Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

• 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.
• 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.
• mlo resistance 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.
• the present invention addresses the need for novel disease control strategies in plants via the application of genetic engineering techniques.
• 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.
• 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.
• 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:
• 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.
• 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.
• 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.
• 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.
• 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.
• said DNA molecules mentioned hereinbefore are modified such that the activity of the endogenous protein is lost.
• said DNA modification results in one, all or a combination of the following changes in the amino acid sequence of the corresponding protein
• 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.
• 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.
• 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.
• the protein is derived from wheat.
• 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.
• the protein is derived from A. thaliana.
• 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.
• 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, wherein the DNA molecule is operably linked to a promoter and to termination signals capable of expressing the DNA molecule in plants.
• the expression cassette is heterologous.
• the promoter and the termination signals are eukaryotic.
• the promoter and termination signals are heterologous with respect to the coding region.
• the invention further provides:
• a vector comprising any one of the expression cassettes described above.
• the vector is used for transformation of the expression cassette in plants.
• 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 or parts of it comprising an isolated DNA molecule of the present invention, wherein said DNA molecule in said expression cassette in expressible in said cell.
• the DNA molecule is not derived from barley.
• the cell is a plant cell.
• 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 or parts of it comprising an isolated DNA molecule of the present invention.
• the DNA molecule is not derived from barley.
• the DNA molecule comprised in the expression cassette is expressible in the plant.
• 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.
• 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.
• fungal pathogens desirably fungal pathogens which infect living epidermal cells
• 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.
• 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.
• 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 in "sense" orientation; or b) expressing in a plant an RNA transcript encoded by any one of the DNA molecules described above 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
• the invention further provides:
• 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:
• 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:
• the mutated copies of the nucleotide sequences of the present invention are used to confer improved resistance to fungal pathogens.
• 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 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
• 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 sequence may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
• a "protein” as defined herein is the entire protein encoded by the corresponding nucleotide sequence, or is a portion of the protein encoded by the corresponding portion of the nucleotide sequence.
• isolated protein is a protein that is encoded by an isolated nucleotide sequence and is therefore not a product of nature.
• An isolated protein may exist in a purified form or may exist in a non-native environment, such as a transgenic host cell, wherein the protein would not normally expressed or would be expressed in a different form or different amount in an isogenic non-transgenic host cell.
• a plant "resistant to a fungal pathogen" has no or lesser symptoms of a fungal infection caused by the fungal , by inhibiting or limiting the ability of the fungal pathogen to grow on the plant. As a consequence, the plant grows better, has higher yields and produces more seeds.
• a protein conferring resistance upon a plant to a fungal pathogen means that the protein is involved in the regulation of plant genetic pathways responsible for resistance of the plant to the fungal pathogen.
• the protein may be a positive regulator in that it enhances resistance of the plant to the fungal pathogen, or the protein may be a negative regulator in that it represses resistance of the plant to the fungal pathogen.
• a particular example of a protein conferring resistance upon a plant to a fungal pathogen is an Mlo protein.
• Mlo protein means herein a member of a family of proteins (the Mlo family) having a substantially similar function in a disease resistance pathway and sharing some structural homology.
• the structural homology can be e.g. that the members of the family share at least one conserved region.
• the term "substantially similar”, when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g. where only changes in amino acids not affecting the polypeptide function occur.
• the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence.
• the percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 80%, more desirably 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.
• substantially similar when used herein with respect to a protein, means a protein corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, e.g. where only changes in amino acids not affecting the polypeptide function occur.
• the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is at least 80%, more desirably 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.
• the percentage of sequence identity is determined using computer programs that are based on dynamic programming algorithms.
• Computer programs that are preferred within the scope of the present invention include the BLAST (Basic Local Alignment Search Tool) search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA.
• Version BLAST 2.0 (Gapped BLAST) of this search tool has been made publicly available on the Internet (currently http://www.ncbi.nlm.nih.gov/BLAST/). It uses a heuristic algorithm which seeks local as opposed to global alignments and is therefore able to detect relationships among sequences which share only isolated regions.
• the scores assigned in a BLAST search have a well-defined statistical interpretation.
• Said programs are preferably run with optional parameters set to the default values.
• gene refers to a coding sequence and associated regulatory sequences wherein the coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
• regulatory sequences are promoter sequences, 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
• “Expression” refers to the transcription and/or translation of an endogenous gene or a transgene in plants.
• expression may refer to the transcription of the antisense DNA only.
• “Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
• the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA that, in the sense or antisense direction, inhibits expression of a particular gene, e.g., antisense RNA.
• the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
• the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
• the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
• the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue or organ or stage of development.
• Heterologous as used herein means "of different natural or of synthetic origin" or represent a non-natural state. For example, if a host cell is transformed with a nucleic sequence derived from another organism, particularly from another species, that gene is heterologous with respect to that host cell and also with respect to descendants of the host cell which carry that gene.
• the transforming nucleic acid may comprise a heterologous promoter, heterologous coding sequence, or heterologous termination sequence. Alternatively, the transforming nucleic acid may be completely heterologous or may comprise any possible combination of heterologous and endogenous nucleic acid sequences.
• heterologous refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. a different copy number, or under the control of different regulatory elements.
• promoter refers to a DNA sequence that initiates transcription of an associated DNA sequence.
• the promoter region may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors.
• Synthetic nucleotide sequence as used herein means a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an artificial sequence that resembles more closely the G+C content and the normal codon distribution of dicot and/or monocot genes is said to be synthetic.
• a regulatory DNA sequence is said to be "operably linked to” or “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence.
• Regulatory elements refer to sequences involved in conferring the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
• plant refers to any plant or part of a plant and particularly to seed plants at any stage of development. Therein are also included cuttings, cell or tissue cultures and seeds.
• plant tissue includes, but is not limited to, whole plants, plant organs, plant seeds, protoplasts, callus, cell cultures, and any groups of plant cells organized into structural and/or functional units.
• a "plant cell” refers to the structural and physiological unit of the plant, comprising a protoplast and a cell wall.
• the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.
• 'Transformation as used herein means introduction of a nucleic acid into a cell.
• a nucleic acid into a cell.
• stable integration of a DNA molecule into the genome of an organism of interest.
• a "selectable marker” is conferred by a gene whose expression in a plant cell gives the cell a selective advantage.
• the selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non- transformed cells.
• the selective advantage possessed by the transformed cells, compared to non-transformed cells may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source.
• Selectable marker gene also refers to a gene or a combination of genes whose expression in a plant cell gives the cell both, a negative and a positive selective advantage.
• a "screenable marker” is conferred by a gene whose expression does not give a selective advantage to a transformed cell, but whose expression makes the transformed cell phenotypically distinct from untransformed cells.
• the present invention relates to DNA molecules encoding Mlo proteins, which confer upon a plant resistance to fungal pathogens.
• the inventors of the present invention are first to identify conserved amino acid sequences among Mlo proteins.
• the conserved amino acid sequences of the present invention are conserved between three Mlo proteins derived from wheat and three Mlo proteins derived from A. thaliana. These amino acid sequences are also conserved in two predicted Arabidopsis Mlo proteins.
• the first conserved amino acid sequence which is set forth in SEQ ID No:1 , comprises 13 amino acids.
• the fourth amino acid in SEQ ID No:1 is either L, V or I
• its fifth amino acid is either V or L
• its seventh amino acid is either F or L
• the thirteenth amino acid in SEQ ID No:1 is not I and is preferably either T, S or A.
• the second conserved amino acid sequence which is set forth in SEQ ID No:2, comprises 14 amino acids.
• the first amino acid in SEQ ID No:2 is not M and is preferably either I, V, S or G.
• Its third amino acid is either F, L or V
• its sixth amino acid is either Y or N
• its seventh amino acid is either A or V
• its eighth amino acid is either L or I
• its tenth amino acid is either T or S.
• the invention encompasses isolated Mlo proteins comprising at least one of the conserved amino acid sequences described above and isolated DNA molecules which encode such Mlo proteins.
• the invention also encompasses isolated Mlo proteins which comprise both conserved sequences set forth in SEQ ID No: 1 and SEQ ID No:2.
• the isolated DNA molecules encoding the Mlo proteins of the present invention are cDNA molecules.
• the DNA molecules encoding Mlo proteins comprising at least one of the conserved amino acid sequences are not derived from barley.
• such DNA molecules are derived from a dicot or from wheat, corn, rice, oats, rye, sorghum, sugarcane, millet, milo, or the palm family.
• a DNA molecule of the present invention is identical or substantially similar to the DNA molecules set forth in SEQ ID No:3, 5 or 7 and in SEQ ID No:9, 11 , 13, 15 or 17 or encode an Mlo protein, which a identical or substantially similar to any one of the Mlo proteins set forth in SEQ ID No:4, 6 or 8 or in SEQ ID No:10, 12, 14, 16, 18.
• the DNA molecules set forth in SEQ ID No:3, 5 or 7 are derived from wheat and encode the Mlo proteins set forth in SEQ ID No:4, 6 or 8, respectively.
• the isolation of such DNA molecules is further illustrated in example 1.
• the DNA molecules set forth in SEQ ID No:9, 11 , 13, 15 or 17 are derived from Arabidopsis and encode the Mlo proteins set forth in SEQ ID No:10, 12, 14, 16 or 18, respectively.
• the isolation of such DNA molecules is further illustrated in example 2.
• the DNA molecule of SEQ ID No:3 encoding a wheat Mlo protein called TrMlol is deposited as strains TrMlol and TrMlol -5 with accession numbers NRRL B-21948 and NRRL B- 21949, respectively.
• TrMlo2 The DNA molecule of SEQ ID No:5 encoding a wheat Mlo protein called TrMlo2 is deposited as strains TrMlo2 and TrMlo2-5 with accession numbers NRRL B-21950 and NRRL B-21951 , respectively.
• the DNA molecule of SEQ ID No:7 encoding a wheat Mlo protein called TrMlo3 is deposited as strains TrMlo3 and TrMlo3-5 with accession numbers NRRL B-21952 and NRRL B-21953, respectively.
• TrMlol and TrMlo3 comprise the full-length cDNAs of the corresponding Mlo genes and also comprise some of the corresponding 5' and 3' untranslated regions.
• TrMlo2 is the longest cDNA clone of the corresponding gene that was recovered. It comprises the entire coding region with exception of the first methionin (start codon) as deduced from a comparison with TrMlol and TrMlo3. TrMlo2 also comprises some of the 3' untranslated region of the corresponding gene.
• the DNA molecule of SEQ ID No:9 encoding an Arabidopsis Mlo protein called CIB10259 is deposited as strain pCIB10259 with accession number NRRL B-21945.
• the DNA molecule of SEQ ID No:11 encoding an Arabidopsis Mlo protein called CIB10295 is deposited as strain pCIB10295 with accession number NRRL B-21946.
• the DNA molecule of SEQ ID No:13 encoding an Arabidopsis Mlo protein called CIB10296 is deposited as strain pCIB1029 with accession number NRRL B-21947.
• CIB10259, CIB10295 and CIB10296 comprise the full-length cDNAs of the corresponding Mlo genes and also comprise and also comprise some of the corresponding 5' and 3' untranslated regions.
• the nucleotide sequences encoding the Arabidopsis Mlo protein family members F19850 and U95973 are obtained from the Genbank. However, for both clones, a predicted amino acid sequence is determined and is found not to match the amino acid sequence predicted in the Genbank entry.
• the Mlo proteins determined by the inventors of the present invention are therefore novel and not obvious. Both newly predicted proteins contain the conserved amino acid sequences set forth in SEQ ID No:1 and 2 and are therefore encompassed by the present invention as well as the isolated cDNAs encoding them.
• An Mlo protein encoded by a DNA molecule of the present invention confer upon a plant resistance to fungal pathogens, desirably fungal pathogens which infect living epidermal plant cells, more desirably fungal pathogens from the order Erysiphales, also known as powdery mildews (Agrios G. (1988) Plant Pathology, Third Edition, Academic Press Inc., in particular p 271).
• an Mlo protein encoded by a DNA molecule of the present invention confer upon a plant resistance from the genus Erysiphe, more preferably the fungal pathogen is Erysiphe graminis.
• the present invention also encompasses recombinant vectors comprising any one of the DNA molecules of this invention.
• such DNA molecules are preferably comprised in an expression cassette comprising regulatory elements for expression of the DNA molecules in a host cell capable of expressing such DNA molecules.
• regulatory elements are usually a promoter and termination signals and preferably also include elements allowing efficient translation of a protein encoded by a DNA molecule of the present invention.
• an expression cassette is heterologous.
• Such vectors are used for transformation of the expression cassette comprising any one of the DNA molecules of this invention into a host cells.
• the expression cassette is stably integrated into the DNA of such host cell.
• the expression cassette is comprised in a vector, which is capable of replication in a host cell and remains in the host cell as an extrachromosomal molecule.
• such extrachromosomal replicating molecule is used amplify the DNA molecules of this invention in a host cell
• a host cell in a preferred embodiment, such host cell is a microorganism, such as a bacteria, in particular E. coli.
• a host cell is an eukaryotic cells, such as e.g. a yeast cell, an insect cell, or a plant cell.
• a DNA molecule of the present invention is modified by incorporation of random mutations in a technique known as in-vitro recombination or DNA shuffling. This technique is described in Stemmer et al., Nature 370: 389-391 (1994) and US Patent 5,605,793 incorporated herein by reference. Millions of mutant copies of the nucleotide sequences are produced based on the original nucleotide sequence described herein and variants with improved properties, such as increased resistance for fungal pathogens or resistance against a wider range of pathogens are recovered.
• the method encompasses forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide comprising the nucleotide sequence of this invention, wherein the template double-stranded polynucleotide has been cleaved into double- stranded-random fragments of a desired size, and comprises the steps of 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; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single- stranded fragments; 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
• the concentration of a single species of double- stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the total DNA.
• the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides.
• the size of the double-stranded random fragments is from about 5 bp to 5 kb.
• the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles.
• the present invention also encompasses cells comprising a DNA molecule of the present invention, wherein the DNA molecule is not in its natural cellular environment.
• such cells are plant cells.
• a DNA molecule of the present invention is expressible in such cells and is comprised in an expression cassette which allow their expression in such cells.
• the expression cassette is stably integrated into the DNA of such host cell.
• the expression cassette is comprised in a vector, which is capable of replication in the cell and remains in the cell as an extrachromosomal molecule.
• the present invention also encompasses a plant comprising the plant cells described above.
• the DNA molecules of the present invention are expressible in the plant, and expression of any one of the DNA molecules of the present invention or of a portion thereof in transgenic plants confers resistance upon the transgenic plant against fungal pathogens.
• 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.
• the present invention therefore also encompasses transgenic plants made resistant to fungal pathogens by the expression of any one of the DNA molecules of the present invention or of a portion thereof.
• Plants transformed in accordance with the present invention may be monocots or dicots and include, but are not limited to, maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis thaliana, and woody plants such as coniferous and deciduous trees, especially maize, wheat, or sugarbeat.
• nucleotide sequence Once a desired nucleotide sequence has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.
• the DNA molecules For their expression in transgenic plants, the DNA molecules may require modification and optimization. It is known in the art that all organisms have specific preferences for codon usage, and the codons in the nucleotide sequence comprised in the DNA molecules of the present invention can be changed to conform with specific plant preferences, while maintaining the amino acids encoded thereby. Furthermore, high expression in plants is best achieved from coding sequences which have at least 35% GC content, and preferably more than 45%.
• Nucleotide sequences which have low GC contents may express poorly due to the existence of ATTTA motifs which may destabilize messages, and AATAAA motifs which may cause inappropriate polyadenylation.
• preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17: 477-498 (1989)).
• the nucleotide sequences are screened for the existence of illegitimate splice sites which cause message truncation.
• sequences adjacent to the initiating methionine may require modification.
• they can be modified by the inclusion of sequences known to be effective in plants.
• Joshi has suggested an appropriate consensus for plants (NAR 15: 6643-6653 (1987)) and Clontech suggests a further consensus translation initiator (1993/1994 catalog, page 210).
• These consensuses are suitable for use with the nucleotide sequences of this invention.
• the sequences are incorporated into constructions comprising the nucleotide sequence, up to and including the ATG (whilst leaving the second amino acid unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibility of modifying the second amino acid of the transgene).
• the DNA molecules in transgenic plants are driven by a promoter shown to be functional in plants.
• the choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species.
• expression in leaves is preferred; for the protection of plants against ear pathogens, expression in inflorescences (e.g. spikes, panicles, cobs, etc.) is preferred; for protection of plants against root pathogens, expression in roots is preferred; for protection of seedlings against soil-borne pathogens, expression in roots and/or seedlings is preferred.
• protection against more than one type of phytopathogen is sought, and thus expression in multiple tissues is desirable.
• dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.
• monocotyledonous promoters for expression in monocotyledons.
• Preferred promoters which are expressed constitutively include promoters derived from Agrobacterium opine synthase genes, e.g. the nos promoter, or a dual promoter from the Agrobacterium Ti plasmid (Velten et al. (1984) EMBO J. 3: 2723-2730), or viral promoters operable in plants, e.g. the CaMV 35S and 19S promoters, and promoters from genes encoding actin or ubiquitin.
• Another preferred promoter is a synthetic promoter, such as the Gelvin Super MAS promoter (Ni et al. (1995) Plant J. 7: 661-676).
• the DNA molecules of this invention can also be expressed under the regulation of promoters which are chemically regulated. This enables the protein conferring fungal diseases to be synthesized only when the crop plants are treated with the inducing chemicals.
• promoters which are chemically regulated. This enables the protein conferring fungal diseases to be synthesized only when the crop plants are treated with the inducing chemicals.
• Preferred technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 and US patent 5,614,395.
• a preferred promoter for chemical induction is the tobacco PR-1a promoter.
• a preferred category of promoters is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. Ideally, such a promoter should only be active locally at the sites of infection, and in this way the protein controlling fungal diseases only accumulates in cells which need to synthesize it to kill the invading insect pest.
• Preferred promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), and Warner et al. Plant J. 3: 191 -201 (1993).
• Preferred tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons.
• a preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)).
• a preferred promoter for root specific expression is that described by de Framond (FEBS 290: 103-106 (1991); EP 0 452 269) and a further preferred root-specific promoter is that from the T-1 gene provided by this invention.
• a preferred stem specific promoter is that described in US patent 5,625,136 and which drives expression of the maize trpA gene.
• Preferred embodiments of the invention are transgenic plants expressing a DNA molecule in a root-specific fashion. Further preferred embodiments are transgenic plants expressing the DNA molecule in a wound-inducible or pathogen infection-inducible manner.
• constructions for expression of the protein in plants require an appropriate transcription terminator to be attached downstream of the heterologous nucleotide sequence.
• terminators are available and known in the art (e.g. tm1 from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of this invention.
• sequences which have been shown to enhance expression such as intron sequences (e.g. from Adh1 and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).
• intron sequences e.g. from Adh1 and bronzel
• viral leader sequences e.g. from TMV, MCMV and AMV.
• DNA molecules may be target expression of the DNA molecules to different cellular localizations in the plant.
• localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be preferred.
• Subcellular localization of transgene encoded enzymes can be undertaken using techniques well known in the art.
• the DNA encoding the target peptide from a known organelle-targeted gene product is manipulated and fused upstream of the nucleotide sequence. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown.
• Vectors suitable for plant transformation are described elsewhere in this specification.
• binary vectors or vectors carrying at least one T- DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be preferred.
• direct gene transfer transformation with a single DNA species or co-transformation can be used (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
• transformation is usually (but not necessarily) undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methatrexate) or a herbicide (glufosinate, glyphosate or a protoporphyrinogen oxidase inhibitor), or a selectable marker which may confer a selective advantage to transformed cells, such as a phospho-mannose isomerase gene.
• a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methatrexate) or a herbicide (glufosinate, glyphosate or a protoporphyrinogen oxidase inhibitor), or a selectable marker which may confer a selective advantage to transformed cells, such as a phospho-mannose isomerase gene.
• selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methatrexate) or a herbicide (glufosinate, glyphosate or a protoporphyr
• the DNA molecules of this invention are directly transformed into the plastid genome.
• Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride era/. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305.
• the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the DNA molecule of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
• the 1 to 1.5 kb flanking regions facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome.
• point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45).
• Plastid expression in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein.
• the present invention also encompasses agricultural products, which comprise transgenic plants made resistant to fungal pathogens by expression of any one of the DNA molecules of the present invention or made resistant to fungal pathogens by any one of the methods described infra. Since such plants are resistant to fungal pathogens, pathogen growth on them is suppressed. Such plants and the agricultural products derived thereof are therefore less likely to contain mycotoxins, which are naturally produced by many fungal pathogens and which can be very toxic to human and to animals. Therefore, such agricultural products have better phytosanitary properties. In a preferred embodiment, such agricultural products are used as feedstuff, as silage, or as food.
• the Mlo proteins encoded by the DNA molecules of the present invention confer upon a plant resistance to a fungal pathogen and it is a preferred object of the present invention to alter the expression of such proteins in their natural host environment. It is a further preferred object of the present invention to alter the stability or the activity of such proteins in their natural environment. Such alterations of the expression, stability, or activity of the proteins encoded by the DNA molecules of the present invention in a plant result in increased resistance of the plant to fungal pathogens.
• a protein encoded by a DNA molecule of the present invention the protein is a negative regulator of resistance of a plant to fungal pathogens, in that it represses genetic pathways in the plant which are responsible for resistance of the plant to fungal pathogens. Therefore, it is a preferred object of the present invention to reduce the expression of the Mlo proteins encoded by the DNA molecules of the present invention in their natural host environment, or to reduce the stability or the activity of such proteins in their natural host environment.
• reduction of the expression of a protein encoded by a DNA molecule of the present invention is obtained by "sense" suppression (referenced in e.g. Jorgensen et al. (1996) Plant Mol. Biol. 31 , 957-973).
• the entirety or a portion of a DNA molecule of the present invention is comprised in an expression cassette, which is introduced in a host cell, preferably a plant cell, in which the DNA molecule is expressible.
• the DNA molecule is inserted in the expression cassette in the "sense orientation", meaning that the 5' end of the DNA molecule is adjacent to the promoter in the expression cassette and that the coding strand of the DNA molecule can be transcribed.
• the DNA molecule is fully translatable and all the genetic information comprised in the DNA molecule of portion thereof is translated into a protein.
• the DNA molecule is partially translatable and a short peptide is translated. In a preferred embodiment, this is achieved by inserting at least one premature stop codon in the DNA molecule, which bring translation to a halt.
• the DNA molecule is transcribed but no translation product is being made. This is usually achieved by removing the start codon, e.g. the "ATG", of the protein encoded by the DNA molecule.
• the expression cassette comprising the DNA molecule or a portion thereof is stably integrated in the genome of the host cell.
• the expression cassette comprising the DNA molecule or a portion thereof is comprised in an extrachromosomal replicating molecule.
• the expression of the gene corresponding to the DNA molecule comprised in the expression cassette is reduced or abolished, leading to reduced levels of the protein or to its absence in the transgenic plants.
• the transgenic plants are resistant to fungal pathogens. "Anti-sense" Suppression
• the reduction of the expression of a protein encoded by a DNA molecule of the present invention is obtained by "anti-sense" suppression.
• the entirety or a portion of a DNA molecule of the present invention is comprised in an expression cassette, wherein the DNA molecule is introduced in a host cell, preferably a plant cell, in which the DNA molecule is expressible.
• the DNA molecule is inserted in the expression cassette in the "anti-sense orientation", meaning that the 3' end of the DNA molecule is adjacent to the promoter in the expression cassette and that the non-coding strand of the DNA molecule can be transcribed.
• the expression cassette comprising the DNA molecule or a portion thereof is stably integrated in the genome of the host cell.
• the expression cassette comprising the DNA molecule or a portion thereof is comprised in an extrachromosomal replicating molecule.
• Several publications describing this approach are cited for further illustration (Green, P. J. et al., Ann. Rev. Biochem. 55:569-597 (1986); van der Krol, A. R. et al, Antisense Nuc. Acids & Proteins, pp. 125-141 (1991 ); Abel, P. P. et al., Proc. Natl. Acad. Sci. USA 86:6949-6952 (1989); Ecker, J. R. et al., Proc. Natl. Acad. Sci. USA 83:5372-5376 (Aug. 1986)).
• At least one genomic copy corresponding to a DNA molecule of the present invention is modified in the genome of the plant by homologous recombination as further illustrated in Paszkowski et al., EMBO Journal 7:4021 -26 (1988).
• This technique uses the property of homologous sequences to recognize each other and to exchange nucleotide sequences between each by a process known in the art as homologous recombination.
• homologous recombination can occur between the chromosomal copy of a nucleotide sequence in a cell and an incoming copy of the nucleotide sequence introduced in the cell by transformation. Specific modifications are thus accurately introduced in the chromosomal copy of the nucleotide sequence.
• the regulatory elements of the gene encoding a protein of the present invention are modified.
• the existing regulatory elements are replaced by different regulatory elements, thus reducing expression of the protein, or they are mutated or deleted, thus abolishing the expression of the protein.
• the coding region of the protein is modified by deletion of a part of the coding sequence of the entire coding sequence, or by mutation. Expression of a mutated protein can also confer upon the plant increased resistance to fungal pathogens.
• a mutation in the chromosomal copy of a DNA molecule is introduced by transforming a cell with a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends.
• An additional feature of the oligonucleotide is the presence of 2'-O- methylation at the RNA residues.
• the RNA/DNA sequence is designed to align with the sequence of a chromosomal copy of a DNA molecule of the present invention and to contain the desired nucleotide change. This technique is further illustrated in US patent 5,501 ,967.
• the RNA coding for a protein of the present invention is cleaved by a catalytic RNA, or ribozyme, specific for such RNA.
• the ribozyme is expressed in transgenic plants and results in reduced amounts of RNA coding for the protein of the present invention in plant cells, thus leading to reduced amounts of protein accumulated in the cells and increased resistance of the plant to fungal pathogens. This method is further illustrated in US patent 4,987,071.
• the activity of the proteins encoded by the nucleotide sequences of this invention is changed. This is achieved by expression of dominant negative mutants of the proteins in transgenic plants, leading to the loss of activity of the endogenous protein.
• the activity of a protein encoded by a DNA molecule of the present invention is inhibited by expressing in transgenic plants nucleic acid ligands, so-called aptamers, which specifically bind to the protein.
• Aptamers are preferentially obtained by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method.
• SELEX Systematic Evolution of Ligands by Exponential Enrichment
• a candidate mixture of single stranded nucleic acids having regions of randomized sequence is contacted with the protein and those nucleic acids having an increased affinity to the target are partitioned from the remainder of the candidate mixture.
• the partitioned nucleic acids are amplified to yield a ligand enriched mixture.
• nucleic acid with optimal affinity to the protein is obtained and is used for expression in transgenic plants.
• This method is further illustrated in US patent 5,270,163.
• Methods for Isolating Nucleotide Sequences Comprising the conserveed Sequences are used for the isolation of other DNA molecules encoding such sequences.
• a mixture of degenerated oligonucleotides that contain at least one possible oligonucleotide encoding a sequences set forth in SEQ ID No:1 , or in SEQ ID No:2 is produced.
• the mixture of oligonucleotides encoding the sequences set forth in SEQ ID No:1 and the mixture of oligonucleotides complementary to sequences encoding the sequences set forth in SEQ ID No:2 are used in a PCR amplification reaction with a template DNA of choice. Mixtures of degenerated oligonucleotides are well known in the art and the degree of degeneration is varied as needed.
• the template DNA is a sample of total DNA from a plant, wherein such DNA sample is obtained by methods well known in the art.
• amplified fragments resulting from the PCR reactions described above are isolated by methods well known in the art and are used to isolate the corresponding full-length cDNAs by screening a cDNA library or by using a RACE protocol, both well known in the art. This method represents a novel and useful strategy to isolate new genes conferring upon plants resistance to fungal pathogens.
• RNA is prepared from leaves of wheat cultivar UC703 and used to program reverse transcription using a Stratagene RT-PCR kit.
• the resulting cDNA is employed in PCR reactions using the following primers:
• TrMlol The fragments are cloned into either pCR2.1 or pCR2.1-TOPO (Invitrogen). Plasmid DNA is prepared from the transformants and subjected to DNA sequencing. Sequencing reveals the existence of three different cDNA sequences with very high similarity to each other. These wheat Mlo genes are called TrMlol , TrMlo2 and TrMlo3.
• An additional wheat Mlo clone is isolated by screening a wheat cDNA library constructed in Lambda-ZAPII vector. To screen this library, mass excision is performed to convert the library into a Bluescript-based collection of cDNA clones. These are grown separately in pools of 80,000 independent clones, and plasmid DNA prepared. PCR reactions are performed on the pooled DNAs using oligonucleotide primers MLO-5 and MLO-15 (see above). Three pools yield a band of the expected size of 650 base pairs. These pools are subsequently fractionated by serial culturing of bacterial clones at lower and lower density, followed at each step by plasmid DNA preparation and PCR on each subpool using primers MLO-5 and MLO-15.
• RACE random amplification of cDNA ends
• MLO-GSP1 5' TGG ACC TCT TCA TGTTCG ATC CCA TCT G 3' (SEQ ID No:26)
• MLO-GSP2 5' CCT GAC GCT GTT CCA GAA TGC GTT TCA 3' (SEQ ID No:27)
• Amplification using primer MLO-GSP1 and the 5' adapter primer provided in the kit results in a DNA fragment of -1300 nucleotides.
• Amplification using primer MLO-GSP2 and the 3' adapter results in a DNA fragment of -600 nucleotides.
• the fragments are cloned into pCR2.1-TOPO and called TrMlol -5, TrMlo2-5 and TrMlo3-5 and comprise the 5' end of the wheat Mlo genes TrMlol , TrMlo2 and TrMlo3, respectively.
• Plasmid DNA is prepared from the clones and the plasmid inserts are sequenced.
• oligonucleotides are designed to amplify a sequence corresponding to the EST from an Arabidopsis cDNA library constructed in plasmid pFL61 (Minet et al. (1992) Gene Nov 16; 121 (2): 393-396.). The oligonucleotides used are:
• N37544-2 5' AGG CTG AAC CAC TGG GGC GCC TCT CAC CAC 3' (SEQ ID No:30)
• the primers N37544-1 and N37544-2 amplify a -500 base pair fragment from DNA prepared from the cDNA library, and the primers T22146-1 and T22146-2 amplify a -250 base pair fragment.
• Primers H76041 -1 and H76041-2 amplify two fragments of -350 and a -300 base pairs.
• the -300 base fragment is of the size predicted from the EST sequence, and is used subsequently to diagnose the presence of a cDNA corresponding to the H76041 EST in the cDNA library.
• DNA from the library is transformed into E. coli, and clones organized into pools of -20,000 clones each.
• DNA from individual pools are screened by PCR using the different primer pairs, and positive pools subsequently subfractionated into smaller and smaller clone numbers. Isolation of individual positive clones is accomplished either by carrying this process through to completion, or in some cases by colony hybridization using EST sequences as probes once pool sizes has reached 200 clones or fewer.
• ESTs N37544 and T22146 a clone corresponding to the EST is successfully isolated, and the insert sequenced.
• the cDNA corresponding to EST N37544 is designated CIB10295 in plasmid pCIB10295 and the plasmid containing the cDNA corresponding to EST T22146 (CIB10296) is designated pCIB10296.
• genomic sequences are available in the form of portions of Arabidopsis BAC clone sequences recently deposited to GenBank.
• the protein sequence predicted to be translated from these genomic sequences, as determined by GenBank does not correspond to the sequence determined by directly sequencing of the cDNAs. Therefore, the amino acid sequence of the genes corresponding to the ESTs N37544 and T22146 are not obvious from the GenBank entry and are only elucidated through cloning and sequencing of the cDNA clones.
• the clone isolated using primers for the H76041 EST is found to contain not the gene for H76041 , but a novel Mlo gene family member as insert. The insert is completely sequenced, and this Mlo gene family member is designated CIB10259 in plasmid pCIB10259.
• Two vectors are constructed for "antisense" expression of barley Mlo gene in wheat. PCR is performed using barley cDNA and the primer pair MLO-5 and MLO-7 (see (1 ) above), and the reaction results in amplification of a 1124 bp fragment which is cloned into pGEM-T (Promega). This fragment is excised from pGEM-T using the enzymes Sacll and Notl. The 1124 base pair fragment is cloned into pBluescript-SK(+).
• the insert is now excised with BamHI and Sad restriction sites and cloned into BamHI-Sacl-digested pCIB9806 (described in patent application 08/838,219) in an orientation in which the Mlo coding sequence runs opposite to the maize ubiquitin promoter.
• This plasmid is designated pCK01.
• PCR is performed using the primer pair MLO-1 (5' ATG TCG GAC AAA AAA GGG GT 3' (SEQ ID No:19)) and MLO-10 (see (1) above), and the reaction results in amplification of a 635 bp fragment which is cloned into pCR2.1 (Invitrogen).
• This fragment is excised from pCR2.1 as an EcoRI fragment, and inserted into pGEM- ⁇ Zf(-) (Promega).
• a 320 nucleotide fragment spanning the naturally-occurring Sad site in Mlo to primer site MLO-10 is excised with Sad and BstXI.
• pCK01 is digested with Sad and BstXI, and the 320 base fragment inserted.
• a 210 nucleotide Sa fragment is excised from the pGEM-9Zf(-) derivative. This fragment contains the 5' end of the Mlo coding sequence, from the primer site MLO-1 to the naturally- occurring Sad site in the Mlo gene.
• the pCK01 derivative is digested with Sa and the 210 base fragment inserted. Clones are analyzed for orientation of the 210 base fragment in the newly-constructed vector by PCR, using primers MLO-1 and MLO-10. Only clones in which the 210 base fragment is inserted in the antisense orientation relative to the ubiquitin promoter yielded a 530 base pair product corresponding to the 5' end of the Mlo coding sequence. The resulting plasmid contained the entire Mlo coding sequence in "antisense" orientation relative to the ubiquitin promoter, and is designated pCK02.
• plasmid pCK02 is digested with BamHI to release the Mlo coding sequence as insert.
• the BamHI fragment is religated back into the pCK02 base vector.
• Colonies with the Mlo coding sequence in reverse orientation relative to pCK02 are identified by Sad digestion, which yields a 1.8 kb fragment in such clones, as opposed to a 210 base fragment in clones of identical configuration as pCK02.
• a clone with the Mlo coding sequence in "sense" orientation relative to the maize ubiquitin promoter is selected as designated pCK03.
• Mlo clones in pCIB10259, pCIB10295 and pCIB10296, along with pCK02 (for the barley Mlo gene) are used in PCR reactions, resulting in bands that carry the full-length gene sequences flanked by BamHI restriction sites.
• the sequences of the primers used are:
• SAS-1 5' GGA TTA AGA TCT AAT GGC 3' (SEQ ID No:35, for pCIB10295)
• SAS-2 5' CAA AGA TCT TCA TTT CTT AAA AG 3' (SEQ ID No:36, for pCIB10295)
• SAS-3 5' GCG GAT CCA TGT CGG ACA AAA AAG G 3' (SEQ ID No:37, for barley Mlo)
• SAS-5 5' GGA TCC ACC ATG GCC ACA AGA TG 3' (SEQ ID No:39, for pCIB10259)
• SAS-6 5' GGA TCC TTA GTC AAT ATC ATT AGC 3' (SEQ ID No:40, for pCIB10259)
• SAS-7 5' GCG GAT CCA TGG GTC ACG GAG GAG A
• the bands are cloned into pCR2.1-TOPO, and the inserts sequenced from the resulting plasmids to confirm the absence of mutations introduced by PCR.
• the plasmids are digested with BamHI, and the inserts purified and cloned into BamHI-digested pPEH28, a shuttle vector containing a copy of the Arabidopsis ubiquitin gene promoter UBQ3 (Norris et al. (1993) Plant Molecular Biology 21 : 895-906) immediately downstream of the BamHI site.
• Clones containing Mlo sequences fused to UBQ3 are identified, and restriction analysis performed to identify clones with the inserts in the "sense” and "antisense” orientations relative to UBQ3.
• a clone with the insert in "sense” orientation and a clone with the insert in "antisense” orientation is digested with Xbal, and the insert purified and cloned into Xbal-digested pCIB200. This places the UBQ3-Mlo gene fusion between T- DNA borders.
• the primer MLO-3 corresponds to a region of the Mlo transgene, while primer ST27 lies within the maize ubiquitin promoter sequence.
• the use of both Mlo gene and ubiquitin promoter primers in PCR eliminates false positives arising from the use of two Mlo primers, which might prime from the chromosomal copy of the Mlo gene present in wheat.
• Plants confirmed to contain Mlo transgenes are subjected to RNA gel blot analysis, to determine whether they contain altered levels of chromosomally-encoded wheat Mlo mRNAs.
• Poly-A + RNAs are prepared from individual transgenic lines, and blotted onto Hybond-N+ filters.
• Blots are probed with a 530 base fragment corresponding to the 5' end of the Mlo gene. This region is absent from the pCK01 clone; therefore, hybridization to antisense RNA expressed from the transgene in transgenic lines containing pCK01 does not occur.
• the probe hybridizes to two bands of differing size.
• a -2.5 kb mRNA corresponding to the antisense transgene is distinguished from the 2.0 kb mRNA derived from wheat chromosomally-encoded Mlo genes. The abundance of the 2.0 kb mRNA is monitored as a measurement of the efficiency of gene suppression achieved by the transgene in individual lines.
• Plants of transgenic and untransformed UC703 (control) wheat lines are grown in the greenhouse until they are two weeks old. Plants are moved to a Percival growth chamber (cycle of 8 hours dark, 16° C, and 16 hours light, 20° C), and inoculated with Erysiphe graminis f. sp. tritici by liberal application of spores. The degree of fungal sporulation is scored two weeks after inoculation. Plants are scored 1 (little to no hyphal growth and no visible sporulation), 2 (some hyphal growth and sporulation, but less control plants) or 3 (hyphal growth and sporulation comparable to controls). Transgenic wheat lines expressing Mlo-constructs show increased resistance to the pathogen. An example for results obtained with the antisense barley Mlo construct is shown below.
• Siblings of Mlo antisense transformants are planted, inoculated with E. graminis, and scored for disease resistance.
• Example 7 Analysis of Arabidopsis Lines Expressing the Mlo Genes
• Derivatives of pCIB200 containing Mlo genes are used to transform Arabidopsis ecotype Ws-O by vacuum infiltration (Bechtold, N., Ellis, J. and Pelletier, G. (1993) C. ft Acad. Sci. Paris 316, 1194-1199).
• Progeny are screened by kanamycin selection to identify transformants.
• plants are identified which expressed Mlo by RNA gel blot analysis.
• For Mlo genes transformants are analyzed for alteration in the steady- state level of mRNA accumulation by RNA gel blot analysis.
• Transformants exhibiting sense or antisense suppression of target genes are assayed for alteration in reaction to the phytopathogenic fungi Erysiphe cichoracearum and Peronospora parasitica, and the bacterial pathogen Pseudomonas syringae pv. tomato. Leaves of transgenic plants are inspected both macroscopically and microscopically using trypan blue staining, to test for the presence of necrosis.
• spores are liberally applied to Arabidopsis rosettes and plants are maintained in a Percival growth chamber at 25°C. Degree of fungal sporulation is scored 10 days after inoculation.
• TrMlo2 GCG CTC GTC ACA CAG ATG GGA TCG
• X1 L, V or I
• X2 V or L
• X3 F or L
• X4 T, S or A.
• X5 I, V, S or G
• X6 F, L or V
• X7 Y or N
• X8 A or V
• X9 L or I
• X10 T or S.
• N A,C,G,T.
• DNA molecules described in this application can be modified for expression in transgenic plant hosts to achieve and optimize or down-regulate their expression.
• the following problems may be encountered and the modification of these DNA molecules can be undertaken using techniques well known in the art.
• Codon Usage The preferred codon usage in some plants differs from the preferred codon usage in some other plant species. Typically plant evolution has tended towards a strong preference of the nucleotides C and G in the third base position of monocotyledons, whereas dicotyledons often use the nucleotides A or T at this position.
• Plant genes typically have a GC content of more than 35%.
• DNA molecules which are rich in A and T nucleotides can cause several problems in plants. Firstly, motifs of ATTTA are believed to cause destabilization of messages and are found at the 3' end of many short-lived mRNAs. Secondly, the occurrence of polyadenylation signals such as AATAAA at inappropriate positions within the message is believed to cause premature truncation of transcription. In addition, monocotyledons may recognize AT-rich sequences as splice sites (see below).
• This analysis can be done for the desired plant species into which the nucleotide sequence is being incorporated, and the sequence adjacent to the ATG modified to incorporate the preferred nucleotides.
• DNA molecules of the present invention may also contain motifs which may be recognized in plants as 5' or 3' splice sites, and be cleaved, thus generating truncated or deleted messages. These sites can be removed using the techniques well known in the art.
• DNA molecules of the present invention may also include molecules that are modified in such a way that the activity of the proteins encoded by the nucleotide sequences of this invention is changed. This is achieved by expression of dominant negative mutants of the proteins in transgenic plants, leading to the loss of activity of the endogenous protein.
• the location of mutations in the Mlo nucleotide sequence leading to the production of such dominant-negative mutations are listed below. Either a single mutation or a combination of the different mutations listed below can be introduced.
• transformation vectors are available for plant transformation, and the DNA molecules of this invention can be used in conjunction with any such vectors.
• the selection of vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the ⁇ pfll gene which confers resistance to kanamycin, paromomycin, geneticin and related antibiotics (Vieira and Messing, 1982, Gene 19: 259-268; Bevan et al., 1983, Nature 304:184-187) the bacterial aac/A gene (Goldschmidt-Clermont, 1991 , Nucl. Acids Res.
• markers to be used include a phosphinothricin acetyltransf erase gene, which confers resistance to the herbicide phosphinothricin (White et al., 1990, Nucl. Acids Res.
• 103: 911-917 a mutant protoporphyrinogen oxidase gene as described in US Patent No. 5,767, 373.
• Selection markers resulting in positive selection such as a phosphomannose isomerase gene, as described in US Patent No 5,767,378, are also used.
• Identification of transformed cells may also be accomplished through expression of screenable marker genes such as genes coding for chloramphenicol acetyl transferase (CAT), ⁇ -glucuronidase (GUS), luciferase, and green fluorescent protein (GFP) or any other protein that confers a phenotypically distinct trait to the transformed cell.
• CAT chloramphenicol acetyl transferase
• GUS ⁇ -glucuronidase
• GFP green fluorescent protein
• the binary vectors pCIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and is constructed in the following manner.
• pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, J Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Vieira & Messing, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology 14: 266-276 (1990)).
• Xhol linkers are ligated to the EcoRV fragment of pCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and the X7o/-digested fragment is cloned into Sa//-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19).
• pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail.
• pCIB2001 is a derivative of pCIB200 which created by the insertion into the polylinker of additional restriction sites.
• Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bell, Avrll, Apal, Hpal, and Stul.
• pCIB2001 in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for >4gro6ac er/um-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the Or/Tand OriV functions also from RK2.
• the pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals. Construction of pCIBIO and Hygromvcin Selection Derivatives thereof
• the binary vector pCIBI 0 contains a gene encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-161 (1987)).
• Various derivatives of pCIBI 0 have been constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).
• Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques which do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of some typical vectors is described.
• pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin).
• the plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278.
• the 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull.
• the new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site.
• the resultant derivative of pCIB246 is designated pCIB3025.
• the GUS gene is then excised from pCIB3025 by digestion with Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060.
• the plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the bar gene from Streptomyces vihdochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)).
• This generated pCIB3064 which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene fro ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, Hindlll, and BamHI.
• This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
• Construction of pSOG19 and pSOG35 pSOG35 is a transformation vector which utilizes the E. coli gene dihydrofolate reductase (DHFR) as a selectable marker conferring resistance to methotrexate.
• DHFR E. coli gene dihydrofolate reductase
• PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adh1 gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10.
• a 250 bp fragment encoding the E. coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pBI221 (Clontech) which comprised the pUC19 vector backbone and the nopaline synthase terminator.
• pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator.
• Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generated the vector pSOG35.
• pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Pstl and EcoRI sites available for the cloning of foreign sequences.
• Gene sequences intended for expression in transgenic plants are firstly assembled in expression cassettes behind a suitable promoter and upstream of a suitable transcription terminator. Promoter Selection
• promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and this selection will reflect the desired location of biosynthesis of a DNA molecule of the present invention.
• the selected promoter may drive expression of the gene under a light-induced or other temporally regulated promoter.
• the selected promoter be chemically regulated. This provides the possibility of inducing the expression of the nucleotide sequence only when desired and caused by treatment with a chemical inducer.
• transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
• intron sequences have been shown to enhance expression, particularly in monocotyledonous cells.
• the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells.
• Intron 1 is found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransf erase gene (Callis et al., Genes Develop 1: 1183-1200 (1987)).
• the intron from the maize bronzel gene had a similar effect in enhancing expression (Callis et al., supra).
• Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
• leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
• leader sequences from Tobacco Mosaic Virus (TMV, the ' ⁇ -sequence'), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski era/. Plant Molec. Biol.
• DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized.
• cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting to cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).
• aminoterminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, aminoterminal sequences in conjunction with carboxyterminal sequences are responsible for vacuolar targeting of gene products (Shinshi er a/. Plant Molec. Biol. 14: 357-368 (1990)).
• the transgene product By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment.
• chloroplast targeting for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the aminoterminal ATG of the transgene.
• the signal sequence selected should include the known cleavage site and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or alternatively replacement of some amino acids within the transgene sequence.
• Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by (Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier, pp 1081-1091 (1982); Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986)). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.
• the choice of targeting which may be required for the insecticidal toxin. This will usually be cytosolic or chloroplastic, although it may is some cases be mitochondrial or peroxisomal.
• the expression of the nucleotide sequence may also require targeting to the ER, the apoplast or the vacuole.
• the above described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell targeting goal under the transcriptional regulation of a promoter which has an expression pattern different to that of the promoter from which the targeting signal derives.
• the present invention encompasses the expression of a DNA molecule under the regulation of any promoter which is expressible in plants, regardless of the origin of the promoter. Furthermore, the invention encompasses the use of any plant-expressible promoter in conjunction with any further sequences required or selected for the expression of the DNA molecule. Such sequences include, but are not restricted to, transcriptional terminators, extraneous sequences to enhance expression (such as introns [e.g. Adh intron 1], viral sequences [e. g. TMV- ⁇ ]), and sequences intended for the targeting of the gene product to specific organelles and cell compartments. Constitutive Expression: the CaMV 35S Promoter
• pCGN1761 contains the 'double' 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone.
• a derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761ENX.
• pCGN1761ENX is useful for the cloning of cDNA sequences or gene sequences (including microbial ORF sequences) within its polylinker for the purposes of their expression under the control of the 35S promoter in transgenic plants.
• the entire 35S promoter-gene sequence-rm/ terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites 5' to the promoter and Xbal, BamHI and Bgll sites 3' to the terminator for transfer to transformation vectors such as those described above in example 35.
• the double 35S promoter fragment can be removed by 5' excision with Hindlll, Sphl, Sail, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter.
• Modification of PCGN1761 ENX bv Optimization of the Translational Initiation Site can be made by the introduction of sequences which may enhance translation. This is particularly useful when genes derived from microorganisms are to be introduced into plant expression cassettes as these genes may not contain sequences adjacent to their initiating methionine which may be suitable for the initiation of translation in plants. In cases where genes derived from microorganisms are to be cloned into plant expression cassettes at their ATG it may be useful to modify the site of their insertion to optimize their expression. Modification of pCGN1761 ENX is described by way of example to incorporate one of several optimized sequences for plant expression (e.g. Joshi, supra). Expression under a Chemically Regulatable Promoter
• the promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers which carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be resequenced to check for amplification errors after the cloning of the amplified promoter in the target vector.
• the chemically regulatable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (see EP 0 332 104, example 21 for construction) and transferred to plasmid pCGN1761 ENX.
• pCIB1004 is cleaved with Ncol and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase.
• the fragment is then cleaved with Hindlll and the resultant PR-1a promoter containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and blunting with T4 polymerase, followed by cleavage with Hindlll and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned.
• actin promoter is a good choice for a constitutive promoter.
• the promoter from the rice Act1 gene has been cloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)).
• a 1.3 kb fragment of the promoter is found to contain all the regulatory elements required for expression in rice protoplasts.
• numerous expression vectors based on the Act1 promoter have been constructed specifically for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991 )).
• promoter containing fragments can be removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761ENX, which is then available for the insertion or specific gene sequences.
• the fusion genes thus constructed can then be transferred to appropriate transformation vectors.
• the rice Acfl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).
• Ubiquitin is another gene product known to accumulate in many call types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - Binet et al. Plant Science 79: 87-94 (1991), maize - Christensen et al. Plant Molec. Biol. 12: 619-632 (1989)).
• the maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926. Further, Taylor et al. (Plant Cell Rep.
• a vector which comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment.
• the ubiquitin promoter is clearly suitable for the expression of a DNA molecule of the present invention in transgenic plants, especially monocotyledons.
• Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences. Root Specific Expression
• a preferred pattern of expression for the nucleotide sequence of the instant invention is root expression. Root expression is particularly useful for the control of soil-borne fungal pathogens.
• a suitable root promoter is that described by de Framond (FEBS 290: 103-106 (1991 )) and also in the published patent application EP 0 452 269. This promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of the nucleotide sequence and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.
• Wound-inducible promoters are particularly suitable for the expression of a DNA molecules of the present invention because they are typically active not just on wound induction, but also at the sites of phytopathogen infection. Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191 -201 (1993)) and all are suitable for use with the instant invention.
• Patent Application WO 93/07278 (to Ciba-Geigy) describes the isolation of the maize trpA gene which is preferentially expressed in pith cells.
• the gene sequence and promoter extend up to -1726 from the start of transcription are presented.
• this promoter or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a DNA molecule of this invention in a pith-preferred manner.
• fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
• Patent Application WO 93/07278 (to Ciba-Geigy) further describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene which is expressed in pollen cells.
• CDPK calcium-dependent protein kinase
• the gene sequence and promoter extend up to 1400 bp from the start of transcription.
• this promoter or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a DNA molecule of this invention.
• fragments containing the pollen-specific promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
• a maize gene encoding phosphoenol carboxylase has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants. Expression with Chloroplast Targeting
• the Dral-Sphl fragment extends from -58 relative to the initiating rbcS ATG to, and including, the first amino acid (also a methionine) of the mature peptide immediately after the import cleavage site, whereas the Tsp509l-Sphl fragment extends from -8 relative to the initiating rbcS ATG to, and including, the first amino acid of the mature peptide.
• these fragment can be appropriately inserted into the polylinker of any chosen expression cassette generating a transcriptional fusion to the untranslated leader of the chosen promoter (e.g.
• the 5' Tsp509l site may be rendered blunt by T4 polymerase treatment, or may alternatively be ligated to a linker or adaptor sequence to facilitate its fusion to the chosen promoter.
• the 3' Sphl site may be maintained as such, or may alternatively be ligated to adaptor of linker sequences to facilitate its insertion into the chosen vector in such a way as to make available appropriate restriction sites for the subsequent insertion of the DNA molecule of this invention.
• the ATG of the Sphl site is maintained and comprises the first ATG of the DNA molecule of this invention.
• Chen & Jagendorf (supra) provide consensus sequences for ideal cleavage for chloroplast import, and in each case a methionine is preferred at the first position of the mature protein. At subsequent positions there is more variation and the amino acid may not be so critical.
• fusion constructions can be assessed for efficiency of import in vitro using the methods described by Bartlett et al. (In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier, pp 1081-1091 (1982)) and Wasmann et al. (Mol. Gen. Genet.

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