.

[0138] SEQ ID NO:2 – Open reading frame encoding wheat Lr67 (resistant) protein.

[0139] SEQ ID NO:3 – cDNA encoding wheat Lr67 (resistant) protein.

[0140] SEQ ID NO:4 – Wheat Lr67 (susceptible) protein

[0141] SEQ ID NO:5 – Open reading frame encoding wheat Lr67 (susceptible) protein.

[0142] SEQ ID NO:6 – cDNA encoding wheat Lr67 (susceptible) protein.

[0143] SEQ ID NO:7 – Arabidopsis Lr67 protein.

[0144] SEQ ID NO:8 – Rice Lr67 protein.

[0145] SEQ ID NO:9 - Vitis vinifera Lr67 protein.

[0146] SEQ ID NO:10 - Gene encoding wheat Lr67 (susceptible) protein. Detailed implementation mode

[0147] Conventional Techniques and Definitions

[0148] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of cell culture, molecular genetics, plant molecular biology, protein chemistry, and biochemistry).

[0149] Unless otherwise stated, the recombinant proteins, 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 from 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 until now), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988) and J.E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates until now).

[0150] The term "and / or", e.g., "X and / or Y", should be understood to mean either "X and Y" or "X or Y", and should be used to provide explicit support for both meanings or for one of them.

[0151] In this specification, the word "comprise" or variants such as "comprises" or "comprising" will be understood to imply the inclusion of the stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0152] Polypeptide

[0153] The present invention relates to polypeptides which, when expressed in plants, confer resistance to one or more biotrophic fungal pathogens on the plants, preferably to one or more or all of leaf rust, stripe rust, stem rust and powdery mildew. The present invention also relates to polypeptides which, when expressed intracellularly, are less effective than polypeptides comprising the amino acids of the sequence provided in SEQ ID NO:4 in transporting glucose across the cell membrane. In addition, the present invention also relates to polypeptides which, when expressed intracellularly, function as sugar transporters. In a preferred embodiment, the polypeptide is encoded by an allele or variant of the Lr67 gene which confers resistance to one or more biotrophic fungal pathogens on the plants. 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 isogenic plants lacking the polynucleotide encoding the polypeptide.

[0154] The term "Lr67" as used herein refers to a family of proteins sharing a high degree of primary amino acid sequence identity, e.g., at least 40%, at least 80%, at least 90% or at least 95% identity with one or more of the amino acid sequences provided in SEQ ID NO: 1, 4 or 7 to 9, preferably SEQ ID NO: 1. The present invention has demonstrated that some variants of the Lr67 protein family, when expressed in plants, confer resistance to one or more biotrophic fungal pathogens on the plants, preferably conferring resistance 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, the variants conferring resistance are referred to herein as Lr67 (resistance) polypeptides, while those not conferring resistance (such as 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 contain glycine at the position corresponding to amino acid number 144 of SEQ ID NO: 1, preferably the polypeptide contains 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.

[0155] "Resistance" as used herein is a relative term, since the presence of the polypeptides of the present invention (i) reduces the disease symptoms in plants containing a resistance-conferring gene (R (resistance) gene), relative to plants lacking the R gene, and / or (ii) reduces pathogen multiplication or spread in plants or in a population of plants containing the R gene. Resistance as used herein is relative to the "susceptible" response of plants to the same pathogen. Generally, the presence of an R gene improves at least one production characteristic of a plant containing the R gene, such as grain yield, when the plant is infected with a pathogen, compared to an isogenic plant that is infected with the pathogen but lacks the R gene. The isogenic plant may have a certain degree of resistance to the pathogen or may be classified as susceptible. Thus, the terms "resistance" and "enhanced resistance" are generally used interchangeably herein. In addition, the polypeptides of the present invention do not necessarily confer complete pathogen resistance, e.g., when certain symptoms still appear in the plant or in the plant population or there is some pathogen multiplication or infection but in reduced numbers. Resistance may occur only at certain stages of plant growth, e.g., in mature plants (fully grown in size) and less mature or not at all mature, in the seedling stage, or at all stages of plant growth by using transgenic strategies to express the Lr67 polypeptide in the plant, providing resistance to the plants of the present invention during their growth and development. Enhanced resistance is determined by some methods known in the art, such as analyzing the number of plant pathogens and / or analyzing plant growth or the amount of plant loss or disease symptoms in the presence of the pathogen, and comparing one or more of these parameters with an isogenic plant lacking the exogenous gene encoding the polypeptide of the present invention.

[0156] As used herein, a "sugar transporter" is a membrane-bound protein that facilitates the movement of sugar across a membrane, such as from outside the cell into the cell, or in the opposite direction from inside the cell to outside the cell, or across the membrane of subcellular organelles within the cell. This facilitation may be active, using an energy source such as moving across the membrane from an ion gradient, or passive. For the Lr67 (susceptible) protein, the sugar can be glucose. In one embodiment, the sugar is a monosaccharide, preferably a hexose monosaccharide or a pentose polysaccharide. In one embodiment, the sugar may be modified, such as a sugar alcohol or a phosphorylated sugar.

[0157] The phrase "is less effective than a polypeptide comprising the amino acids of the sequence provided in SEQ ID NO:4 in transporting glucose across the cell membrane" as used herein means that the polypeptide of the present invention has a glucose-transporting ability into cells, such as yeast cells or plant cells, that is less than 50%, or less than 25%, or less than 10% of that of a polypeptide comprising the amino acids of the sequence provided in SEQ ID NO:4. This can be readily determined as described herein (see, for example, Figure 11 and the related experimental details).

[0158] By "substantially purified polypeptide" or "purified polypeptide", we mean a polypeptide that has generally been separated from the lipids, nucleotides, other peptides, and other impurity molecules to which it is bound in its native state. Preferably, the substantially purified polypeptide is at least 90% separated from other components to which it is bound in its native state. In one embodiment, the polypeptide of the present invention has an amino acid sequence other than that of the Lr67 polypeptide that occurs in its native state, i.e., it is an amino acid sequence variant.

[0159] The transgenic plants and host cells of the present invention may contain an exogenous polynucleotide encoding the polypeptide of the present invention. In these cases, the plants and cells produce a recombinant polypeptide. In the context of a polypeptide, the term "recombinant" when produced by a cell refers to a polypeptide encoded by an exogenous polynucleotide, the polynucleotide of which has been introduced into the cell or a progenitor cell by recombinant DNA or RNA techniques (such as, for example, transformation). Generally, the cell contains a non-endogenous gene that causes a change in the 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.

[0160] The terms "polypeptide" and "protein" are generally used interchangeably.

[0161] The % identity of polypeptides was determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program), using a gap creation penalty = 5 and a gap extension penalty = 0.3. The query sequence was at least 400 amino acids long, and GAP analysis was performed on two sequences over a region of at least 400 amino acids. More preferably, the query sequence was at least 500 amino acids long, and GAP analysis was performed on two sequences over a region of at least 500 amino acids. Even more preferably, GAP analysis was performed on two sequences over their full length, which is approximately 514 amino acid residues for the Lr67 polypeptide.

[0162] As used herein, a "bioactive fragment" is a portion of a polypeptide of the invention that retains the defined activity of the full-length polypeptide, such as one or both of the following: (i) when expressed in a plant, such as wheat, confers (enhanced) activity against one or more biotrophic fungal pathogens, preferably confers (enhanced) activity against one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew, and (ii) when expressed intracellularly, the polypeptide functions effectively as a sugar transporter, preferably less effectively than a polypeptide comprising the amino acids of the sequence provided in SEQ ID NO: 4 in transporting glucose across the cell membrane. Bioactive 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 bioactive fragment retains at least 50%, at least 75%, or at least 90% of the activity of the full-length protein. In one embodiment, the bioactive fragment comprises 12 transmembrane domains.

[0163] With respect to the defined polypeptides, it is understood that higher % identity numbers than those provided above will encompass preferred embodiments. Thus, where applicable, and in accordance with the lowest % identity numbers, preferably, the polypeptide comprises an amino acid sequence having 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% identity to the relevant named SEQ ID NO.

[0164] In one embodiment, the polypeptide of the 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 its natural state.

[0165] The phrase "at the position corresponding to amino acid number" or variants thereof used herein refers to the relative position of an amino acid compared to the surrounding amino acids. In this regard, in some embodiments, when aligned with, for example, SEQ ID NO:1, the polypeptide of the invention may have deletion or substitution mutations that alter the relative positioning of the amino acids. For example, as Figure 9 shown, amino acid number 178 of wheat Lr67 (resistance) corresponds to amino acid number 179 of the homologous Arabidopsis Lr67 protein.

[0166] Amino acid sequence mutants of the polypeptide of the invention can be prepared by introducing appropriate nucleotide variations into the nucleic acid of the 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. Combinations of deletions, insertions, and substitutions can occur in the final construct, provided that the final peptide product possesses the desired characteristics. Preferably, the amino acid sequence mutant relative to the reference wild-type polypeptide has only one, two, three, four, or fewer than 10 amino acid changes.

[0167] The mutated (altered) polypeptide can be prepared using any technique known in the art, for example, using directed or rational design strategies (see below). Products derived from the mutated / altered DNA can be easily screened using the techniques described herein to determine whether they possess one or more of the following characteristics: (i) when expressed in a plant, such as wheat, confer one or more (enhanced) resistances to biotrophic fungal pathogens, preferably confer one or more or all (enhanced) resistances to leaf rust, stripe rust, stem rust, and powdery mildew, and (ii) when expressed intracellularly, the encoded polypeptide is less effective than a polypeptide comprising the amino acids of the sequence provided in SEQ ID NO:4 in transporting glucose across the cell membrane, and (iii) when expressed intracellularly, the polypeptide functions effectively as a sugar transporter. For example, with respect to (i), the method may include producing transgenic plants expressing the mutated / altered DNA and determining the effect of the pathogen on plant growth.

[0168] When designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on the characteristics to be modified. The site of the mutation can be modified individually or continuously, for example, by (1) first making substitutions with conservative amino acid selections and then more radical selections depending on the result to be achieved, (2) deleting the target residue, or (3) inserting other residues at adjacent positions.

[0169] The range of amino acid sequence deletion is generally from about 1 to 15 residues, more preferably about 1 to 10 residues, and typically about 1 to 5 contiguous residues.

[0170] Substitution mutants remove at least one amino acid residue from the polypeptide and insert a different residue in its place. It is desirable that it retains a certain activity, preferably non-substitution or only conservative substitutions at amino acid positions that are highly conserved in the relevant protein family. Examples of conservative substitutions are shown in Table 1 under the heading "Exemplary Substitutions".

[0171] In a preferred embodiment, the mutant / variant polypeptide has one or two or three or four conservative amino acid changes compared to the naturally occurring polypeptide. Table 1 provides details of conservative amino acid changes. In a preferred embodiment, the changes are not within one or more motifs that are highly conserved among the different polypeptides provided herein, and / or are not within the 12 transmembrane helices of the Lr67 polypeptide. As known to those skilled in the art, it can be reasonably predicted that such minor changes will not alter the activity of the polypeptide upon recombinant cell expression.

[0172] The primary amino acid sequence of the polypeptides of the present invention can be used to design variants / mutants based on comparison with closely related sugar transporter polypeptides (e.g., as Figure 9 and 10 shown). As can be understood by those skilled in the art, residues that are highly conserved within closely related proteins are less likely to be altered than less conserved residues, particularly non-conservative substitutions and activity retention (see above).

[0173] 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 cell ligands, etc. Polypeptides may be post-translationally modified in cells, for example by phosphorylation, which regulates their activity. These modifications can be used to increase the stability and / or biological activity of the polypeptides of the present invention.

[0174] Table 1. Exemplary Substitutions.

[0175]

[0176] Directed Evolution

[0177] In directed evolution, random mutagenesis is applied to the protein, and a selection scheme is used to pick out mutants with desired properties, e.g., increased activity. Then the next round of mutagenesis and selection is applied. Typically, directed evolution strategies include three steps:

[0178] 1) Diversification: Mutations and / or random recombination of genes encoding proteins of interest are induced to create a library of large gene variants. Variant gene libraries can be generated by error-prone PCR (see, e.g., Leung, 1989; Cadwell and Joyce, 1992) from a library of DNase I-digested fragments prepared from a parental template (Stemmer, 1994a; Stemmer, 1994b; Crameri et al., 1998; Coco et al., 2001), from degenerate oligonucleotides (Ness et al., 2002, Coco, 2002) or from a mixture of both, or even from an undigested parental template (Zhao 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 recombinant parental sequences by homologous or non-homologous recombination, (Ostermeier et al., 1999; Volkov et al., 1999; Sieber et al., 2001). Variant gene libraries can also be generated by subcloning the gene of interest into a suitable vector, transforming the vector into a "mutator gene" system such as E. coli XL-1red (Stratagene), and propagating the transformed bacteria for an appropriate number of generations. Variant gene libraries can also be generated by DNA shuffling of the gene of interest (i.e., homologous recombination of a selected library of mutant genes by random fragmentation and reassembly in vitro), as extensively described by Harayama (1998).

[0179] 2) Selection: Screening or selection is used to test for the presence of mutants (variants) in the library that possess the desired property. Screening allows for the manual identification and isolation of high-performance mutants, while selection automatically eliminates all non-functional mutants. Screening may include screening for the presence of known conserved amino acid motifs. Alternatively, or in addition, screening may include expressing the mutant polynucleotide in a host organism or a part thereof and assaying the activity level.

[0180] 3) Amplification: The variants identified in selection or screening are replicated many-fold so that the researcher can sequence their DNA to see what mutations have occurred.

[0181] These three steps together are referred to as one "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, biochemical methods are used to characterize all the evolved protein or polynucleotide mutants.

[0182] Rational design

[0183] Based on the known information of protein structure and folding, proteins are rationally designed. This can be accomplished by de novo design or by redesign based on natural scaffolds (see, e.g., Hellinga, 1997; and Lu and Berry, Protein Structure Design and Engineering, Handbook of Proteins 2, 1153-1157 (2007)). Protein design generally involves identifying sequences that fold into a given or target structure, which can be done using computer models. Computer protein design algorithms search the sequence-conformation space for sequences that are low in 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 the protein. These energy functions typically include a combination of molecular mechanics, statistical (i.e., knowledge-based), and 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.

[0184] Polynucleotides and Genes

[0185] 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, such as formed by an automated synthesizer, which may be conjugated 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 modified nucleotides not found in nature, all of which are well known to those skilled in the art. The polymer may be single-stranded, essentially double-stranded, or partially double-stranded. Base pairing as used herein refers to the standard base pairing between nucleotides, including G:U base pairing. "Complementary" means that two polynucleotides are capable of base pairing (hybridizing) along a portion of their lengths or the full length of one or both of them. "Hybridized polynucleotides" means that the polynucleotide base pairs with its complement. The term "polynucleotide" as used herein is interchangeable with the term "nucleic acid". Preferred polynucleotides of the present invention encode the polypeptides of the present invention.

[0186] "Isolated polynucleotide" means that if the polynucleotide is found in nature, the polynucleotide has generally been isolated from the polynucleotide sequences to which it is bound or linked in its natural state. Preferably, if the isolated polynucleotide is found in nature, it is at least 90% isolated from other components to which it is bound in its natural state. Preferred polynucleotides are not naturally occurring, for example, by covalently linking two shorter polynucleotide sequences in a manner not found in nature (chimeric polynucleotides).

[0187] This 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 that contains a protein-coding region or that is transcribed but not translated in a cell, as well as associated non-coding and regulatory regions. These associated regions are typically located at a distance of about 2 kb on either side of the coding or transcribed region, adjacent to the 5'-end and 3'-end. In this regard, a gene may include control signals such as promoters, enhancers, termination, and / or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals, in which case the gene is referred to as a "chimeric gene". Sequences located at the 5'-end of the coding region and present on the mRNA are referred to as 5'-untranslated sequences. Sequences located at the 3'-end or downstream of the coding region and present on the mRNA are referred to as 3'-untranslated sequences. The term "gene" includes genes in cDNA and genomic form.

[0188] As used herein, the "Lr67 gene" refers to a nucleotide sequence that is homologous to the 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, preferably conferring resistance to one or more or all of leaf rust, stripe rust, stem rust, and powdery mildew (see, for example, SEQ ID NO:3). The Lr67 gene includes naturally occurring alleles or variants present in cereals such as wheat, as well as artificially produced variants.

[0189] The genomic form or clone of a gene containing a transcriptional region can be interrupted by non-coding sequences referred to as "introns" or "intervening regions" or "intervening sequences", which may be homologous or heterologous relative 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; thus, introns are not present in messenger RNA (mRNA). Introns can 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 the RNA sequence present in mature mRNA or mature mRNA when the RNA molecule is not translated. The mRNA is used to specify the sequence or order of amino acids in the nascent polypeptide during translation. The term "gene" includes synthetic or fusion molecules that encode all or part of the proteins of the present invention described herein, as well as the complementary nucleotide sequences of any of the foregoing. A gene can be introduced into a suitable vector for extrachromosomal maintenance in a cell or, preferably, for integration into the host genome.

[0190] As used herein, a "chimeric gene" refers to any gene that contains covalently linked sequences not found in nature. Typically, a chimeric gene contains regulatory sequences and transcriptional sequences or protein-coding sequences that are not found together in nature. Thus, a chimeric gene may contain regulatory sequences and coding sequences derived from different sources, or regulatory sequences 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 can be operably linked to a promoter or polyadenylation / termination region heterologous to the Lr67 gene, thereby forming a chimeric gene. The term "endogenous" as used herein is used to denote a substance that is normally present or produced in an unaltered plant at the same developmental stage as the plant under study. An "endogenous gene" is 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 by recombinant DNA technology. The terms "exogenous polynucleotide" or "exogenous polynucleotides" or "heterologous polynucleotide" etc. refer to any nucleic acid that has been introduced into the genome of a cell by experimental manipulation.

[0191] An exogenous or foreign gene may be a gene inserted into a non-natural organism, a natural gene introduced into a new location within a natural host, or a chimeric gene. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The term "genetically modified" includes the introduction of a gene into a cell by transformation or transduction, the mutation of a gene in a cell, or the alteration or regulation of the regulation of a gene within a cell or organism or its progeny that has had these actions performed on it.

[0192] 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 containing a non-endogenous polynucleotide that causes a change in the yield of the encoded polypeptide, such as an exogenous polypeptide that increases the expression of an endogenous polypeptide, or a cell that does not produce a polypeptide in its natural state. The 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 that have been subsequently purified and separated from at least some other components and produced within these cells or cell-free systems. An exogenous polynucleotide (nucleic acid) may be a continuous stretch of nucleotides that occurs in nature, or may contain two or more continuous stretches of nucleotides from different sources (naturally occurring and / or synthetic) that are ligated to form a single polynucleotide. Generally, this chimeric polynucleotide contains at least one open reading frame encoding a polypeptide of the present invention that is operably linked to a promoter that is suitable for driving the transcription of the open reading frame within the cell of interest.

[0193] The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program), with a gap creation penalty = 5 and a gap extension penalty = 0.3. The query sequence length is at least 1,200 nucleotides, and the GAP analysis aligns the two sequences within a region of at least 1,200 nucleotides. More preferably, the query sequence length is at least 1,500 nucleotides, and the GAP analysis aligns the two sequences within a region of at least 1,500 nucleotides. Even more preferably, the GAP analysis aligns the full lengths of the two sequences.

[0194] Regarding the defined polynucleotides, it should be understood that numbers representing higher % identities than those provided above will encompass preferred embodiments. Thus, where applicable, and in accordance with the lowest value of the % identity numbers, preferably, the polynucleotide comprises a polynucleotide sequence having 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% identity to the relevant named SEQ ID NO.

[0195] In yet another embodiment, the invention relates to polynucleotides that are substantially identical to the polynucleotides specifically described herein. As used herein, with respect to polynucleotides the term "substantially identical" means a substitution of one or a few (e.g., 2, 3 or 4) nucleotides while maintaining at least one activity of the native protein encoded by the polynucleotide. Additionally, the term includes an addition or deletion of nucleotides that results in an increase or decrease in the size of the native protein encoded, while maintaining at least one activity of the native protein encoded by the polynucleotide by one or a few (e.g., 2, 3 or 4) amino acids.

[0196] The invention also relates to the use of oligonucleotides, for example in methods of screening for the polynucleotides of the invention, or in methods of encoding the polypeptides of the invention. As used herein, "oligonucleotide" is a polynucleotide having a length of up to 50 nucleotides. This minimum value of the oligonucleotide size is the size required for the formation of a stable hybrid between the oligonucleotide and the complementary sequence of the nucleic acid molecule of the invention. They can be RNA, DNA or any combination or derivative thereof. Generally, 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, this minimum value of the oligonucleotide size is the size required for the formation of a stable hybrid between the oligonucleotide and the complementary sequence of the target nucleic acid molecule. Preferably, the oligonucleotide length is at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides. The oligonucleotides of the invention used as probes are typically conjugated to a label, such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.

[0197] 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. The 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.

[0198] The polynucleotides and oligonucleotides of the present invention include those that hybridize to one or more of the sequences provided in SEQ ID NO:2, 3, 5, or 6 under stringent conditions. As used herein, stringent conditions are those (1) employing low ionic strength and high temperature for washing, for example, 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% NaDodSO4 at 50°C; (2) employing a denaturing agent such as formamide in hybridization at 42°C, for example, 50% (v / v) formamide and 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5, and 750 mM NaCl, 75 mM sodium citrate; or (3) employing 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate in 0.2x SSC and 0.1% SDS at 42°C.

[0199] The polynucleotides of the present invention may have one or more mutations, i.e., deletions, insertions, or substitutions of nucleotide residues, compared to the naturally occurring molecule. Mutants can 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 that are close to the genome of the reference polynucleotide or oligonucleotide as defined herein, and / or molecules that are capable of hybridizing thereto. For example, variants may include additional nucleotides (such as 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 substituted without affecting the ability of the oligonucleotide to hybridize to the target region. In addition, variants can be readily designed that hybridize tightly to regions of the plant genome (e.g., within 50 nucleotides) at the positions where the specific oligonucleotides defined herein hybridize. In particular, this includes polynucleotides that encode the same polypeptide or amino acid sequence but have nucleotide sequences that vary due to genetic code redundancy. The terms "polynucleotide variant" and "variant" also include naturally occurring allelic variants.

[0200] Nucleic Acid Construct

[0201] The present invention includes nucleic acid constructs comprising the polynucleotides of the present invention, as well as vectors and host cells containing these, methods for producing and using them, and their uses. The present invention refers to elements that are operably linked or connected. "Operably linked" or "operably connected", etc. mean that polynucleotide elements are connected in a functionally related manner. Generally, operably linked nucleic acid sequences are ligated continuously, continuously and in frame, and when necessary, two protein-coding regions are joined. When RNA polymerase transcribes two coding sequences into a single RNA, the coding sequences are "operably linked to" another coding sequence, and if translated, then it is translated into a single polypeptide having amino acids originating from the two coding sequences. The coding sequences do not need to be continuous with another sequence, as long as the expressed sequences can ultimately be processed to produce the desired protein.

[0202] As used herein, the terms "cis-acting sequence", "cis-acting element", or "cis-regulatory region", or "regulatory region", or similar terms shall mean any nucleotide sequence that, when in an appropriate position and linked to an expressible genetic sequence, is capable of regulating, at least in part, the expression of the genetic sequence. Those skilled in the art will know that cis-regulatory regions can activate, silence, enhance, inhibit, or alter 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 present invention, the cis-acting sequence is an activator sequence that enhances or stimulates the expression of an expressible genetic sequence.

[0203] "Operably connecting" a promoter or enhancer element to a transcribable polynucleotide means placing the transcribable polynucleotide (such as a protein-coding polynucleotide or other transcript) under the control 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 its variant at a distance from the transcription start site of the transcribable polynucleotide, approximately the same distance as between the control promoter and the protein-coding region in the natural environment; that is, the gene from which the promoter originated. As is known in the art, some variants can be accommodated within this distance without loss of function. Similarly, the preferred positioning of regulatory sequence elements (such as operons, enhancers, etc.) relative to the transcribable polynucleotide to be placed under its control is defined by the positioning of the element in the natural environment; that is, the gene from which it originated.

[0204] As used herein, "promoter" or "promoter sequence" refers to a region of a gene, generally upstream (5') of the RNA-coding region, which controls the initiation and level of transcription in a cell of interest. A "promoter" includes the transcriptional regulatory sequences of a classical genomic gene, such as TATA box and CCAAT box sequences, as well as other regulatory elements (i.e., upstream activating sequences, enhancers and silencers) that respond to developmental and / or environmental stimuli or change gene expression in a tissue-specific or cell-type specific manner. A promoter often, but not necessarily (e.g., some PolIII promoters), is located upstream of the structural gene whose expression it regulates. In addition, regulatory elements containing a promoter are often located within 2 kb of the transcriptional start site of a gene. A promoter may contain other specific regulatory elements located further distal from the start site to further enhance expression in a cell, and / or to alter the timing or inducibility of expression of the operably linked structural gene.

[0205] A "constitutive promoter" refers to a promoter that directs the expression of an operably linked transcribed sequence in many or all tissues of an organism such as a plant. As used herein, the term constitutive does not necessarily indicate that a gene is expressed at the same level in all cell types, but indicates that the gene is expressed in a wide range of cell types, although some variation in levels is often detectable. As used herein, "selective expression" refers to expression that occurs almost exclusively within a particular organ (e.g., in a plant), such as endosperm, embryo, leaf, fruit, tuber or root. In a preferred embodiment, the promoter selectively or preferentially expresses in the leaves and / or stems of a plant, preferably a cereal plant. Thus, selective expression may be contrasted with constitutive expression, which refers to expression in many or all tissues of a plant under most or all conditions the plant experiences.

[0206] Selective expression may also result in compartmentalization of the gene expression product in a particular plant tissue, organ or developmental stage. Compartmentalization in a particular subcellular location (e.g., plastid, cytosol, vacuole or apoplast space) may be achieved by inclusion of a signal polypeptide in the structure of the gene product for transport to the desired cellular compartment, or for semi-autonomous organelles (plastids and mitochondria) by direct integration of the transgene with appropriate regulatory sequences into the genome of the organelle.

[0207] A "tissue-specific promoter" or "organ-specific promoter" is a promoter that preferentially expresses in one tissue or organ as compared to many other tissues or organs, preferably most, if not all other tissues or organs in, e.g., a plant. Typically, the promoter expresses at a level 10-fold higher in the specific tissue or organ than in other tissues or organs.

[0208] In one embodiment, the promoter is a stem-specific promoter, a leaf-specific promoter, or a promoter that directs gene expression in the aerial parts of the plant (at least the stem and leaves) (green tissue-specific promoter) such as the ribulose-1,5-bisphosphate carboxylase (RUBISCO) promoter.

[0209] Examples of stem-specific promoters include, but are not limited to, those described in US 5,625,136 and Bam et al. (2008).

[0210] The promoters contemplated by the present invention may be derived from the host plant to be transformed or may originate from alternative sources that are functional in the region where they are located in the host plant. Other sources include Agrobacterium T-DNA genes, such as the gene promoters for nopaline, octapine, mannopine, or other opine promoters biosynthesis, tissue-specific promoters (see, 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 mono- and dicotyledonous plants are well known in the art (see, 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 promoters (CaMV 35S, 19S). Non-limiting methods for assessing promoter activity are disclosed by Medberry et al. (1992, 1993), Sambrook et al. (1989, ibid.), and US 5,164,316.

[0211] Alternatively or in addition, the promoter may be an inducible promoter or a developmentally regulated promoter capable of driving the expression of the introduced polynucleotide at an appropriate developmental stage such as in a plant. Other cis-acting sequences that may be employed include transcriptional and / or translational enhancers. Enhancer regions are well known to those skilled in the art and include the ATG translation initiation codon and adjacent sequences. When included, the initiation codon may be in frame with the coding sequence associated with the exogenous or heterologous polynucleotide to ensure translation of the entire sequence (if it is to be translated). The translation initiation region may be provided by the source of the transcriptional initiation region, or by the exogenous or heterologous polynucleotide. The sequence may also originate from the source of the promoter selected to drive transcription and be specifically modified to increase the translation of the mRNA.

[0212] The nucleic acid construct of the present invention may contain a 3' untranslated sequence of about 50 to 1000 nucleotide base pairs, which includes a transcription termination sequence. The 3' untranslated sequence may contain a transcription termination signal, which may or may not include a polyadenylation signal and any other regulatory signals capable of affecting mRNA processing. The polyadenylation signal functions to add a polyadenylate tract to the 3' end of the mRNA precursor. Although variations are not common, polyadenylation signals are generally recognized by the presence of homology to the canonical form of 5'AATAAA-3'. Transcription termination sequences that do not include a polyadenylation signal include terminators of PolI or PolIII RNA polymerase that contain a stretch of 4 or more thymidine. Examples of suitable 3' untranslated sequences are 3' transcribed untranslated regions that contain a polyadenylation signal from the octopine synthase (ocs) gene or the nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et al., 1983). Examples of suitable 3' untranslated 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.

[0213] Since the DNA sequence inserted at the transcription start site and the start of the coding sequence, i.e., the untranslated 5' leader sequence (5'UTR), can affect gene expression if it is translated and transcribed, a specific leader sequence can also be employed. Suitable leader sequences include sequences that contain sequences selected for guiding the optimal expression of foreign or exogenous DNA sequences. 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 improper initiation of translation.

[0214] Vector

[0215] The present invention includes the use of vectors for the manipulation and transfer of genetic constructs. A "chimeric vector" refers to a nucleic acid molecule, preferably a DNA molecule such as originating from a plastid, phage, or plant virus, into which a nucleic acid sequence 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 the target cell or tissue or progenitor cell or its tissue, or capable of integration into the genome of the defined host so that the cloned sequence may be replicated. Thus, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a linear or closed circular plastid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, when introduced into a cell, the vector may be integrated into the genome of the recipient cell and replicated along with the chromosome into which it has been integrated. The vector system may include a single vector or plastid, two or more vectors or plastids together containing the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of vector will generally depend on the compatibility of the vector with the cell into which it is to be introduced. The vector may also include selectable markers such as antibiotic resistance genes, herbicide resistance genes, or other genes for selecting suitable transformants. Examples of such genes are well known to those skilled in the art.

[0216] The nucleic acid constructs of the present invention may be introduced into vectors such as plastids. Plastid vectors generally include additional nucleic acid sequences that provide convenient selection, amplification, and transformation of expression cassettes in both 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. The additional nucleic acid sequences include an origin of replication that provides autonomous replication of the vector, a selectable marker gene, preferably encoding antibiotic or herbicide resistance, a unique multiple cloning site for insertion of the nucleic acid sequence or gene encoded in the nucleic acid construct, and sequences that enhance transformation of eukaryotic and prokaryotic (especially plant) cells.

[0217] A "marker gene" refers to a gene that confers a different phenotype to the cell expressing the marker gene and thus allows the transformed cell to be distinguished from cells that do not have the marker. Selectable marker genes confer properties that allow them to be selected based on resistance to a selection agent (e.g., herbicide, antibiotic, radiation, heat, or other treatments that are damaging to non-transformed cells). Screenable marker genes (or reporter genes) confer properties that can be identified by observation or assay, i.e., by "screening" (e.g., β-glucuronidase, luciferase, GFP, or other enzymes that are not active in non-transformed cells). The marker gene and the nucleotide sequence of interest need not be linked.

[0218] For the purposes of facilitating the identification of transformants, ideal nucleic acid constructs include selectable or screenable marker genes, such as, or also, exogenous or heterologous polynucleotides. The actual choice of the marker is not critical as long as it is functional (i.e., selective) in combination with the selected plant cells. It is not necessary to ligate the marker gene and the exogenous or heterologous polynucleotide of interest, since co-transformation of unlinked genes is also an effective method in plant transformation, e.g., as described in US 4,399,216.

[0219] 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 the selection of plant transformants include, but are not limited to, the hyg gene encoding resistance to hygromycin B; the neomycin phosphotransferase (nptII) gene conferring resistance to kanamycin, paromomycin, G418; the glutathione-S-transferase gene from rat liver conferring resistance to glutathione-derived herbicides, as described in EP 256223; the glutamine synthetase gene that confers resistance to glutamine synthetase inhibitors such as phosphinothricin when overexpressed, e.g., as described in WO 87 / 05327, the acetyltransferase gene from Streptomyces viridochromogenes conferring resistance to the selection agent phosphinothricin, e.g., as described in EP275957, the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine, e.g., as described by Hinchee et al. (1988), the bar gene conferring resistance to bialaphos, e.g., as described in WO91 / 02071; the nitrilase gene from Klebsiella ozaenae conferring resistance to bromoxynil such as bxn (Stalker et al., 1988); the dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al., 1988); the mutant acetolactate synthase gene (ALS) that confers resistance to imidazolinones, sulfonylureas or other ALS-inhibiting compounds (EP154,204); the mutant anthranilate synthase gene conferring resistance to 5-methyltryptophan; or the dalapon dehalogenase gene conferring resistance to herbicides.

[0220] Preferred selectable markers include, but are not limited to, the uidA gene encoding β-glucuronidase (GUS), an enzyme for which various chromogenic substrates are known; the β-galactosidase gene encoding an enzyme for which chromogenic substrates are known; the aequorin gene for use in calcium-sensitive bioluminescence detection (Prasher et al., 1985); the green fluorescent protein gene (Niedz et al., 1995) or derivatives thereof; the luciferase (luc) gene permitting bioluminescence detection (Ow et al., 1986), and others known in the art. As used herein, a "reporter gene" refers to a molecule that provides an assayable recognizable signal in accordance with its chemical properties to facilitate the identification of promoter activity by the protein product.

[0221] Preferably, the nucleic acid construct is stably incorporated into the genome of, for example, a plant. Thus, the nucleic acid includes appropriate elements allowing the molecule to be incorporated into the genome, or the construct is placed in an appropriate vector capable of integrating into the chromosomes of a plant cell.

[0222] One embodiment of the invention includes a recombinant vector comprising at least one polynucleotide molecule of the invention inserted into any vector capable of delivering a nucleic acid molecule to a host cell. Such vectors contain heterologous nucleic acid sequences, i.e., nucleic acid sequences found not to be naturally adjacent to the nucleic acid molecules of the 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 plastid.

[0223] Many vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described, e.g., in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Suppl., 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Generally, plant expression vectors include, for example, one or more plant genes cloned under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors also contain a promoter regulatory region (such as 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, RNA processing signals, a transcription termination site, and / or a polyadenylation signal.

[0224] The protein level of the present invention can be regulated by increasing the expression level of the nucleotide sequence encoding the protein in plant cells or by decreasing the expression level of the gene encoding the protein in plants, thereby resulting in modified pathogen resistance. The expression level of a gene can be regulated by altering the copy number per cell, for example, by introducing a synthetic gene construct comprising the coding sequence and its operably linked transcriptional control elements that are functional in the cell. Multiple transformants may be selected and screened for those with favorable levels and / or transgenic expression specificity due to the influence of endogenous sequences around the transgenic integration site. The favorable levels and forms of transgenic expression are those that result in substantial modification of pathogen resistance or other phenotypes. Alternatively, individual lines with altered pathogen resistance or other phenotypes related to pathogen resistance may be screened from mutagenized seed populations or plant populations from breeding programs.

[0225] Recombinant Cell

[0226] Another embodiment of the present invention includes recombinant cells or progeny cells thereof comprising host cells transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method of inserting the nucleic acid molecule into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. The recombinant cells can remain as single cells or can grow into tissues, organs, or multicellular organisms. The transformed nucleic acid molecules of the present invention can remain episomal or can integrate into one or more sites within the chromosome of the transformed (i.e., recombinant) cell in a manner that retains their ability to be expressed. Preferred host cells are plant cells, more preferably cereal plant cells, more preferably barley or wheat cells, and even more preferably wheat cells.

[0227] Transgenic Plant

[0228] As used herein, the term "plant" refers to the whole plant, and refers to any entity of the plant kingdom, but as an adjective, refers to any substance 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. The germinated seeds from which the plants, roots, and seedlings 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, which 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 tissues, ground tissues, etc.), cells, and their progeny. As used herein, the term "plant cell" refers to a cell obtained from a plant or a cell in a plant, and includes protoplasts or other cells derived from a plant, gamete-producing cells, and cells capable of regenerating into a whole plant. Plant cells may be cells in culture. "Plant tissue" refers to differentiated tissues in a plant or undifferentiated tissues obtained from a plant ("graft") or derived from immature or mature embryos, seeds, roots, seedlings, fruits, tubers, pollen, tumor tissues (crown gall), and aggregates of different forms 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, plant parts, and products containing these.

[0229] As used herein, the term "seed" refers to the "mature seed" of a plant, which is either ready to be harvested or has been harvested from the plant, usually commercially harvested in the field, or a "developing seed" that appears in the plant after fertilization and before the establishment of seed dormancy and before harvest.

[0230] As used in the present invention, a "transgenic plant" refers to a plant containing a nucleic acid construct not found in the wild-type plant of the same species, variety, or cultivar. That is, a transgenic plant (transformed plant) contains genetic material (transgene) that was not present before transformation. The transgene may include genetic sequences obtained from or derived from plant cells, other plant cells, or non-plant sources, or synthetic sequences. Generally, the transgene is introduced into the plant source by artificial manipulation, e.g., by transformation, but any method recognized in the art as a technique may be used. The genetic material is preferably stably integrated into the genome of the plant. The introduced genetic material may contain sequences that exist in the natural state in the same species but in a rearranged order or with a different arrangement of elements, such as antisense sequences. Plants containing these sequences are included in the "transgenic plants" herein.

[0231] "Non-genetically modified 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 (transgene) that has been introduced, such that their progeny will not segregate for the desired phenotype.

[0232] As used herein, the term "compared to isogenic plants", or similar phrases, refers to plants that are isogenic to the transgenic plant but do not have the transgene of interest. Preferably, the corresponding non-transgenic plants have 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 plants of the same cultivar or variety that have been transformed with an "empty vector" construct, which may be non-transgenic plants. "Wild type", as used herein, refers to cells, tissues or plants that have not been modified according to the present invention. Wild type cells, tissues or plants may be used as controls to compare the expression level of the exogenous nucleic acid or the degree and nature of the property modification with the modified cells, tissues or plants, as described herein.

[0233] Transgenic plants, as defined in the context of the present invention, include the progeny of plants that have been genetically modified using recombinant techniques, wherein the progeny contain the transgene of interest. Such progeny may be obtained by self-pollination of the original transgenic plant or by crossing this plant with other plants of the same species. This generally regulates the production of at least one protein as defined herein in the desired plant or plant organ. Transgenic plant parts include all parts and cells of a plant that contain the transgene, such as cultured tissues, callus and protoplasts.

[0234] Plants contemplated for use in the practice of the present invention include monocotyledonous and dicotyledonous plants. Target plants include, but are not limited to, the following: cereals (e.g., wheat, barley, rye, oats, rice, maize, sorghum and related crops); grapes; sugar beets (sugar beet and fodder beet); pome fruits, stone fruits and soft fruits (apple, pear, plum, peach, almond, cherry, strawberry, raspberry, blackberry); leguminous plants (broad bean, lentil, pea, soybean); oil crops (rape or other Brassica, mustard, poppy, olive, sunflower, safflower, flax, coconut, castor oil plant, cocoa bean, peanut); cucurbitaceous plants (gourd, cucumber, melon); fiber plants (cotton, flax, hemp, jute); citrus fruits (orange, lemon, grapefruit, tangerine); vegetables (spinach, lettuce, asparagus, cabbage, carrot, onion, tomato, potato, pepper); Lauraceae (avocado, cinnamon, camphor) or plants such as maize, tobacco, nut, coffee, sugar cane, tea, vine, hops, turf, banana and natural rubber plants, as well as ornamental plants (flowers, shrubs, broad-leaved trees and evergreen trees such as conifers). Preferably, the plant is a cereal plant, more preferably wheat, rice, maize, triticale, oats or barley, even more preferably wheat.

[0235] As used herein, the term "wheat" refers to any species of the genus Triticum, including its precursors, and progeny produced by its hybridization with other species. Wheat includes "hexaploid wheat", which has a genomic constitution of AABBDD and consists of 42 chromosomes; and "tetraploid wheat", which has a genomic constitution of AABB and consists of 28 chromosomes. Hexaploid wheat includes common wheat (T. aestivum), spelt wheat (T. spelta), macha wheat (T. macha), club wheat (T. compactum), Indian dwarf wheat (T. sphaerococcum), vavilov wheat (T. vavilovii), and their interspecies hybrids. A 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 their interspecies hybrids. In addition, the term "wheat" includes potential precursors of hexaploid or tetraploid Triticum species, such as for the A genome, Triticum urartu, Triticum monococcum, or Triticum boeoticum, for the B genome, Aegilops speltoides, and for the D genome, Triticum tauschii (also known as Aegilops squarrosa or Aegilops tauschii). Particularly preferred precursors are those of the A genome, and even more preferably the precursor of the A genome is Triticum monococcum. Wheat cultivars useful in the present invention may belong to, but are not limited to, any of the species listed above. Also covered are plants produced using Triticum as a parent in a sexual cross with a non-Triticum species (such as rye [Secale cereale]) by conventional techniques, non-Triticum species including but not limited to triticale.

[0236] As used herein, the term "barley" refers to any species of the genus Hordeum, including its precursors, and progeny produced by its hybridization with other species. Preferred commercially grown barley species of plants such as barley (Hordeum vulgare) or lines or cultivars or varieties suitable for food commodity production.

[0237] "Transgenic plants", as defined in the context of the present invention, include plants (and parts and cells of plants) that have been genetically altered using recombinant techniques and their progeny, the recombinant techniques enabling the production of at least one polypeptide of the present invention in the desired plant or plant organ. 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).

[0238] In a preferred embodiment, the transgenic plants are homozygous for each gene (transgene) that has been introduced, such that their progeny do not segregate for the desired phenotype. The transgenic plants can also be heterozygous for the introduced transgenes, such as, for example, in the F1 progeny, which are grown from hybrid seeds. Such plants can provide advantages well known in the art such as hybrid vigor.

[0239] As used herein, "other genetic markers" can be any molecule linked to a desired trait of the 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, grain quality, dormancy characteristics, grain color, deoxynivalenol acidity in seeds, plant height, flour color, etc. Examples of such genes are the stripe rust resistance genes Yr10 or Yr17, nematode resistance genes such as Cre1 and Cre3, alleles at glutenin loci that determine dough strength such as Ax, Bx, Dx, Ay, By, and Dy alleles, and the Rht genes that determine semi-dwarf growth habit and thus lodging resistance.

[0240] Four general methods of directly delivering genes into cells have been described as follows: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, for example, WO 87 / 06614, US 5,472,869, 5,384,253, WO 92 / 09696, and WO 93 / 21335) and gene gun (see, for example, 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).

[0241] Accelerating methods that can be used include, for example, particle bombardment. An example of a method for delivering a transformed nucleic acid molecule into a plant cell is particle 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 acid and delivered into cells by means of a propulsive force. Exemplary particles include those composed of tungsten, gold, platinum, etc. In addition to being an effective method for reproducibly transforming monocotyledonous plants, a particular advantage of particle bombardment is that neither protoplast isolation nor susceptibility to Agrobacterium infection is required. A particle 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 scutella or callus can be arranged on solid media.

[0242] In another alternative embodiment, plastids can be stably transformed. The disclosed methods for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker, and targeting the DNA to 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).

[0243] Agrobacterium-mediated transfer is a system widely applicable for introducing genes into plant cells, since DNA can be introduced into whole plant tissues, thus bypassing the need for regeneration of intact plants from protoplasts. It is well known in the art to use Agrobacterium-mediated plant integration vectors for introducing DNA into plant cells (see, for example, US 5,177,010, US 5,104,310, US 5,004,863, US 5,159,135). In addition, the integration of T-DNA is a relatively precise method that rarely results in rearrangements. The DNA region to be transferred is delimited by border sequences, and the intervening DNA is often inserted into the plant genome.

[0244] Agrobacterium transformation vectors are capable of replicating in both E. coli and Agrobacterium, as described for ease of manipulation (Klee et al., Plant DNA Infectious Agents, Hohn and Schell, (eds.), Springer-Verlag, New York, (1985): 179-203). In addition, technological advancements in vectors for Agrobacterium-mediated gene transfer have improved gene arrangements and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide-encoding genes. The described vectors have a convenient multiple cloning site flanked by a promoter and a polyadenylation site for direct expression of the inserted polypeptide-encoding gene, and the vectors are suitable for the purposes herein. In addition, Agrobacterium containing an assembled or unassembled Ti gene can be used for transformation. This is a method of choice on those plant varieties where Agrobacterium-mediated transformation is efficient due to the convenience and defined nature of gene transfer.

[0245] Transgenic plants formed by the Agrobacterium transformation method typically contain a single genetic locus on one chromosome. Such transgenic plants can be referred to as hemizygous for the added gene. More preferably, transgenic plants that are homozygous for the added structural gene; i.e., transgenic plants containing two added genes, one at the same locus on each chromosome of the chromosome pair. Homozygous transgenic plants can be obtained by the following steps: allowing independently segregating transgenic plants containing a single added gene to undergo sexual mating (selfing), germinating some of the resulting seeds and analyzing the resulting plants for the gene of interest.

[0246] It should also be understood that two different transgenic plants can be mated to produce offspring containing two independently segregating foreign genes. Selfing of suitable progeny can produce plants that are homozygous for both foreign genes. Backcrossing to the parental plant and crossing with non-transgenic plants, such as vegetative 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 (ed.) American Society of Agronomy, Madison Wis. (1987).

[0247] Methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments are used to achieve transformation of plant protoplasts. Application of these systems to different plant variants depends on the ability to regenerate specific plant varieties from protoplasts. Illustrative methods for regenerating cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

[0248] Other methods of cell transformation can also be used, and these methods include, but are not limited to, introducing DNA into a plant by directing DNA transfer into pollen, directing DNA injection into the reproductive organs of a plant, or by directing DNA injection into immature embryo cells followed by drying the embryo and rehydrating it to introduce the DNA into the plant.

[0249] The regeneration, development, and cultivation of plants from single 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 includes the following steps: selecting the transformed cells and culturing these individual 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.

[0250] The development or regeneration of plants containing exogenous, foreign genes is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Alternatively, the pollen obtained from the regenerated plants is crossed with plants grown from seeds of agriculturally important varieties. Conversely, the pollen of these important variety plants is used to pollinate the regenerated plants. The transgenic plants of the present invention containing the desired exogenous nucleic acid are cultivated using methods well known to those skilled in the art.

[0251] Methods for transforming dicotyledonous plants, mainly by using Agrobacterium tumefaciens, and for 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).

[0252] Methods for transforming cereal plants such as wheat and barley to introduce genetic modifications into plants by introducing exogenous nucleic acids, as well as methods for regenerating plants from protoplasts or immature embryos of plants are well known in the art. For example, see CA 2,092,588, AU 61781 / 94, AU 667939, US 6,100,447, WO 97 / 048814, US 5,589,617, US 6,541,257, and other methods are described in WO 99 / 14314. Preferably, transgenic wheat or barley plants are produced by Agrobacterium tumefaciens-mediated transformation procedures. A vector carrying the desired nucleic acid construct may be introduced into a tissue culture plant or a renewable wheat cell or a suitable plant system such as a protoplast of an explant. Renewable wheat cells are preferably derived from the scutellum of immature embryos, mature embryos and callus derived therefrom, or meristems.

[0253] To confirm the presence of the transgene in transgenic cells and plants, polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Depending on the nature of the transgene expression product, any of a variety of methods can be used to detect the product, including Western blot analysis and enzyme assays. A particularly effective method for quantifying protein expression and detecting replication in different plant tissues is to use a reporter gene such as GUS. Once transgenic plants are obtained, the plants can be cultivated to produce plant tissues or parts with the desired phenotype. The plant tissues or plant parts can be harvested, and / or the seeds can be collected. The seeds can be used as a source for cultivating other plants that contain tissues or parts with the desired characteristics.

[0254] Marker-Assisted Selection

[0255] Marker-assisted selection is a recognized method for selecting heterozygous plants when backcrossing to a recurrent parent in classical breeding programs. In each backcross generation, the population of plants will be heterozygous for the gene of interest, which is 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, e.g., seedlings and testing for the desired trait with specific markers, plants can be selected for further backcrossing while concentrating energy and resources on fewer plants. To further accelerate the backcross program, embryos of immature seeds (25 days after flowering) can be excised and grown in a nutrient medium under sterile conditions, rather than the immature seeds. This process, known as "embryo rescue", is used in combination with DNA extraction at the three-leaf stage and analysis of at least one Lr67 allele or variant conferring resistance of the plant 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 rapid selection of plants carrying the desired trait, which can be grown to maturity in a greenhouse or field for subsequent further backcrossing to the recurrent parent.

[0256] 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, hybridization of nucleic acids with suitable labeled probes, single-strand conformation analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalytic nucleic acid cleavage or combinations thereof (see, e.g., Lemieux, 2000; Langridge et al., 2001). The present invention also includes the use of molecular marker techniques to detect polymorphisms in alleles of, e.g., the Lr67 gene, which confers resistance of the plant to one or more biotrophic fungal pathogens, preferably to one or more or all of leaf rust, stripe rust, stem rust and powdery mildew. Such methods include the detection or analysis of restriction fragment length polymorphisms (RFLP), RAPD, amplified fragment length polymorphisms (AFLP) and satellite (simple sequence repeat, SSR) polymorphisms. Closely 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).

[0257] In one embodiment, the linked locus of marker-assisted selection is within at least 1 cM or 0.5 cM or 0.1 cM or 0.01 cM of the gene encoding the polypeptide of the present invention.

[0258] "Polymerase chain reaction" ("PCR") is a reaction in which replicated copies are formed from a target polynucleotide, using a "primer pair" or "primer set" consisting of "upstream" and "downstream" primers, a polymerization catalyst such as a DNA polymerase and usually a thermostable polymerase. For example, methods of PCR known in the art are taught in "PCR" (M.J. McPherson and S.G Moller (eds.), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCR can be carried out on cDNA obtained from reverse transcribed mRNA isolated from plant cells expressing the Lr67 gene or allele, which confers resistance to one or more biotrophic fungal pathogens on the plant, preferably conferring resistance 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 carried out on genomic DNA isolated from the plant.

[0259] Primers are oligonucleotide sequences that are capable of hybridizing to a target sequence in a sequence-specific manner and are extended during the PCR process. An amplicon or PCR product or PCR fragment or amplification product is an extended product containing the primer and a newly synthesized copy of the target sequence. A multiplex PCR system contains multiple sets of primers, which results in the simultaneous production of more than one amplicon. Primers can match the target sequence exactly or they may contain internally mismatched bases, which can result in the introduction of a restriction enzyme or a catalytic nucleic acid recognition / cutting site in the specific target sequence. Primers can also contain additional sequences and / or contain modified or labeled nucleotides to facilitate the capture or detection of the amplicon. Repeated cycles of DNA thermal denaturation, primer annealing to its complementary sequence and extension of the annealed primer by the polymerase result in the exponential amplification of the target sequence. The term target or target sequence or template refers to the nucleic acid sequence that is amplified.

[0260] Methods for the direct sequencing of nucleotide sequences are well known to those skilled in the art and examples thereof are found in Ausubel et al., (ibid.) and Sambrook et al., (ibid.). Sequencing can be carried out by any suitable method, for example, dideoxy sequencing, chemical sequencing or variations thereof. Direct sequencing has the advantage of determining changes at any base pair of a specific sequence.

[0261] TILLING

[0262] The plants of the present invention (Targeting Induced Local Lesions IN Genomes, TILLING) can be produced using a process called TILLING. In the first step, mutations (such as new single-base changes) are induced in a plant population by treating seeds (or pollen) with a chemical mutagen, and then the plants are grown for one generation so that 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 accessed repeatedly over time.

[0263] 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 the population may carry the same polymorphism) and induced SNPs (i.e., only rare plant individuals may display such a mutation). After heteroduplex formation, the use of an endonuclease (such as Cel I) that can recognize and cleave mismatched DNA is crucial for detecting new SNPs in the TILLING population.

[0264] Using this method, many plants are screened to identify any individuals with single-base changes and small insertions or deletions (1 - 30 bp) in a specific region of any gene or genome. The size of the genomic fragments assayed ranges from 0.3 to 1.6 kb. In an 8-fold pool, with 96 lanes per assay and 1.4 kb fragments (ignoring the fragment ends, which are problematic for noise SNP detection), this combination allows screening of up to 1 million base pairs of genomic DNA in each assay, making TILLING a high-throughput technology.

[0265] TILLING is further described in Slade and Knauf (2005) and Henikoff et al. (2004).

[0266] In addition to allowing efficient detection of mutations, the high-throughput TILLING technology is also ideal for detecting natural polymorphisms. Thus, the number and location of polymorphic sites are revealed by forming heteroduplexes with a known sequence to interrogate unknown homologous DNA. Nucleotide changes and small insertions and deletions, including at least some with variable repeat numbers, can all be identified. This has been termed Ecotilling (Comai et al., 2004).

[0267] Each SNP is recorded by its approximate position within some nucleotides by SNP. Thus, the haplotype-based mobility archives each haplotype. Using aliquots of the same amplified DNA used for the mismatch-cleavage assay, sequence data is obtained with a relatively small incremental effort. By approaching the polymorphism with sequencing primers, either the left sequencing primer or the right sequencing primer of a single reaction is selected. Sequencher software performs multiple alignments and discovers base changes, which are confirmed by gel bands in each case.

[0268] Performing Ecotilling is cheaper than the method currently used to discover most SNPs - whole sequencing. Plates containing ecological DNA arrays are screened, rather than DNA pools from mutagenized plants. Since detection is performed on gels with near base-pair resolution and a uniform background pattern in each lane, bands of the same size can be matched, thus discovering and genotyping SNPs in a single step. In this way, the final sequencing of SNPs is simple and effective, and DNA sequencing can be directly performed using the same PCR product aliquots used for screening, which makes it even simpler and effective.

[0269] Plant / Cereal Processing

[0270] The grain / seeds of the present invention, preferably grains and more preferably wheat grains, or other plant parts of the present invention, can be processed using techniques known in the art to produce food ingredients, foods or non-food products.

[0271] In one embodiment, the product is whole grain flour such as ultra-fine ground whole grain flour, or flour made from 100% grain. Whole grain flour includes refined flour components (refined flour or refined flours) and grit content (ultra-fine ground grit content).

[0272] Refined flour is the flour prepared, for example, by milling and screening clean grains such as wheat or barley grains. The particle size of refined flour is described as flour in which no less than 98% of the particles pass through the holes of a wire mesh specified as not larger than "212 microns (US wire 70)". The grit portion includes at least one of bran and germ. For example, the germ is the embryonic plant found within the grain kernel. The germ includes lipids, fiber, vitamins, proteins, minerals and phyto-nutrients such as flavonoids. Bran includes several cell layers and has a large amount of lipids, fiber, vitamins, proteins, minerals and phyto-nutrients such as flavonoids. In addition, the grit portion may include the aleurone layer, which also includes lipids, fiber, vitamins, proteins, minerals and phyto-nutrients, such as flavonoids. Although the aleurone layer is technically considered part of the endosperm, it exhibits many of the same characteristics as bran and is therefore usually removed together with bran and germ during the milling process. The aleurone layer contains proteins, vitamins and phyto-nutrients such as ferulic acid.

[0273] In addition, the coarse fraction may be blended with refined flour components. The coarse fraction can be blended with refined flour components to form whole grain flour, thereby providing whole grain flour with higher nutritional value, higher fiber content, and higher antioxidant capacity compared to refined flour. For example, different levels of the coarse fraction or whole grain flour can be used to replace refined flour or whole grain flour in bakery products, snack products, and food products. The whole grain flour of the present invention (i.e., ultra-fine milled whole grain flour) may be sold directly to consumers for use in their homemade bakery products. In an exemplary embodiment, the granulation of the whole grain flour is such that 98% of the particles are less than 212 microns by weight of the whole grain flour.

[0274] In yet another embodiment, enzymes found in the bran and germ of the whole grain flour and / or coarse fraction are inactivated to keep the whole grain flour and / or coarse fraction stable. Stabilization is a process of inactivating the enzymes found in the bran and germ layers using steam, heat, radiation, or other treatments. The stabilized flour retains its cooking characteristics and has a longer shelf life.

[0275] In additional embodiments, the whole grain flour, coarse fraction, or refined flour may be an ingredient (composition) of a physical product and can be used in food products. For example, the food product can be a bagel, cookie, bread, bun, croissant, dumpling, English muffin, muffin, pita bread, quickbread, refrigerated / frozen dough, dough, baked beans, tortilla, chili, burrito, tamale, tortilla, pot pie, ready-to-eat cereal, ready-to-eat meal, filling, microwaveable meal, brownie, cake, cheesecake, coffee cake, cookie, dessert, pastry, sweet bread, confectionery, pie crust, pie filling, baby food, baking mix, batter, mix, blended juice, meat brightener, meat substitute, flavoring, soup mix, gravy, roux, salad dressing, soup, yogurt, noodle, pasta, ramen, chow mein noodles, lo mein noodles, ice cream inclusion, sherbet, ice cream cone, ice cream sandwich, cracker, crouton, doughnut, egg roll, puffed food, fruit and grain bar, microwaveable snack product, nutrition bar, pancake, value-added bakery product, saltine cracker, pudding, oatmeal-based product, snack fry, snack food, snack mix, waffle, pizza crust, animal food or pet food.

[0276] In an alternative embodiment, whole grain flour, refined flour, or grits content is an ingredient of the nutritional supplement. For example, the 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 grits content of the present invention includes vitamins, minerals, amino acids, enzymes, and fiber. For example, the grits portion contains concentrated amounts of dietary fiber as well as other essential nutrients such as vitamin B, selenium, chromium, manganese, magnesium, and antioxidants, which are essential for a healthy diet. For example, 22 grams of the grits content of the present invention provides 33% of an individual's daily recommended fiber intake. The nutritional supplement can include any known nutritional components that contribute to the overall health of an individual, examples including but not limited to vitamins, minerals, other fiber components, fatty acids, antioxidants, amino acids, polysaccharides, proteins, lutein, ribose, omega-3 fatty acids, and / or other nutritional components. The supplement can be provided in the following ways, but is not limited to these forms: ready-to-drink mixes, ready-to-drink beverages, nutritional bars, crackers, cookies, wafers, gel capsules, capsules, chewables, chewable tablets, and pills. One embodiment provides a fiber supplement in the form of a flavored drink mix or malt-type beverage, which may be particularly attractive as a children's fiber supplement.

[0277] In an additional embodiment, a milling process can be used to make multi-grain flour or multi-grain grits content. For example, the bran and germ from one type of grain can be ground and mixed with the ground endosperm or whole grain cereal flour of another type of grain. Or, the bran and germ from one type of grain can be ground and mixed with the ground endosperm or whole grain cereal flour of another type of grain. It is contemplated that the present invention encompasses mixing any combination of one or more bran, germ, endosperm with the whole grain flour of one or more grains. This multi-grain method can be used to make custom flour and take advantage of the quality and nutritional content of multiple types of cereal grains to make flour.

[0278] It is contemplated that the whole grain flour, grits content, and / or cereal products of the present invention can be produced by milling methods known in the art. An exemplary embodiment involves grinding the grain in a single stream without separating the endosperm, bran, and germ of the grain into separate streams. The cleaned and conditioned grain is fed into a first-pass grinder such as a hammer mill, roller mill, pin mill, impact mill, disk mill, air mill, notch mill, etc. After the grain is ground, it exits and is fed to a sieve. Additionally, it is contemplated that the whole grain flour, grits content, and / or cereal products of the present invention can be modified or enhanced by many other methods such as fermentation, instantizing, extrusion, encapsulation, baking, roasting, etc.

[0279] Wheat grain germination

[0280] The malt liquor-based beverages provided by the present invention relate to alcoholic beverages (including distilled beverages) and non-alcoholic beverages produced by using some or all of malt liquor as their starting material. Examples include beer, low-malt beer (low-malt liquor beer beverage), whisky, low-alcohol malt liquor-based beverages (e.g., malt liquor-based beverages containing less than 1% alcohol), and non-alcoholic beverages.

[0281] Malting is a process of controlling steeping and germination, followed by drying of grains such as barley and wheat grains. This series of events is important for synthesizing many enzymes that cause grain modification, which is a process mainly for depolymerizing the cell walls of dead endosperm and mobilizing grain nutrients. In subsequent drying methods, odors and colors are produced by chemical browning reactions. Although malt liquor is mainly used in beverage production, it can also be used in other industrial processes, such as as a source of enzymes in the baking industry, or as a flavoring and coloring agent in the food industry, such as in the form of malt liquor or malt liquor powder, or directly as malt liquor syrup, etc.

[0282] In one embodiment, the present invention relates to a method for producing a malt liquor composition. The method preferably includes the following steps:

[0283] (i) Providing grains, such as the barley or wheat grains of the present invention,

[0284] (ii) Steeping the grains,

[0285] (iii) Allowing the steeped grains to germinate under predetermined conditions and

[0286] (iv) Drying the germinated grains.

[0287] For example, malt liquor can be produced by any of the methods described by Hoseney (Principles of Cereal Science and Technology, 2nd Edition, 1994: American Association of Cereal Chemists, St. Paul, Minn.). However, the present invention also uses any other method suitable for producing malt liquor, such as methods for producing specialty malt liquor, including but not limited to, methods for roasting malt liquor.

[0288] Malt liquor is mainly used for brewing beer, but is also used for producing distilled spirits. Brewing includes producing wort, primary and secondary fermentation, and post-treatment. First, the malt liquor is milled and stirred into water and heated. During this "mashing" period, the enzymes activated during malting degrade the starch of the grains into fermentable sugars. The produced wort is clarified, yeast is added, and the mixture is fermented and post-treated.

[0289] Examples

[0290] Example 1. The Lr67 / Yr46 Gene is a Mature Plant Resistance Gene Different from Lr34 / Yr18 and Lr46 / Yr29

[0291] Introduction

[0292] Wheat rust resistance genes are divided into two major categories, which are called seedling and adult plant resistance (APR) genes. Seedling resistance genes can be detected phenotypically after the rust pathogen acts on the plant and are observed during the seedling stage and in adult plants; thus they confer a resistant phenotype throughout all stages of plant growth. In contrast, APR is generally not detectable during the seedling stage but can be detected during the later growth stages of seedlings and often serves as the resistance of plants growing in the field to the pathogen. Conversely, some APR genes are induced to express during the seedling stage by altering the growth temperature and light conditions, but under all growth conditions, these genes do not express during the seedling stage. In addition, seedling resistance genes usually exhibit major effect phenotypes and different infection types, while most APR genes partially affect different levels of disease severity. Race specificity of seedling resistance genes is more common. Among the few APR genes that have been studied in wheat, most appear to be cultivar non-specific, and the number of clearly cultivar-specific ones is limited. Those in the non-cultivar-specific category with partial resistance are related to the slow rusting phenotype first described by Caldwell (1968). Generally, compared with susceptible plants, slow rusting resistance shows a longer latent period, fewer and smaller uredinia within two weeks after inoculation.

[0293] One of the well-characterized non-cultivar-specific genes is the adult-plant leaf rust resistance gene Lr34 (WO2010 / 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 types isolated from wild wheat), especially those of Chinese origin (Borghi 2001; Kolmer et al., 2008). An important feature of Lr34 is that no virulence of the wheat leaf rust pathogen has been reported to date, and the effect of enhanced rust resistance when the Lr34 gene is combined with other cultivar-specific leaf rust resistance genes in wheat cultivars contributes to the durability of wheat cultivars with the Lr34 gene combination (Kolmer, 1996). However, variability in the degree of rust population development among leaf rust isolates of the Lr34 gene has been reported (Bender and Pretorius, 2000). The co-segregation of Lr34 with the adult-plant stripe rust resistance gene Yr18 in dual rust resistance in many wheat backgrounds (McIntosh 1992; Singh 1992) has contributed to the continued widespread use of Lr34 / Yr18 germplasm in wheat breeding. Subsequent observations that the Lr34 / Yr18 locus also contributes to partial resistance to adult-plant powdery mildew (Pm38) - highlight the multi-pathogen 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 methods were used to study the multi-pathogen resistance locus containing Lr34 on wheat chromosome 7DS (Spielmeyer et al., 2008). Susceptible mutants were recovered that showed no loss of DNA markers in the QTL interval on 7DS. These mutants subsequently showed point mutations where the chemical mutagen had created single base substitutions. Additional mutants generated by γ-ray irradiation had single base deletions in the gene encoding an ATP-binding cassette (ABC) transporter at the multi-pathogen resistance locus (Krattinger et al., 2009). In addition to the ABC transporter, 6 other genes co-segregated with the resistance locus and were genetically and physically tightly linked. However, none of these mutants (8 individual mutants) had alterations in the additional genes. Thus, mutagenic alterations in the ABC transporter alone were sufficient to confer complete loss of resistance to leaf rust, stripe rust, and powdery mildew encoded by Lr34 / Yr18 / Pm38. Combining haplotype analysis and high-resolution mapping confirmed that a single gene, the ABC transporter, confers all three resistances (Krattinger et al., 2009).

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

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

[0297] Studies of mapping populations from crosses containing RL6077, using multiple isolates of wheat leaf rust and stripe rust from Canada, Mexico, and Australia, confirmed the co-segregation of each APR to chromosome 4DL (the short arm of chromosome 4D, the D genome in bread wheat) (Herrera-Foessel et al., 2011; Hiebert et al., 2010). These APR genes 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 presence of a leaf tip necrosis phenotype (Ltn), slow rusting leaf rust infection, and simple sequence repeat (SSR) marker alleles linked to Lr67 on 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 from crosses involving the Avocet susceptible parent for mature plant leaf rust and stripe rust. These RI families were derived from crosses of Avocet x RL6077, Avocet x Sujata, and Avocet x NP876. An F2 family was also generated from a Thatcher x Thatcher+Lr67 (RL6077) cross. These RI families were then used in genetic mapping studies and mutagenesis experiments as follows.

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

[0301] To identify DNA sequences located at or tightly linked to the Lr67 locus, 15 recombinant inbred lines (RILs) (each from resistant (Lr67R) and susceptible (Lr67S) phenotypes) were selected and subjected to extensive AFLP and SSR analyses. Genomic complexity - reduced libraries of pooled DNA from 15 resistant isogenic lines based on the PstI restriction enzyme were evaluated for SNP - based markers. Sequences from wheat BAC clones containing SSRs tightly linked to Lr67 (Hererra et al., 2011) were also used as anchor points for comparative genomics approaches to identify homologous genes from the rice and Brachypodium genomes ( Figure 1 ). Then the collinear region between chromosome 1 of Brachypodium and chromosome 3 of rice was used to develop DNA markers to amplify corresponding sequences from wheat and Aegilops, which are tightly linked to the D - genome progenitor of wheat chromosome 4DL ( Figure 1 ).

[0302] AFLP and SNP of genomic complexity - reduced libraries produced from Lr67 - linked markers generated DNA markers, but they did not co - segregate with all 15 isogenic resistant and susceptible recombinant inbred lines. However, DNA fragments isolated from different parts of the chaperone - protein - encoding gene containing the Hsp70 (heat shock protein, see Figure 1 ) domain, isolated by comparative genomics methods, were completely linked to 15 susceptible recombinant inbred lines and were absent in 15 resistant lines ( Figure 2)。The degree of association of the Hsp70 chaperone was confirmed within 500 recombinant inbred lines, and the degree of association of the Hsp70 chaperone was demonstrated to show complete correlation with the Lr67 locus. Genomic DNA blot analysis provided additional evidence that the gene encoding the Hsp70 chaperone was deleted in the Lr67-resistant parental lines, RL6077, Sujata, and NP876. Given that a loss-of-function susceptible mutant had been isolated from Lr67, the deletion of the Hsp70 chaperone gene in resistant plants was insufficient to confer the Lr67-resistant phenotype. Therefore, all predicted gene sequences were searched in the vicinity of Hsp70 (within 21 kb), and three other genes were identified by comparative genomics. They are genes in the rice or Brachypodium genome maps that have protein domains annotated as encoding proteins of the Sec14B cytoplasmic factor family, monosaccharide transporter (MST - closely linked to sugar transporter - SUT), and protein interaction / binding protein (PIP).

[0303] Determine the nucleotide sequences of the corresponding genes in several wheat varieties and Lr67 mutants. 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) found in the SUT gene were unique to wheat containing Lr67 ( Figure 4 ), while the insertion / deletion polymorphism of PIP was not diagnostic for Lr67. The SNP in the SUT gene was co-segregated with Lr67 adult-plant rust within 520 recombinant inbred lines. To ensure that there was no additional gene sequence loss or unknown rearrangement in the 4DL region of wheat chromosome containing Lr67 that harbored the corresponding syntenic regions of Brachypodium (chromosome 1) and rice (chromosome 3), the equivalent region from Aegilops (D-genome progenitor) was analyzed. A 70-kb sequence contig showed the presence of Hsp70, SUT, and PIP within a 21-kb fragment in the contig, and there were no other predicted genes in this region. When analyzing 1152 plants from the F2 progeny population of the Thatcher x Thatcher + Lr67 cross, no recombination was detected between these three genes.

[0304] Analysis of mutants in which the Lr67 phenotype was inactivated revealed the deletion of three γ mutants for some of the SUT, PIP, and markers identified by comparative genomics within the Lr67 region. Figure 3, as shown by the dotted line (missing). However, 4 of the mutants (2 EMS mutants and 2 mutants from γ-radiation, namely, γ318 and γ1239) retained the SUT and PIP genes co-segregating with Lr67. Sequence analysis of the SUT gene in these mutants revealed only single nucleotide changes and small deletions in the SUT gene, but none in PIP. Each mutation change in the 4 mutants occurred in the amino acids found in the conserved region of the amino acid hexose transporter ( Figure 4 ). Four additional mutants obtained by sodium azide mutagenesis of RL6077 also showed single nucleotide changes that altered the amino acid composition of the SUT gene. Based on co-segregation and mutation analysis, the inventors inferred that the SUT gene is sufficient to confer the Lr67 resistance phenotype. When testing the full range of rust and powdery mildew, 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 conclusively identified as the Lr67 gene.

[0305] The Lr67 gene encodes a protein containing 514 amino acids ( Figure 5) 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 as: 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. Each of the following amino acid regions is predicted by the same program: the 19 amino acids at the N-terminus, 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 TM helices 8 and 9, and the 42 amino acids at the C-terminus are located on the inner side of the membrane, and the other amino acids are located on the outer side of the membrane. The protein is a member of the Major Facilitator Superfamily (MFS), which is a large and diverse group of secondary transporters that includes uniporters, cotransporters, and antiporters, and facilitates the transport of various compounds, including sugars, across the cytoplasmic or inner cell membrane. Homologous polypeptides are identified by querying the amino acid sequence of SEQ ID NO:1 in the NCBI protein database, as well as identifying multiple homologs. The amino acid sequence of Lr67 has approximately 89 - 93% identity with homologous polypeptides in several cereals including rice, and approximately 80% identity with the 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 in 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 the plant. The polypeptide encoded by the A-genome homolog of the Lr67 gene has an amino acid sequence that is 507 / 514 (98.6%) identical to SEQ ID NO:1, including glycine at position 144 and valine at position 387, which is a typical susceptible Lr67 polypeptide. This result indicates to the inventors that the resistant Lr67 polypeptide may act 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 difference between the polypeptides encoded by the Lr67 resistant and susceptible alleles is defined by two nucleotide changes that create the SNPs used in the Lr67 diagnostic markers. These two nucleotide changes result in amino acid changes, as referenced in SEQ ID NO:1, from a conserved glycine to an arginine in the predicted fourth transmembrane domain (position 144) and from a valine to a leucine in the tenth transmembrane domain (position 387) ( Figure 6 、 7 、8). These two nucleotide changes are rare in wheat and are not found in the D genome progenitors or in most commercial wheat varieties, except for a few that carry Lr67. Thus, the Lr67 resistance likely originated after the formation of hexaploid wheat by hybridization from its diploid progenitor wheat. When analyzing over 1000 wheat landraces from a wide geographic origin and a wide range of advanced lines with two Lr67 diagnostic markers, the mutations that cause Lr67 are very rare and are located in a subset of landraces from the Indo-Gangetic Plain.

[0309] In homologs of other plant species, such as the sugar transporter 13 in Arabidopsis and cereals, without exception, do not contain an arginine at the position corresponding to amino acid 144 of SEQ ID NO:1, or a leucine at the position corresponding to amino acid 387. Invariably, the homologs have a glycine at the position corresponding to amino acid 144 and are almost invariably valine at position 387 (see, for example, the alignments provided in Figure 9 and 10 ). Thus, these two amino acids are highly conserved in the SUT polypeptide, and the mutation of one or both of them indicates that the altered functional amino acids are the cause of resistance to rust or powdery mildew pathogens.

[0310] Example 3. Glucose Uptake Study of Lr67(SUT) Expressed in Yeast - Variation of Polypeptide Sequences

[0311] The demonstration that the Lr67 gene encodes a protein showing homology to known sugar transporters led the inventors to test the sugar transport function of the Lr67 polypeptide in yeast cells.

[0312] Experimental Procedure

[0313] The following experiments were completed using the protein coding region (resistant and susceptible alleles) encoding the Lr67 polypeptide cloned into the yeast expression vector pRS416. This vector contains the constitutive ADH1 promoter and CYC1 terminator for the expression of the inserted coding region. The yeast strain used was a hexose transport defective mutant of Saccharomyces cerevisiae named EBY.VW4000. Using radiolabeled 14C] Glucose uptake was determined by incorporation of radiolabeled material measured by liquid scintillation counting.

[0314] Results

[0315] Glucose uptake over time

[0316] Yeast cells transformed with genetic constructs expressing the resistant (Lr67(res)) or susceptible (Lr67(sus)) alleles of Lr67 were incubated with 100 μM 14 C] glucose for 10 minutes. Glucose uptake was measured at 2-minute intervals. Yeast cells expressing the Lr67 susceptible allele were shown to transport glucose at a faster rate than yeast cells expressing the Lr67 resistant allele or the empty vector ( Figure 11 ). In fact, cells expressing the Lr67 resistant allele did not show detectable glucose transport activity above the control, although this assay was only carried out for 10 minutes and was not sensitive.

[0317] Lr67(sus) glucose uptake kinetics

[0318] Lr67(sus) uptake of 14 C] glucose showed classical Michaelis–Menten saturation kinetics with respect to glucose concentration. Double-reciprocal (Lineweaver–Burk) plots were used to transform the data for linear regression analysis. Lr67(sus) was shown to have a high affinity for glucose, with a K m of 73 μM and a V max of 3.02 nmol min -1 g FW -1 . ( Figure 12 )

[0319] Amino acid substitution rate analysis

[0320] As described above, there are two SNP differences between the susceptible and resistant alleles of Lr67 at the nucleotide sequence. This creates two amino acid substitutions in the protein product, which include glycine to arginine at position 144 (G144R) and valine to leucine at position 387 (V387L). To test whether these amino acid substitutions, individually or together, affect the glucose uptake rate of LR67, the amino acids at positions 144 and 387 in the LR67 resistant allele were separately converted to the equivalent amino acids present in the LR67 susceptible allele by mutagenesis of the cloned gene (i.e., R144G and L387V).

[0321] Yeast cells transformed with Lr67(sus) or Lr67(res) or Lr67(res)R144G and Lr67(res)L387V were incubated with 100 μM 14 C] glucose for 10 minutes. Yeast cells expressing Lr67(res)R144G showed the ability to transport glucose at a higher rate than yeast cells expressing Lr67(res) or Lr67(res)L387V ( Figure 13 ), but not to the full extent of Lr67(sus). This indicates that position 144 is the more important of the two alternative amino acids for rust resistance function, and that the glycine-to-arginine conversion (G144R) or vice versa (R144G) alters the glucose transport rate of the Lr67 polypeptide. However, the addition of the second amino acid substitution (L387V) also contributes to the glucose transport rate.

[0322] Since deletion mutations in Avocet x RL6077 created by gamma irradiation that removed Lr67 resulted in susceptible alleles for rust and powdery mildew, the inventors concluded that the Lr67 (resistance) polypeptide must have an active function in wheat cells, and more likely hexose transport as a sugar other than glucose. That is, the conversion of the Lr67 susceptible allele to the resistant allele in wheat plants such as Avocet requires arginine at position 144, and is improved by the presence of leucine at position 387.

[0323] Example 4. Production of Transgenic Plants

[0324] Experiments were conducted using the cloned gene and amino acid variants of the genetic construct introduced into transgenic wheat and other plants such as cereals using standard Agrobacterium-mediated techniques to transform Fielder variety wheat plants, to increase resistance to fungal pathogens such as rust and powdery mildew. Experiments can also be done to modify the genes encoding the Arabidopsis and grape homologs of Lr67 to encode mutant polypeptides having arginine at position 144 and leucine at position 387, and to convert them to resistant polypeptides in order to provide resistance genes for these and other plant species.

[0325] Materials and Methods

[0326] 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 contains the full-length genomic sequence of the Lr67 gene and includes a 1318 bp native promoter region and a 1512 bp native terminator sequence containing the 3’ untranslated region. The Lr67 fragment was inserted into the binary transformation vector pWBVec8. The Lr67 binary vector was transformed into the Agrobacterium (A. tumefaciens) strain AGL-1 and used to produce stably transformed barley plants (cv. Golden Promise) as described by Tingay et al. (1997).

[0327] Results

[0328] Eight independent transgenic plants transformed with the Lr67 transgene were identified. All eight plants that had undergone the tissue culture steps involved in the transformation but lacked the Lr67 transgene, as well as the wild-type plants, 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 culturing were carried out on mature plants and seedlings of the transgenic barley grown in a humidity chamber using the Puccinia hordei pathotype 4653P+ (Sydney University PBIC culture number 990492), which is avirulent on plants with the resistance genes Rph3, 5, 7, 10, 11, 14, and 15 and virulent on lines with the resistance genes Rph1, 2, 4, 6, 8, 9, 12, 13, and 19.

[0329] Uredospores were observed on the leaves of the control plants but not on the positive Lr67 transgenic plants in the T0 generation. All of the Lr67 transgenic plants exhibited premature leaf senescence compared to the control plants, similar to the leaf senescence phenotype described as leaf tip necrosis, which is a characteristic feature of wheat Lr67-mediated resistance. When testing the T1 generation of the plants, when testing the seedlings, all plants containing Lr67 showed a leaf rust resistance phenotype, while all plants lacking the Lr67 gene showed leaf rust susceptibility. These results confirm that the isolated Lr67 gene is functionally active and sufficient to confer resistance to barley leaf rust. These experiments also extended the range of pathogen species to which Lr67 confers resistance to include Puccinia hordei in addition to Puccinia striiformis, Puccinia triticina, Puccinia graminis, and Blumeria vulgaris.

[0330] Those skilled in the art will understand that many variations and / or modifications can 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. Accordingly, this embodiment should be considered illustrative in all respects and not restrictive.

[0331] This application claims the priority of AU 2013903161 filed on August 21, 2013, the content of which is hereby incorporated by reference in its entirety.

[0332] Publications discussed and / or cited herein are hereby incorporated by reference in their entirety.

[0333] Any discussion of documents, acts, materials, devices, strategies, terms, etc. included in this specification is for the purpose of providing context for the present invention only. This does not represent an admission that any or all of these things form part of the prior art base or are common general knowledge in the relevant field of the present invention existing before the priority date of each claim of this application.

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Claims

1. A method for producing flour, the method comprising; i) obtaining cereal grains having a genetically modified gene encoding an Lr67 polypeptide, the Lr67 polypeptide being as shown in SEQ ID NO:1, ii) grinding the grains, iii) screening the ground grains, and iv) recovering the flour; wherein the cereal grains are from a wheat plant or a barley plant.

2. The method according to claim 1, wherein the flour is whole wheat flour.

3. The method according to claim 1, wherein the plant resists one or more biotrophic fungal pathogens selected from the group consisting of: Blumeria graminis f. sp. tritici Blumeria graminis Fusarium graminearum Fusarium graminearum Bipolaris sorokiniana Bipolaris sorokiniana Erysiphe graminis f. sp. tritici Puccinia graminis Puccinia graminis f. sp. tritici Puccinia striiformis Puccinia striiformis Puccinia hordei Puccinia hordei and Puccinia triticina Puccinia recondita f. sp . tritici .

4. A method for producing malt liquor, the method comprising; i) obtaining cereal grains having a genetically modified gene encoding an Lr67 polypeptide, the Lr67 polypeptide being as shown in SEQ ID NO:1, ii) soaking the grains, iii) germinating the soaked grains iv) drying the germinated grains, and v) recovering the malt liquor; wherein the cereal grains are from a wheat plant or a barley plant.

5. The method according to claim 4, wherein the plant resists one or more biotrophic fungal pathogens selected from the group consisting of: Blumeria graminis Blumeria graminis f. sp. tritici , Fusarium graminearum Fusarium graminearum , Bipolaris sorokiniana Bipolaris sorokiniana , Blumeria graminis Erysiphe graminis f. sp. tritici , Puccinia graminis Puccinia graminis f. sp. tritici , Puccinia striiformis Puccinia striiformis , Puccinia hordei Puccinia hordei and Puccinia triticina Puccinia recondita f. sp . tritici .

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