GENES CONTROLLING DISEASES
The invention describes nucleotide sequences encoding proteins controlling resistance of plants to fungal diseases. The invention also relates to plants resistant to fungal diseases, and to methods of making plants resistant to fungal diseases.
Fungal diseases are responsible for yearly losses of about $ 9.1 bn on agricultural crops in the US, and are caused by a large variety of biologically diverse pathogens. Different strategies have traditionally been used to control them. Resistance traits have been bred into agriculturally important varieties, thus providing various levels of resistance against either a narrow range of pathogen isolates or races, or against a broader range. However, this involves the long and labor intensive process of introducing desirable traits into commercial lines by genetic crosses and, due to the risk of pests evolving to overcome natural plant resistance, a constant effort to breed new resistance traits into commercial lines is required. Alternatively, fungal diseases have been controlled by the application of chemical fungicides. This strategy usually results in efficient control, but is also associated with the possible development of resistant pathogens and can be associated with a negative impact on the environment. Moreover, in certain crops, such as barley and wheat, the control of fungal pathogens by chemical fungicides is difficult or impractical.
Recent techniques have allowed a better understanding of the interactions between plants and their pathogens at the molecular level and mechanisms of resistance have been partially unraveled. While a large portion of this molecular characterization has been conducted in the model plant Arabidopsis, resistance mechanisms have also begun to be elucidated in economically important crops.
The powdery mildews are a major disease affecting most plant species and have been widely studied. They are characterized by spots or patches of a white to grayish growth on plant tissues, which correspond to the mycelium and cleistothecia of the fungus. Powdery mildews are caused by several species of fungi of the order Erysiphales. For example, Erysiphe graminis causes the powdery mildew of cereals and grasses. While powdery mildews are hard to control in most crops, barley lines resistant to most known isolates of the pathogen are available. It was shown that mutations at a single locus, the Mlo locus, are responsible for resistance phenotype. The mechanism of mlo resistance has been partially elucidated; it involves the formation of large cell wall appositions, called papillae, at the contact sites with the pathogen, which mainly contain callose, but also carbohydrates,
phenols and proteins. In mlo plants, cell wall appositions prevent the penetration of the pathogen, thus providing resistance.
Unfortunately, this powerful tool to control powdery mildews is restricted to barley. In the view of the problems caused by fungal diseases in agriculture, in particular by powdery mildews, there remains an unfulfilled need for new and effective strategies to control these types of pathogens in other crops, which are economically attractive to the farmers and environmentally acceptable.
The present invention addresses the need for novel disease control strategies in plants via the application of genetic engineering techniques. In particular, this invention relates to control strategies against powdery mildew, preferably in economically important crops. The present invention relates to isolated DNA molecules encoding Mlo proteins, wherein such Mlo proteins confer resistance of plants to fungal pathogens. In particular, the invention relates to Mlo proteins containing conserved amino acid sequences that the inventors of the present invention are first to discover, and to the isolated DNA molecules encoding such Mlo proteins. The present invention is also drawn to vectors for expression of the DNA molecules of the present invention in plants. The present invention further relates to transgenic plants comprising any one of the DNA molecules of the present invention. The present invention also describes agricultural products with improved phytosanitary properties comprising transgenic plants resistant to fungal pathogens by expression of any of the DNA molecules of the present invention. The present invention also further relates to methods of making plants resistant to fungal diseases by altering the expression in transgenic plants of proteins encoded by the endogenous copies of the genes corresponding to any one of the DNA molecules of the present invention or altering the activity or the stability of proteins encoded by the endogenous copies of the genes corresponding to any one of the DNA molecules of the present invention. Such transgenic plants are desirably resistant to pathogens that infect living epidermal plant cells, more desirably to fungi from the order Erysiphales, also known as powdery mildews, preferably from the genus Erysiphe, the causing agent of powdery mildew, more preferably the plants are resistant to Erysiphe graminis. The present invention further describes a method to isolate DNA molecules encoding proteins having the same or a similar function as the DNA molecules of the present invention and encoding the conserved amino acid sequences set forth in the present invention.
The present invention thus provides new and effective strategies to control fungal diseases in economically important crops, potentially reducing amounts of chemicals applied to crops and reducing the risk of appearance of pathogens resistant to control agents.
The invention thus provides:
A DNA molecule encoding an Mlo protein conferring upon a plant resistance to fungal pathogens, wherein said protein comprises at least one amino acid sequence identical or substantially similar to an amino acid sequence set forth in SEQ ID No:1 or SEQ ID No:2, wherein said DNA molecule is preferably a cDNA molecule. Preferably, the DNA molecule is derived from wheat. In a preferred embodiment, the DNA molecule is preferably not derived from barley and is derived from a plant which is either a dicot or from a group of plants consisting of wheat, corn, rice, oats, rye, sorghum, sugarcane, millet, milo, and the palm family. In a preferred embodiment, the DNA molecule of the present invention is identical or substantially similar to any one of the nucleotide sequences set forth in SEQ ID No: 3, SEQ ID No: 5 or SEQ ID No:7, or encodes an Mlo protein identical or substantially similar to an Mlo protein set forth in SEQ ID No: 4, SEQ ID No: 6 or SEQ ID No: 8. In a more preferred embodiment, the DNA molecule comprising the nucleotide sequences set forth in SEQ ID No: 3, SEQ ID No: 5 or SEQ ID No: 7 is derived from wheat. In another preferred embodiment, the DNA molecule of the present invention is identical or substantially similar to any one of the nucleotide sequences set forth in SEQ ID No: 9, SEQ ID No: 11 , SEQ ID No: 13, SEQ ID No: 15 or SEQ ID No: 17, or encodes an Mlo protein identical or substantially similar to an Mlo protein encoded by any one of the nucleotide sequences set forth in SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No:16 or SEQ ID No:18. In a more preferred embodiment, the DNA molecule comprising the nucleotide sequences set forth in SEQ ID No: 9, SEQ ID No: 11 , SEQ ID No: 13, SEQ ID No: 15 or SEQ ID No: 17 is derived from Arabidopsis thaliana. In another preferred embodiment, said DNA molecules mentioned hereinbefore are modified such that the activity of the endogenous protein is lost. In one particular embodiment of the present invention, said DNA modification results in one, all or a combination of the following changes in the amino acid sequence of the corresponding protein
— Trp (163) to Arg
— frameshift after Pro (396) — -frameshift after Trp (160)
— Met (1) to lie
— Gly (227) to Asp
— Met (1) to Val
— Arg (11) to Trp
— missing Phe (183), Thr (184)
— Val (31) to Glu
— Ser (32) to Phe
— Leu (271) to His.
In a further preferred embodiment, the fungal pathogens desirably infect living epidermal cells, more desirably the fungal pathogens are from the order Erysiphales, also known as powdery mildews, preferably from the genus Erysiphe and more preferably the fungal pathogen is Erysiphe graminis.
In a further embodiment, the isolated DNA molecule is antisense to an isolated molecule as described above, e.g. antisense to a DNA molecule, e.g. a cDNA molecule, encoding an Mlo protein comprising at least one amino acid sequence identical or substantially similar to an amino acid sequences set forth in SEQ ID No:1 or SEQ ID No:2, especially antisense to a DNA molecule identical or substantially similar to a DNA molecule set forth in SEQ ID Nos:3, 5, 7 ,9, 11 , 13, 15 or 17 and encoding an Mlo protein identical or substantially similar to an Mlo protein set forth in SEQ ID Nos:4, 6, 8, 10, 12, 14, 16 or 18.
The invention further provides:
A protein comprising at least one amino acid sequence identical or substantially similar to an amino acid sequence set forth in SEQ ID No: 1 or SEQ ID No:2, wherein said protein is an Mlo protein and confers upon a plant resistance to fungal pathogens. The protein is preferably not derived from barley and is derived from a plant which is either a dicot or from a group of plants consisting of wheat, corn, rice, oats, rye, sorghum, sugarcane, millet, milo, and the palm family. Preferably, the DNA molecule is derived from wheat. In a preferred embodiment, the protein of the present invention is encoded by a nucleotide sequence identical or substantially similar to any one of the nucleotide sequences set forth in SEQ ID No: 3, SEQ ID No: 5 or SEQ ID No:7 or is identical or substantially similar to any one of the Mlo proteins set forth in SEQ ID No: 4, SEQ ID No: 6 or SEQ ID No: 8. In a more preferred embodiment, the protein is derived from wheat. In another preferred embodiment, the protein of the present invention is encoded by a nucleotide sequence identical or substantially similar to any one of the nucleotide sequences set forth in SEQ ID No: 9, SEQ ID No: 11 , SEQ ID No: 13, SEQ ID No:15 or SEQ ID No: 17 or is identical or substantially similar to any
one of the Mlo proteins set forth in SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16 or SEQ ID No: 18. In a more preferred embodiment, the protein is derived from A. thaliana. In another preferred embodiment, the fungal pathogens desirably infect living epidermal cells, more desirably the fungal pathogens are from the order Erysiphales, also known as powdery mildews, preferably from the genus Erysiphe and more preferably the fungal pathogen is Erysiphe graminis. In a further embodiment, the present invention also encompasses mutated forms or truncated forms of proteins encoded by any of the DNA molecules described above.
The invention further provides:
An expression cassette comprising any one of the DNA molecules described above, e.g. a cDNA as described above or a portion thereof, wherein the DNA molecule is operably linked to a promoter and, optionally, to termination signals capable of expressing the DNA molecule in plants. In a preferred embodiment, the expression cassette is heterologous. In a further preferred embodiment, the promoter and the termination signals are eukaryotic. In a further preferred embodiment, the promoter and termination signals are heterologous with respect to the coding region.
In a preferred embodiment, an expression cassette of the present invention comprises a maize ubiquitin promoter operably linked to a gene encoding a Mlo protein, preferably derived from wheat, operably linked to termination signals, prefeably derived from the CaMV 35S transcript. Preferably, a maize ubiquitin intron 1 is comprised between the maize ubiquitin promoter and the gene encoding a Mlo protein and an inverted PEPC intron is comprised between the gene encoding a Mlo protein and the termination signals. In a preferred embodiment, a full-length gene encoding a Mlo protein is used. In another preferred embodiment, a fragment of a gene encoding a Mlo protein, for example a fragment of approximately 1.1 kb in length, is used.
The invention further provides:
A vector comprising any one of the expression cassettes described above. In a preferred embodiment, the vector is used for transformation of the expression cassette in plants. In another preferred embodiment, the vector of the present invention is used for amplification of any one of the DNA molecules described above.
The invention further provides:
A cell comprising an expression cassette described above or parts of it comprising an isolated DNA molecule of the present invention or a portion thereof, wherein said DNA molecule in said expression cassette in expressible in said cell. In a preferred embodiment, the DNA molecule is not derived from barley. In another preferred embodiment, the cell is a plant cell. In a further preferred embodiment, the expression cassette is stably integrated in the genome of the cell or is included in a self-replicating vector and remains in the cell as an extrachromosomal molecule.
The invention further provides:
A plant comprising an expression cassette described above or parts of it comprising an isolated DNA molecule of the present invention. In a preferred embodiment, the DNA molecule is not derived from barley. In another preferred embodiment, the DNA molecule comprised in the expression cassette is expressible in the plant. In another preferred embodiment, the DNA molecule is stably integrated in the plant genome or is included in a self-replicating vector and remains in the cell as an extrachromosomal molecule. In another preferred embodiment, the plant is resistant to fungal pathogens, desirably fungal pathogens which infect living epidermal cells, more desirably the fungal pathogens are from the order Erysiphales, also known as powdery mildews, preferably from the genus Erysiphe and more preferably the fungal pathogen is Erysiphe graminis.
The inventions also relates to the seed for such a plant, which seed is optionally treated (e.g. primed or coated) and/or packaged, e.g. placed in a bag with instructions for use.
The invention further provides:
Agricultural products comprising a plant comprising an isolated DNA molecule of the present invention or a portion thereof. In a preferred embodiment, the agricultural product is used as e.g. feed, food, or silage and does not contain mycotoxins produced by fungal pathogens, such as e.g. aflatoxins. Therefore, the agricultural product has improved phytosanitary properties.
The invention further provides:
A method for making a plant resistant to a fungal pathogen comprising the step of: a) expressing in a plant an RNA transcript encoded by any one of the DNA molecules described above or a portion thereof in "sense" orientation; or
b) expressing in a plant an RNA transcript encoded by any one of the DNA molecules described above or a portion thereof in "anti-sense" orientation; or c) expressing in a plant a ribozyme capable of specifically cleaving a messenger RNA transcript encoded by an endogenous gene corresponding to any one of the DNA molecules described above; or d) expressing in a plant an aptamer specifically directed to an endogenous protein encoded by a gene corresponding to any one of the DNA molecules described above; or e) expressing in a plant a mutated or a truncated form of any one of the DNA molecules described above, so that it can act as a dominant negative mutant; or f) modifying by homologous recombination in a plant at least one chromosomal copy of the gene corresponding to any one of the DNA molecules described above; or g) modifying by homologous recombination in a plant at least one chromosomal copy of the regulatory elements of a gene corresponding to any one of the DNA molecules described above.
The invention further provides:
A plant obtained by any one of the method described immediately above including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag with instructions for use. In another preferred embodiment, the plant obtained is resistant to fungal pathogens, desirably fungal pathogens which infect living epidermal cells, more desirably the fungal pathogens are from the order Erysiphales, also known as powdery mildews, preferably from the genus Erysiphe and more preferably the fungal pathogen is Erysiphe graminis.
The invention further provides:
An agricultural product with improved phytosanitary properties obtained by any one of the methods described immediately above.
The invention further provides:
A method for isolating DNA molecules encoding Mlo proteins comprising the steps of: a) mixing a degenerated oligonucleotide encoding at least six amino acids of SEQ ID
No:1 and a degenerated oligonucleotide complementary to a sequence encoding at least six amino acids of SEQ ID No:2, with DNA extracted from a plant under conditions allowing hybridization of said degenerated oligonucleotides to said DNA; and
b) amplifying a DNA fragment of said plant DNA, wherein said DNA fragment comprises a at its left and right ends nucleotide sequences that can anneal to said degenerated oligonucleotides in step a); and c) obtaining a full-length cDNA clone comprising the DNA fragment of step b).
The invention further provides:
A method for producing mutated copies of the nucleotide sequences of the present invention by uin-vitro recombination" or "DNA shuffling". The mutated copies of the nucleotide sequences of the present invention are used to confer improved resistance to fungal pathogens. In a preferred embodiment, the mutant copies of the nucleotide sequences of the present invention are used to confer resistance to a broader range of pathogens. One such method is described below:
A method for mutagenizing a DNA molecule according to the present invention, wherein said
DNA molecule has been cleaved into double-stranded-random fragments of a desired size, and comprising the steps of: a) adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded template polynucleotide; b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and d) repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double- stranded polynucleotide from the third step of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide.
The present invention further provides methods of altering the expression of a gene encoding a Mlo protein in a plant cell using sense and antisense RNA fragments of the gene. Importantly, such sense and antisense RNA fragments are capable of forming a double- stranded RNA molecule. The invention also relates to plant cells obtained using such
methods, to plants derived from such cells, to the progeny of such plants, to seeds derived from such plants and to agricultural products with improved phytosanitary properties obtained by any one of the methods described immediately above.
The invention further provides:
A method comprising introducing into a plant cell a first DNA sequence capable of expressing in said cell a sense RNA fragment of a gene encoding a Mlo protein, and a second DNA sequence capable of expressing in said cell an antisense RNA fragment of said gene encoding a Mlo protein, wherein said sense RNA fragment and said antisense RNA fragment are capable of forming a double-stranded RNA molecule, wherein the expression of a gene encoding a Mlo protein in said plant cell is altered. Preferably, the gene the expression of which in said cell is altered comprises the first or second DNA sequence. Preferably, the DNA sequences are stably integrated in the genome of the plant cell.
Further preferred embodiments of the present invention are listed below:
1. the first DNA sequence and the second DNA sequence are comprised in two different DNA molecules. In a preferred embodiment, the first DNA molecule further comprises a first promoter operably linked to the first DNA sequence and the second DNA molecule further comprises a second promoter operably linked to the second DNA sequence;
2. the first DNA sequence and the second DNA sequence are comprised in one DNA molecule. In a preferred embodiment, the DNA molecule further comprises a promoter operably linked to said first or said second DNA sequence. Preferably the promoter is operably linked to the second DNA sequence capable of expressing in the cell an antisense RNA fragment of the gene encoding a Mlo protein;
2.1. the first DNA sequence and the second DNA sequence are comprised in the same DNA strand of the DNA molecule;
2.1.1. the sense RNA fragment and the antisense RNA fragment are comprised in one RNA molecule. Preferably, the RNA molecule is capable of folding such that said RNA fragments comprised therein form a double-stranded region;
2.1.2. the sense RNA fragment and the antisense RNA fragment are comprised in two RNA molecules. In this case, the first DNA sequence and the second DNA sequence are preferably operably linked to a bi-directional promoter or, alternatively, the first DNA sequence is operably linked to a first promoter and the second DNA sequence is operably
linked to a second promoter, wherein the first promoter and the second promoter comprise the same promoter or comprise different promoters;
2.2. the first DNA sequence and the second DNA sequence are comprised in complementary strands of said DNA molecule. In a preferred embodiment, the first DNA sequence is the complementary DNA strand of the second DNA sequence in said DNA molecule. In this case, the DNA molecule further comprises a first promoter operably linked to said first or second DNA sequence. In a preferred embodiment, the DNA molecule further comprises a first site-specific recombination site between said first promoter and said first or second DNA sequence and a second site-specific recombination site at the 3'end of said first DNA sequence, wherein said first and second site-specific recombination sites are preferably in inverted orientation to each other and are capable of inverting said first or second DNA sequence between said first and second site-specific recombination sites in presence of a site-specific recombinase. In a further preferred embodiment and as a result of said inverting said first promoter is capable of expressing said second DNA sequence. The plant cell preferably further comprises a site-specific recombinase capable of recognizing said site- specific recombination sites. In yet another preferred embodiment, the DNA molecule further comprises a first promoter operably linked to said first DNA sequence and a second promoter operably linked to said second DNA sequence, wherein the first promoter and the second promoter comprise the same promoter or comprise different promoters. The first and second DNA sequences used in the present invention are derived from any gene encoding a Mlo protein. In a preferred embodiment, such gene encodes a Mlo protein which comprises at least one of the sequences set forth in SEQ ID No:1 or SEQ ID No:2. In another preferred embodiment, a gene encoding a Mlo protein is derived from a monocotyledonous plant, preferably wheat, barley or rice. In another preferred embodiment, the gene is identical or substantially similar to any one of the nucleotide sequences set forth in SEQ ID No:3, SEQ ID No:5 or SEQ ID No:7, or encodes an Mlo protein identical or substantially similar to any one of the amino acid sequences set forth in SEQ ID No:4, SEQ ID No:6 or SEQ ID No:8. In a further preferred embodiment, the gene encoding a Mlo protein is derived from a dicotyledonous plant, preferably Arabidopsis and is preferably identical or substantially similar to any one of the nucleotide sequences set forth in SEQ ID No:9, SEQ ID No:11 , SEQ ID No: 13, SEQ ID No: 15 or SEQ ID No: 17, or encodes an Mlo protein identical or substantially similar to any one of the amino acid sequences set forth in SEQ ID No:10, SEQ ID No:12, SEQ ID No:14, SEQ ID No:16 or SEQ ID No:18.
In a further preferred embodiment, a promoter used in the present invention is a heterologous promoter, for example a tissue specific promoter, a developmentally regulated promoter, a constitutive promoter or an inducible promoter. Optionally, the promoter is a divergent promoter capable of initiating transcription of DNA sequences on each side of the promoter. In yet another preferred embodiment, the DNA sequence further comprises a linker between the DNA sequences encoding said the sense and antisense RNA fragments. The linker comprises, e.g. an expression cassette comprising a functional gene, e.g. a selectable marker gene or regulatory sequences, e.g. intron processing signals.
The invention further provides:
A plant cell obtained by the method described immediately above, a plant and the progeny thereof derived from the plant cell, and seeds derived from the plant.
The invention further provides:
An agricultural product with improved phytosanitary properties obtained by a method described immediately above.
The invention therefore provides:
A method comprising introducing into a plant cell a sense RNA fragment of a gene encoding a Mlo protein and an antisense RNA fragment of said gene encoding a Mlo protein, wherein said sense RNA fragment and said antisense RNA fragment are capable of forming a double-stranded RNA molecule, wherein the expression of a gene encoding a Mlo protein in said cell is altered. In a preferred embodiment, the RNA fragments are comprised in two different RNA molecules. In another preferred embodiment, the RNA fragments are mixed before being introduced into said cell. In another preferred embodiment, the RNA fragments are mixed before being introduced into said cell under conditions allowing them to form a double-stranded RNA molecule. In another preferred embodiment, the RNA fragments are introduced into said cell sequentially. In yet another preferred embodiment, the RNA fragments are comprised in one RNA molecule. In such case, the RNA molecule is preferably capable of folding such that said RNA fragments comprised therein form a double-stranded RNA molecule.
The invention further provides:
A plant cell obtained by the method described immediately above, a plant and the progeny thereof derived from the plant cell, and seeds derived from the plant.
The invention further provides:
An agricultural product with improved phytosanitary properties obtained by a method described immediately above.
The invention also further provides:
A plant cell comprising the sense and antisense RNA fragments of the present invention, wherein the expression of a gene encoding a Mlo protein in said plant cell is preferably altered by said RNA fragments, a plant and the progeny thereof derived from the plant cell, and seeds derived from the plant. In a preferred embodiment, such a plant is resistant to a fungal pathogen, preferably to a fungal pathogen that infects living epidermal cells, preferably from the order Erysiphales, preferably from the genus Erysiphe, preferably the plant is resistant to Erysiphe graminis. In a preferred embodiment, a plant of the present invention is a monocotyledonous plant, preferably wheat.
The invention also further provides:
A plant cell comprising the two DNA sequences of claim 47, wherein the expression of a gene encoding a Mlo protein in said plant cell is altered, when said DNA sequences are expressed, a plant and the progeny thereof derived from the plant cell, and seeds derived from the plant. In a preferred embodiment, the plant is resistant to a fungal pathogen, preferably to a fungal pathogen that infects living epidermal cells, preferably from the order Erysiphales, preferably from the genus Erysiphe, preferably the plant is resistant to Erysiphe graminis. In a preferred embodiment, a plant of the present invention is a monocotyledonous plant, preferably wheat.
The invention also further provides:
A method for making a plant resistant to a fungal pathogen comprising expressing in said plant a sense RNA fragment of a gene encoding a Mlo protein and an antisense RNA fragment of said gene encoding a Mlo protein, wherein said sense RNA fragment and said antisense RNA fragment are capable of forming a double-stranded RNA molecule. Any gene encoding a Mlo protein is used in the present method. In a preferred embodiment, such gene encodes a Mlo protein which comprises at least one of the sequences set forth in SEQ ID
No:1 or SEQ ID No:2. In another preferred embodiment, such gene is derived from a monocotyledonous plant, preferably wheat, barley or rice. In another preferred embodiment, the gene is identical or substantially similar to any one of the nucleotide sequences set forth in SEQ ID No:3, SEQ ID No:5 or SEQ ID No:7, or encodes an Mlo protein identical or substantially similar to any one of the amino acid sequences set forth in SEQ ID No:4, SEQ ID No:6 or SEQ ID No:8. In a further preferred embodiment, the gene encoding a Mlo protein is derived from a dicotyledonous plant, preferably Arabidopsis and is preferably identical or substantially similar to any one of the nucleotide sequences set forth in SEQ ID No:9, SEQ ID No:11 , SEQ ID No:13, SEQ ID No:15 or SEQ ID No:17, or encodes an Mlo protein identical or substantially similar to any one of the amino acid sequences set forth in SEQ ID No:10, SEQ ID No:12, SEQ ID No:14, SEQ ID No:16 or SEQ ID No:18. In a preferred embodiment, the plant is a monocotyledonous plant, preferably wheat.
The invention further provides:
A plant obtained by a method described immediately above including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag with instructions for use. In another preferred embodiment, the plant obtained is resistant to fungal pathogens, desirably fungal pathogens which infect living epidermal cells, more desirably the fungal pathogens are from the order Erysiphales, also known as powdery mildews, preferably from the genus Erysiphe and more preferably the fungal pathogen is Erysiphe graminis.
The invention further provides:
An agricultural product with improved phytosanitary properties obtained by a method described immediately above.
The invention also further provides:
A DNA construct comprising a first DNA sequence capable of expressing in a cell a sense RNA fragment of a gene encoding a Mlo protein and a second DNA sequence capable of expressing in said cell an antisense RNA fragment of said gene encoding a Mlo protein, wherein said sense RNA fragment and said antisense RNA fragment are capable of forming a double-stranded RNA molecule. In a preferred embodiment, the expression of a gene encoding a Mlo protein in said cell is altered. In another preferred embodiment, the cell is a plant cell.
ln another preferred embodiment, the DNA construct further comprises a promoter operably linked to said first or said second DNA sequence. In a further preferred embodiment, a promoter used in the present invention is a heterologous promoter, for example a tissue specific promoter, a developmentally regulated promoter, a constitutive promoter or an inducible promoter. Optionally, the promoter is a divergent promoter capable of initiating transcription of DNA sequences on each side of the promoter. In an alternative embodiment, the DNA construct further comprises a first promoter operably linked to said first DNA sequence and a second promoter operably linked to said second DNA sequence. In another alternative embodiment, a DNA construct of the present invention further comprises a bidirectional promoter operably linked to said first DNA sequence and to said second DNA sequence.
The invention also further provides:
An expression cassette comprising any one of the DNA constructs described immediately above operably linked to a promoter. In a preferred embodiment, the promoter in the expression cassette comprises a rice actin promoter. In a further preferred embodiment, the promoter is operably linked to the second DNA sequence capable of expressing in a cell an antisense RNA fragment of a gene encoding a Mlo protein. A gene encoding a Mlo protein used in the expression cassette is preferably derived from wheat, preferably from a wheat TrMlo-2 gene, and, for example, DNA sequences of approximately 1.1 kb in length are used. In another preferred embodiment, a spacer, preferably of about 28 nucleotides, is inserted between the second DNA sequence and the first DNA sequence. Optionally, the expression cassette further comprises termination signals, preferably derived from the nos gene, and operably linked to the 3' end of the first DNA sequence capable of expressing in a cell a sense RNA fragment of the gene encoding a Mlo protein.
The invention also further provides:
A vector comprising any one of the expression cassettes described above, plant cells comprising such vectors, plants derived from such cells, the progeny of such plants and seeds derived from such plants.
DEFINITIONS
An "isolated DNA molecule" is a nucleotide sequence that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleotide