WO1998032325A1 - Procedes et compositions a deux composants induisant la letalite cellulaire vegetale - Google Patents

Procedes et compositions a deux composants induisant la letalite cellulaire vegetale Download PDF

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Publication number
WO1998032325A1
WO1998032325A1 PCT/US1998/001315 US9801315W WO9832325A1 WO 1998032325 A1 WO1998032325 A1 WO 1998032325A1 US 9801315 W US9801315 W US 9801315W WO 9832325 A1 WO9832325 A1 WO 9832325A1
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Prior art keywords
plant
cell
polypeptide
expression cassette
promoter
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PCT/US1998/001315
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English (en)
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Neal Gutterson
Ed Ralston
Diane Burgess
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Dna Plant Technology Corporation
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Priority to JP53214698A priority Critical patent/JP2002514065A/ja
Priority to EP98904662A priority patent/EP1006780A4/fr
Priority to AU62482/98A priority patent/AU744675B2/en
Priority to CA002277817A priority patent/CA2277817A1/fr
Publication of WO1998032325A1 publication Critical patent/WO1998032325A1/fr

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    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8263—Ablation; Apoptosis
    • 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
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8217—Gene switch
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates generally to methods for preventing the growth of specific cells in a multi-cellular eukaryote, particularly plant cells. Recombinantly modified plant cells for expression of cytotoxic genes are also provided.
  • One key objective of plant genetic engineering is to create novel traits either through expression of an introduced gene or by silencing of an endogenous gene.
  • One use of targeted gene expression is the elimination of specific plant cells through the production of an enzyme that is lethal to the cell.
  • an enzyme that is lethal to the cell In order to eliminate only a specific set of cells, it is necessary that expression of a potentially lethal function be controlled precisely such that the cell-lethal function is expressed only in the cell ' s targeted for elimination and in no others.
  • Several different approaches have now been attempted to create novel plant traits using a single component cell lethality system. In one-component cell lethality systems, specific cell types are targeted for elimination using a single promoter driving expression of a cytotoxic gene product.
  • promoters that are active in specific tissues or under specific conditions. For example, male sterility has been demonstrated using promoters active in tapetal tissue. A number of different promoters have been identified that are expressed in tapetal tissue and other tissue. A further example is the use of cell lethality to create disease resistance via a hypersensitive response. A number of promoters have been characterized by different workers that are induced upon pathogen infection. A further example is the attempt to create nematode resistance by killing giant cells, the specific root cells upon which cyst and root knot nematodes feed. A number of promoters have been characterized by different workers that are induced in giant cells, but again sufficient promoter specificity has been difficult to achieve.
  • the workers cite the difficulty of creating transgenic plants using a construct containing a ribonuclease coding sequence (obtained from Bacillus amyloliquifaciens , barnase) under the control of a promoter induced upon infection with the fungus Phytophthora infestans.
  • Their solution to the difficulty was to express a protective function, barstar, under the control of a constitutive promoter that would hopefully protect non-infected cells, but allow infected cells to be killed.
  • a further example is the attempt to create nematode resistance by killing the specific root cells upon which cyst and root knot nematodes feed, as described in WO 92/21757, WO 93/10251, WO 93/18170, WO 94/10320, and WO 94/17194.
  • a number of promoters have been characterized by different workers that are induced in specialized nematode feeding cells, but again sufficient promoter specificity has been difficult to achieve.
  • WO 93/10251 the difficulty of obtaining sufficient promoter specificity is addressed through expression of a protective function in cells other than the target cells.
  • WO 96/26283 Another example of the protective approach is described in WO 96/26283, which described the production of male sterility using the tapetal specific promoter TA29 from tobacco to program expression of barnase.
  • the protective function for the barnase protein is the barstar protein, whose expression is sought in non-target tissues.
  • protective functions are not available.
  • DT diphtheria toxin
  • EF-2 elongation factor
  • the tk gene product is a conditional cell-lethal function, requiring the presence of a nucleoside analog such as ganciclovir for lethality.
  • a nucleoside analog such as ganciclovir for lethality.
  • Brady et al. Proc. Natl. Acad. Sci., USA 91:365-69 (1994) describe the use of this approach for specific ablation of human immunodeficiency virus Tat-expressing cells following introduction of a tk gene whose expression is under control of the Tat protein and treatment with ganciclovir.
  • the requirement of "twofold specificity" has been recognized. Panchal et al., Nature Biotechnol. 14:852-56 (1996).
  • the approach taken by Panchal et al. was to use immunorecognition of the surface of cancer cells as the first level of specificity, and specific protease activities of cancer cells as the second level of specificity.
  • male sterile plants can be produced.
  • the production of male sterile plants is particularly useful in producing Fl hybrids.
  • Fl hybrid plants are used extensively in most areas of agriculture because of their improved traits, such as increased yield, disease or low temperature resistance.
  • Fl hybrids are often produced by a manual process of emasculation of the intended female of the cross, to prevent self pollination, followed by application of pollen taken from the male of the cross to the stigma of the female of the cross.
  • the production of such hybrids is labor intensive, which contributes greatly to the increased cost of hybrid seed.
  • WO 96/26283 describes the production of male sterility using the tapetal specific promoter TA29 from tobacco to program expression of the ribonuclease, barnase.
  • U.S. Patent No. 5,409,823 discloses use of transactivators to control expression of gene products which disrupt formation of pollen. Since it is often desirable that the male fertility be restored in either the male sterile line or the Fl hybrid, attempts have been made to produce plants in which male sterility can be made conditional. Examples of this approach include WO 93/25695 and WO 97/13401. What is needed in the art are compositions and methods which provide selective elimination or inhibition of growth of a selected cell type in an organism, for example, in a plant. The present invention provides these and other advantages.
  • the present invention is directed to a plant cell.
  • the plant cell comprises a first expression cassette comprising a first non-constitutive plant promoter operably linked to a polynucleotide encoding a first polypeptide and a second expression cassette comprising a second non-constitutive plant promoter operably linked to a polynucleotide encoding a second polypeptide.
  • At least the first or the second expression cassette is heterologous to the cell.
  • the first and second promoters have different but overlapping specificities such that the first and second polypeptides are expressed in the same cell.
  • the presence of the first and second polypeptides in the same cell impairs cellular function.
  • the first and second polypeptides each comprise a separate subsequence of a single functional polypeptide.
  • the functional polypeptide can be a ribonuclease such as Barnase, or Tl.
  • the functional polypeptide be modified to have enhanced stability.
  • the enhanced stability barnase is bn3-2 and bn5-2.
  • the first polypeptide is an avirulence gene product derived from a plant pathogen and the second polypeptide is a resistance gene product associated with the avirulence gene.
  • first polypeptide can be avr9 and the second polypeptide cfo9.
  • the functional polypeptide can be a nuclease or colicin.
  • the first or the second promoter is a tissue-specific promoter such as when each is functional in seeds or tapetal cells.
  • the first or second promoter is induced following interaction with a plant pathogen or pest.
  • the present invention relates to a plant cell comprising a first expression cassette comprising a first plant promoter operably linked to a polynucleotide encoding a first polypeptide and a second expression cassette comprising a second plant promoter operably linked to a polynucleotide encoding a second polypeptide.
  • the first and second polypeptides each comprise a separate subsequence of a single functional polypeptide. Often, the functional polypeptide impairs cellular function.
  • the first and second promoters have different but overlapping specificities such that the first and second polypeptides are expressed in the same cell.
  • the present invention relates to a method for modifying the cellular function of a plant cell.
  • the method comprises the steps of introducing into the cell a first expression cassette comprising a first non-constitutive plant promoter operably linked to a polynucleotide encoding a first polypeptide and a second expression cassette comprising a second non-constitutive plant promoter operably linked to a polynucleotide encoding a second polypeptide, wherein the first and second promoters have different but overlapping specificities such that the first and second polypeptides are expressed in the same cell.
  • the first or the second expression cassettes can be introduced into the plant cell through a sexual cross.
  • the present invention relates to a method of modifying cellular function in a plant cell.
  • the method comprises the steps of introducing into a plant cell a first expression cassette comprising a first plant promoter operably linked to a polynucleotide encoding a first non-functional polypeptide and a second expression cassette comprising a second plant promoter operably linked to a polynucleotide encoding a second non-functional polypeptide, wherein the first and second polypeptides each comprise a separate subsequence of a single functional polypeptide.
  • the first and second promoters have different but overlapping specificities such that the first and second polypeptides are expressed in the same cell.
  • the present invention is directed to a method for preventing the growth of a eukaryotic cell.
  • the method comprises the steps of introducing into the cell, a first expression cassette comprising a first tissue-specific plant promoter operably linked to a polynucleotide encoding a first polypeptide and a second expression cassette comprising a second tissue-specific plant promoter operably linked to a polynucleotide encoding a second polypeptide, wherein the first and second promoters are functional in the cell and presence of the first and second polypeptides in a cell impairs cellular function.
  • the cell is a mammalian cell.
  • the mammalian cell can be in a non-human animal.
  • the first and second expression cassettes are introduced into the cell using a retroviral vector.
  • the present invention further provides plants containing a plant cell comprising a first and a second expression cassette located at the same locus on each of two homologous chromosomes.
  • One expression cassette comprises a first plant promoter operably linked to a first polynucleotide sequence encoding a first polypeptide.
  • a recombinase site e.g. , a lox site is present between the first promoter and the first polynucleotide sequence.
  • a second expression cassette comprises the first plant promoter inoperably linked to the first polynucleotide sequence, wherein an intervening expression cassette is flanked by two recombinase sites and situated between the first promoter and the first polynucleotide sequence of the second expression cassette.
  • the intervening expression cassette comprises a second plant promoter operably linked to a second polynucleotide sequence encoding a second polypeptide. The presence of the first and second polypeptides in a cell is lethal to the cell.
  • the first and second polypeptide can be selected from a number of proteins, which when present together are lethal to a cell.
  • one polypeptide can be a transactivator protein which activates expression of the other expression cassette which encodes a polypeptide which is lethal to plant cells (e.g. , a ribonuclease).
  • the polypeptides can be an avirulence gene product derived from a plant pathogen and a plant resistance gene product associated with the avirulence gene (e.g. , AVR9 and CF9).
  • the promoters in the two expression cassettes preferably provide tissue specific expression of one or both of the polypeptides.
  • the target cells are tapetal cells.
  • plants of the invention can be prepared by introducing into a plant the expression cassettes described above.
  • FIGURES Figure 1 shows how the allelic variants of p-Mon-Avr9 or TA29-Cf9 containing sublines (A, and A 2 ) are created using the cre/lox recombinase system and their use in producing male steriles.
  • ALS acetolactate synthase.
  • Figure 2 shows the production of allelic variants of the invention using the cre/lox system and AVR9/CF9.
  • ALS acetolactate synthase.
  • the present invention provides methods for inhibiting the growth or killing of specified eukaryotic cells in multicellular eukaryotic organisms, particularly plants. More specifically, the present invention relates to plant cells comprising at least two expression cassettes operably linked to polynucleotides which when expressed provide a desired effect to specific tissue or cell types. The desired effect can impair cellular function or can be beneficial to the cell (e.g. , resistance to plant pathogens or pests).
  • the expression cassettes can occupy the same or different loci of chromosome homologs, or be located on different chromosomes or elsewhere as part of the genome.
  • the present invention provides novel methods for producing plants which can utilize the multi- component system described herein, and embraces the plants so produced and methods of their use.
  • the two or more component lethality/inhibitory methods of the present invention employs two or more promoters having overlapping but distinct tissue-specific expression.
  • This system provides a greater degree of control in targeting a desired function to selected cells than one- component systems.
  • Two or more component systems will typically be optimized by consideration of the following: 1) the desired effect should be cell autonomous; 2) the desired effect should not be dependent upon any other cellular function; 3) the desired effect should be a sensitive function of the level of the associated components.
  • Methods of the present invention provide means to maintain inbred lines in a hybrid system in which there is no inhibitory or lethality expressed in either inbred line. However, crossing these inbred lines yields a hybrid having the inhibitory or lethal phenotype.
  • the present invention also provides a means to produce a lethal or inhibitory effect in a highly tissue-specific, condition-specific, or developmental stage-specific manner, using two different promoters with overlapping specificity to obtain the necessary specificity.
  • the resulting invention has utility, for example, in creating and maintaining male sterile and female sterile plants. Definitions
  • plant includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same.
  • plant organs e.g., leaves, stems, roots, etc.
  • the class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
  • operably linked includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the RNA sequence which is typically transcribed into a polypeptide.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • an "expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a target cell.
  • the expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the expression cassette portion of the expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.
  • chromosome homolog or “homologous chromosome” refers to two or more chromosomes that can pair during meiosis.
  • Each homologue is a duplicate of a chromosome contributed by the male or female parent.
  • Homologous chromosomes contain the same linear sequence of genes, each gene (or allele) is present at the same locus on each homolog.
  • Two polynucleotide sequences e.g. , two expression cassettes of the invention are said to be at the "same locus" if the two sequences are genetically mapped to the same locus as determined, for instance, by frequency of crossover events between the two loci.
  • lethal or “impairs cellular function” includes reference to polynucleotide(s) or polypeptide(s) that are cytotoxic to an extent that kills cells or inhibits cell division or differentiation.
  • lethal or “impairs cellular function” includes reference either to 1) the disruption of a cell through perturbation of some function of the cell or by degradation of a component of the cell, or 2) to the prevention of continued growth of a cell through perturbation of some function of the cell or degradation of some component of the cell.
  • typical cellular functions in the context of the instant invention are protein synthesis, RNA synthesis, maintenance of osmotic competence, lipid synthesis, DNA synthesis.
  • Typical cellular components subject to degradation in the context of the instant invention are proteins, carbohydrates, membranes, deoxyribonucleic acids, ribonucleic acids.
  • beneficial includes reference to polynucleotide(s) or polypeptide(s) that impart a protective effect to a cell.
  • a beneficial effect includes resistance to environmental stresses including, but not limited to, plant pathogens, pests, drought, heavy metals, and salt.
  • heterologous is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its original form.
  • a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form.
  • a heterologous expression cassette is one that comprises at least one element not endogenous to the species or sub-species in which it is introduced.
  • polynucleotide and “nucleic acid” includes reference to both double stranded and single stranded DNA or RNA.
  • a polynucleotide can be a gene subsequence or a full length gene (cDNA or genomic). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res . 19:5081 (1991); Ohtsuka et al. , J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al. , Mol. Cell. Probes 8:91-98 (1994)).
  • the term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations, " which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:
  • enhanced stability refers to a polypeptide that has been modified so that one or more of the amino acids has been changed relative to the wild type polypeptide. Such modifications provide enhanced stability to the polypeptide, either alone or in combination with another polypeptide.
  • Enhanced stability includes, e.g., enhanced thermal stability, enhanced activity at lower concentrations, enhanced active site activity, and the like.
  • “functional” includes reference to an activity sufficient to produce a desired effect. Thus, for example, a promoter functional in a specified cell will drive expression to the desired levels. A “functional polypeptide” will have the activity to achieve a desired result, such as cell inhibition or death.
  • a functional polypeptide will provide the cell with a beneficial or therapeutic effect, such as resistance to plant pests or disease.
  • a functional polypeptide e.g. , a ribonuclease such as barnase
  • an active expression cassette produces a "functional polypeptide" as defined herein.
  • a particular protein or “functional polypeptide” includes the naturally occurring protein or a protein produced in a cell that has the substantially the same activity as the naturally ocurring protein.
  • “Functional polypeptides" of the invention also include modified polypeptides (with amino acid substitutions, both conservative and non-conservative) that have the same activity as a wild-type or unmodified polypeptide.
  • promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that initiate transcription only in certain tissues, such as leaves, roots, fruit, seeds, tapetal tissue, anthers, stigmas, or flowers. Such promoters are referred to as “tissue specific” .
  • a “cell type” specific promoter is primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
  • inducible promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. A “constitutive” promoter is a promoter which is active under most environmental conditions.
  • the present invention has use over a broad range of types of plants, including species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum
  • the expression cassettes of the multi-component cell lethality system of the present invention are DNA or RNA constructs which can be cloned and/or synthesized by any number of standard techniques.
  • An expression cassette will typically comprise transcriptional and translational initiation regulatory sequences which will direct the transcription of the polynucleotide encoding a non-lethal polypeptide in the intended tissues of the transformed plant.
  • Such nucleic acid constructs may be introduced into the genome of the desired plant host by a variety of conventional techniques. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g. , Weising et al. Ann. Rev. Genet. 22:421-477 (1988).
  • the DNA or RNA nucleic acid construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the nucleic acid constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
  • the nucleic acid constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobactenum tumefaciens host vector. The virulence functions of the Agrobactenum tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
  • Agrobactenum tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, e.g., Horsch et al. Science, 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).
  • Microinjection techniques are known in the art and well described in the scientific and patent literature.
  • the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. EMBO J. 3:2717-2722 (1984).
  • Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA
  • the expression cassettes of the present invention can comprise a marker gene which confers a selectable phenotype on plant cells.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulforon or Basta.
  • Plant cells of the present invention comprise two or more expression cassettes.
  • an expression cassette of the present invention is in a cell in which the promoter is functional, the promoter drives expression of the operably linked polynucleotide.
  • Such an expression is a functional expression cassette.
  • two "functional transcripts" are made, at least one of which is typically translated to a "functional polypeptide”.
  • cell function is disrupted such that the cell is inhibited or killed.
  • neither of the expression cassettes is capable of causing the desired result.
  • the presence in the cell of all functional expression cassettes of the two or more component system yields an inhibitory or cytotoxic effect upon the cell.
  • the two or more (multi) component system of the invention yields a beneficial effect, e.g. , resistance to a plant pathogen or pest.
  • one of the expression cassettes is able to express its operably linked polynucleotide in the absence of the other collaborating expression cassette.
  • the collaborating expression cassette requires the presence of the polypeptide expressed by its partner expression cassette to become functional; typically the product of this inducible expression cassette is by itself lethal or inhibitory.
  • the polypeptide may be an activator polypeptide as in the tet repressor/VP16 activator fusion as discussed in Weinmann et al. , The Plant Journal, 5(4):559-569 (1994).
  • each of the expression cassettes are located at the same locus on each chromosome of a homologous pair.
  • a particularly preferred means of introducing each expression cassette at the same locus employs the cre/lox recombinase system. Bayley et al., Plant Molecular Biology 18:353-361 (1992).
  • the cre/lox system allows the introduction of a precursor expression cassette which can subsequently be manipulated via ere recombinase to remove a subsequence of that precursor cassette. In this way alternate alleles can be made. Accordingly, the recombinase allows the formation of the two expression cassettes employed in the two-component lethality system of the present invention.
  • Other recombinase systems include the Saccharomyces cervisiae FLP/FRT, lambda att/Int, R recombinase of Zygosaccharomyces rouxii, and Mu Gin recombinase.
  • one of the polynucleotides encoding a non-lethal peptide of the present invention could be located on a transposable element such as Ds from maize which could be excised by crossing to a line carrying a transposase (see, e.g. , Carroll et al. Genetics 139:407-420 (1995)).
  • an expression cassette which can subsequently be manipulated via a recombinase to remove an intervening subsequence of that cassette is introduced into a plant.
  • the intervening expression cassette is positioned between recombinase sites in the same orientation, such that the promoter of the expression cassette is inoperably linked to a coding sequence.
  • the intervening expression cassette is excised from the expression cassette such that the promoter is now operably linked to the corresponding coding sequence.
  • the recombinase can be used to insert an intervening expression cassette into a recombinase site in an expression cassette previously introduced into the plant.
  • the intervening subsequence is inserted into the expression cassette such that the promoter is now inoperably linked to the corresponding coding sequence.
  • alternate functional cassettes can be introduced to a particular locus in the genome.
  • One of the cassettes will have within it a non-functional expression cassette and will also have within it a functional intervening expression cassette.
  • the other of the cassettes will be a functional expression cassette (without the intervening sequence).
  • the intervening sequence also includes an intervening expression cassette encoding a second polypeptide.
  • the presence of polypeptides from both expression cassettes is lethal to the cell.
  • an initial plant line is created which contains a non-functional expression cassette interrupted by an intervening expression cassette flanked by recombinase sites in the same orientation (e.g. , lox sites).
  • This initial line is then crossed with a second plant containing the appropriate recombinase (e.g. , ere).
  • Plants in the Fl generation will contain a functional expression.
  • Fl plants containing a functional cassette are then selfed according to standard techniques to produce a homozygous line containing a functional expression cassette encoding the first polypeptide.
  • the original transformant is also selfed to produce a second line in which the first expression cassette remains non-functional.
  • the two lines are then crossed to produce plants heterozygous at the locus.
  • an initial plant line is created which contains a functional expression cassette containing a recombinase site (e.g. , a lox site) between the promoter and the structural gene.
  • a plasmid containing an intervening expression cassette, and a second recombinase site is introduced into tissue or cells (e.g. , protoplasts) from the initial plant along with a second plasmid encoding the appropriate recombinase (e.g. , ere).
  • tissue or cells e.g. , protoplasts
  • the second plant line is then regenerated from the transformed tissue or cells.
  • a recombination event between the plasmid DNA containing the intervening expression cassette and the first expression cassette will result in insertion of the plasmid DNA containing the intervening expression cassette into the first expression cassette, thereby rendering the first expression cassette non- functional and thereby yielding the second expression cassette.
  • the two lines are then crossed to produce plants containing the two cassettes at the same locus on each of the two homologous chromosomes heterozygous at the locus. Cells in which both expression cassettes are functional will be eliminated.
  • it is desirable to use combinations of mutant lox sites to increase the relative efficiency of the insertion event or excision. Mutant lox sites e.g. , lox 66 and lox 77 ) are described by Albert et al. Plant J. 6:649-659 (1995)),
  • two alternative alleles at one locus can be created encoding each one of the polypeptides of the present invention which, when homozygous, yield alternate sublines (e.g. , sublines A x and A_).
  • Crossing of sublines A, and A 2 yield the heterozygote line, A. Since the alternate alleles of line A are at the same locus they will segregate away from each other upon crossing the line A heterozygote with a line which is absent either of these alleles, line B.
  • the resulting hybrids will have only one functional expression cassette of the multi-component system per cell and, consequently, these cells will not yield a desired lethal or beneficial result (e.g., the male sterile line A described above will be 100% male fertile).
  • sublines A ! and A 2 each having one expression cassette of the two-component system, can be created using a recombinase system as described above, preferably the cre/lox system.
  • the initial transformant will have a first polynucleotide, often encoding a first polypeptide, operably linked to a first promoter and inserted in opposite orientation between a second promoter which drives expression of a second polypeptide. Lox sites are placed in the same orientation on both sides of the second expression cassette insert.
  • Subline A 2 is created by crossing line Aj to a line carrying a recombinase which recognizes the recombination sites (e.g., the lox sites) and excises the intervening sequence allowing the first promoter to drive expression of the first polynucleotide to which it is operably linked. When made homozygous by breeding methods well known to those of skill in the art, this will be used as line A 2 .
  • Hybrid line A is formed by crossing sublines A x and A 2 to yield a plant comprising a functional multi-component system of the present invention.
  • a tet operator-Ms polynucleotide is inserted in opposite orientation between a tapetal-specific promoter such as TA29 and the tet repressor/VP16 (Act) activator. Lox sites are placed in the same orientation on both sides of these first promoter-first polynucleotide inserts.
  • a j the regulatory protein Act is not expressed since the tet operator-Ms gene is present between the TA29 and Act (the regulatory protein).
  • Subline A 2 which lacks the insert as a result of ere, expresses Act, the activator of the tet operator. Crossing of A, to homozygous subline A 2 results in expression of Ms and results in 100% male sterile hybrids.
  • a pMon-Avr9 gene is flanked by lox sites and inserted in opposite orientation between a tapetal-specific promoter such as TA29 and Cf9.
  • a tapetal-specific promoter such as TA29 and Cf9.
  • TA29 tapetal-specific promoter
  • Another subline is created in which the insert is removed by a recombinase and the TA29 promoter drives expression of Cf9. Crossing of these two lines results in a hybrid which is male sterile due to the joint expression of Avr9 and Cf9 in tapetal cells.
  • Promoters employed in the expression cassettes of the present invention can be chosen to function in identical sets of tissue types simultaneously, or at different stages of development or of the cell cycle. However, the individual promoters are generally chosen such that they will function in multiple, different, and overlapping subsets of cells. Accordingly, the promoters of the present invention typically have "different but overlapping specificities. " The overlap of these different subsets is that collection of cells where all expression cassettes of the multi-component system are present and functional. The presence in a cell of all of the complete set of functional expression cassettes of the multi-component system will result in a desired effect on the organism. Thus, for example, a single functional expression cassette within a cell yields a non- lethal/non- inhibitory phenotype.
  • promoters can be used with the multi-component system of the present invention.
  • Methods for identifying promoters with a particular expression pattern in terms of, e.g., tissue type, cell type, stage of development, and/or environmental conditions, are well known in the art.
  • a typical step in promoter isolation methods is identification of gene products that are expressed with some degree of specificity in the target tissue.
  • methodologies are: differential hybridization to cDNA libraries; subtractive hybridization; differential display; differential 2-D gel electrophoresis; isolation of proteins known to be expressed with some specificity in the target tissue. Such methods are well known to those of skill in the art.
  • the amino acid sequence for at least a portion of the identified protein it is necessary to obtain the amino acid sequence for at least a portion of the identified protein, and then to use the protein sequence as the basis for preparing a nucleic acid that can be used as a probe to identify either genomic DNA directly, or preferably, to identify a cDNA clone from a library prepared from the target tissue. Once such a cDNA clone has been identified, that sequence can be used to identify the sequence at the 5' end of the transcript of the indicated gene. For differential hybridization, subtractive hybridization and differential display, the nucleic acid sequence identified as enriched in the target tissue is used to identify the sequence at the 5' end of the transcript of the indicated gene.
  • any of these sequences identified as being from the gene transcript can be used to screen a genomic library prepared from the target organism.
  • Methods for identifying and confirming the transcriptional start site are well known in the art. In the process of isolating promoters expressed under particular environmental conditions or stresses, or in specific tissues, or at particular developmental stages, a number of genes are identified that are expressed under the desired circumstances, in the desired tissue, or at the desired stage. Further analysis will reveal expression of each particular gene in one or more other tissues of the plant. If the cell lethality function is only formed upon association of two different gene products, then it is only necessary to identify two promoters with activity in the desired tissue or condition but that do not have activity in any other common tissue.
  • a region of suitable size is selected from the genomic DNA that is 5' to the transcriptional start, or the translational start site, and such sequences are then linked to a partial coding sequence as described above. If the transcriptional start site is used as the point of fusion, any of a number of possible 5' untranslated regions can be used in between the transcriptional start site and the partial coding sequence. If the translational start site at the 3' end of the specific promoter is used, then it is linked directly to the methionine start codon of a partial coding sequence.
  • promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site.
  • TATAAT TATA box consensus sequence
  • a promoter element with a series of adenines surrounding the trinucleotide G (or T) N G. J. Messing et al. , in Genetic Engineering in Plants, pp. 221-227 (Kosage, Meredith and Hollaender, eds. 1983).
  • TATAAT TATA box consensus sequence
  • a promoter element with a series of adenines surrounding the trinucleotide G (or T) N G. J. Messing et al. , in Genetic Engineering in Plants, pp. 221-227 (Kosage, Meredith and Hollaender, eds. 1983).
  • a polyadenylation region should be included.
  • the polyadenylation region can be derived from the 3' end of a natural gene, from a variety of other plant genes
  • Modification of the promoter characterized as described herein can be done using any of a number of methods well known in the art. For example, specific enhancer sequences can be added to the promoter to increase the expression level or to modify the expression pattern. Further, an intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Examples of such promoters include TA29 from tobacco (Mariani et al. , Nature, 347:737-41, (1990)), 127a, 108, 92b, 101B, and 5B from tomato (Chen and Smith, Plant Physiol. , 101 : 1413 (1993), Smith et al. Mol. Gen. Genet.
  • Anther-specific promoters could also be used such as ones isolated by Twell et al. (Mol. Gen. Genet. , 217:240-45, (1991)).
  • Anther-specific promoters could also be used such as ones isolated by Twell et al. , Mol. Gen. Genet. 217:240-45 (1991) or Scott et al. , Plant Mol. Biol. 17: 195-207 (1991). Seed coat specific promoters, such as the pT218 promoter (Fobert et al., The Plant Journal 6:567-77 (1994)) or the pWM403 promoter could also be used in the present invention. Tissue-specific promoters for a range of different tissues have been identified, including roots, sepals, petals, and vascular elements.
  • promoters induced upon pathogen infection have been identified, such as the prp-1 promoter (Strittmatter et al., Bio/Technology 13:1085-90 (1995)). Promoters induced in specialized nematode feeding structures have been identified (disclosed in patent applications WO 92/21757, WO 93/10251, WO 93/18170, WO 94/10320, WO 94/17194).
  • the promoter in one of the expression cassettes is a promoter inducible by the gene product of the second expression cassette.
  • the gene product of the inducible expression cassette is, by itself, lethal to the plant cell. Tapetal-specific expression of genes such as ribonucleases (e.g. , Barnase), or premature expression of ⁇ -1,3 glucanases in the tapetum, have been shown to produce male sterility. Examples of other lethal polypeptides and nucleic acids are set forth below.
  • the second expression cassettes may encode a repressor/activator fusion protein.
  • telomeres are a tet artificial promoter comprising at least one tet operator and a TATA -box (as described by Weinman et al.).
  • TnpA binding protein from maize Spm when fused to an activator domain such as VP16, can be used to transactivate the Spm promoter (Schlappi et al,
  • activator domains which can be employed in the present invention include the acid domains from Vpl, ABB, PvAlf, HAP4, and GCN4. Non- acidic activator domains can also be used, such as proline-rich domains, serine/threonine-rich domains, and glutamine-rich domains.
  • Transactivator polypeptides are not limited to repressor/activator fusions, but include naturally occurring transactivator polypeptides such as the transcriptional activator polypeptides expressed by geminiviruses.
  • TGMV Tomato Golden Mosaic Virus
  • ACMV African Cassava Mosaic Virus
  • Each expression cassette of the present invention is individually functional but the product of each cassette alone does not provide the desired effect. It takes the combination of all transcripts (typically translated into polypeptides) from the individual expression cassettes to result in the desired phenotype. Such transcripts are individually are non-functional.
  • lethal or inhibitory transcripts can provide sense or antisense suppression, or lethal or inhibitory transcripts can be translated into a prozyme which is activated upon processing by a specific protease which is the product of the other expression cassette.
  • Prozymes can be artificially created by linking a desired "pro" region to an active enzyme through a linker containing recognition sequences of a desired protease.
  • proteases useful in the invention include proteases from poty viruses such as the NIa proteinase from tobacco etch virus or tobacco vein mottling virus (see, e.g. , Parks and Dougherty Virology 182: 17-27 (1991)).
  • the expression cassettes of the present invention can also jointly provide a beneficial effect to a cell.
  • individually each expression cassette encodes a transcript which is non-functional or encodes a non-functional polypeptide.
  • the presence of both transcripts or their encoded products in a cell e.g. , both monomers of a heterodimeric protein
  • the present invention provides lethal as well as restorative or therapeutic benefits to desired cells.
  • Polypeptides of the present invention can consist of separate functional proteins from distinct loci, or the polypeptides can be derived from overlapping or non- overlapping subsequences of a single functional protein which provides for the desired phenotype when co-expressed in a cell. Additionally polypeptides of the present invention can consist of separate monomers of a lethal dimeric protein. In some embodiments the polypeptides will be a prozyme and the specific protease which processes the prozyme and renders it inhibitory or lethal.
  • the multi-component system of the present invention is a two-component system.
  • the two-component (two peptide) system in which the two components are derived from a one-component (single protein) can generally be derived from any single protein that has a cell-lethal or inhibitory function (depending only upon the protein folding constraints of the initial protein).
  • the two peptides are from non-overlapping or minimally overlapping (e.g., 50, 35, 20, 15, 10, 5 or less) subsequences from a single inhibitory or cytotoxic protein.
  • the peptides produced reassociate in the target cell reconstituting the function of the single peptide from which the 2 partial peptides are derived.
  • the secondary and tertiary structure ' of a host of proteins and the processes of protein folding are known to those of skill and provide the basis for designing two-component peptide systems from a single protein.
  • the 2 peptides will relate to the starting protein as 1) unmodified peptides that comprise the entire original protein, with the addition of a methionine or the conservative replacement of an amino acid with a methionine at the point of separation of the 2 peptides; 2) modified peptides as in (1) with the additional replacement of some amino acids by other amino acids designed to enhance the stability of the peptides and reassociated peptide complex; 3) modified peptides that comprise less than the full protein in toto; 4) peptides that are derived from only a portion of the original protein, where the portion of the original protein encodes a suitable function.
  • non-functional polynucleotides or their encoded polypeptides can be achieved by a number approaches well known to the skilled artisan.
  • these polynucleotides or polypeptides when co-expressed in a cell, can confer lethality or some other desired function.
  • These peptide subsequences, taken together, can be related to the original peptide as comprising the total protein sequence of the original functional protein, or as comprising a portion of the total protein sequence only. To ensure that sufficient temperature stability is retained in the now dimeric active protein, it may be necessary to incorporate specific amino acid changes into the partial coding sequences.
  • the amino acid changes can be determined by examination of the original protein and the known amino acid interactions based on the protein structure as revealed through a range of physical techniques.
  • the amino acid changes can be determined by random mutagenesis and screening of a combinatorial library of protein products.
  • the amino acid changes can be determined by completely random mutagenesis and selection, using chemical treatments, PCR- induced mutagenesis, or other similar mutagenic treatments known to those skilled in the art.
  • the partial coding sequences derived from the original protein coding sequences is selected to retain activity of the reconstituted protein as well as a suitable level of stability with respect to environmental perturbations such as temperature changes.
  • Several general routes can be taken to determining effective partial coding sequences.
  • polypeptides include avirulence/resistance gene combinations which lead to a hypersensitive response and cell death.
  • Examples of this system are the AVR elicitor polypeptides from Cladosporium fulvum and the corresponding resistance genes, Cf from Lycopersicon (e.g. , Cf2/Avr2, Cf4/Avr4, Cf5IAvr5, and Cf9/Avr9, see, Jones et al. Science 266:789- 793 (1994) and Hammond-Kosack and Jones Plant Cell 8: 1773-1791 (1996)).
  • a preferred combination is CJ9/Avr9.
  • a hypersensitive response is elicited in cells expressing both Avr9 and Cf9 and results in cell death.
  • the AVR peptide is linked to a sequence targeting it to the apoplast (see, e.g. , Hammond-Kosack et al. Proc. Natl. Acad. Sci. USA 91:10445-10449).
  • Other avirulence/resistance gene combinations include the tomato Pto gene and the Pseudomonas syringae avrPto avirulence gene (Martin et al. Science 262: 1432 (1993), the RPS2 gene of Arabidopsis thaliana confers resistance to P. syringae that express the avrRpt2 avirulence gene (Bent et al. Science 265: 1856-1860 (1994)), and the tobacco N gene and TMV replicase (Padgett et al. Molecular Plant Microbe Interactions 10709-715 (1997)).
  • Polypeptides of the present invention can also be derived from overlapping or non-overlapping subsequences of a single functional protein which provides for the desired phenotype when co-expressed in a cell. Additionally polypeptides of the present invention can consist of separate monomers of a lethal dimeric protein. In some embodiments the polypeptides will be a prozyme and a protease which processes the prozyme and renders it inhibitory or lethal.
  • pancreatic ribonuclease A can be cleaned by subtilisin between residues 20 and 21, yielding a large and a small peptide, neither of which retains any activity, as essential catalytic residues are present in each peptide fragment.
  • subtilisin between residues 20 and 21
  • the small peptide binds to the larger fragment and activity is reconstituted.
  • staphylococcal nuclease can be resolved into three peptide fragments following partial proteolysis.
  • Proteases initially cleave an intact protein at exposed residues, often ones that are part of exposed loops not involved in specific domains. Following partial proteolysis and analysis of the resulting peptide fragments by polyacrylamide gel electrophoresis to confirm that a simple digestion resulted, residual activity is evaluated. If activity is retained, the peptides are separated to determine whether neither peptide retains activity separately, and subsequently whether activity can be reconstituted upon remilling. Sequencing of the amino and carboxy termini of the two (or more) fragments reveals how to engineer the partial coding sequences in the instant invention. ii. Sequence conservation-based design
  • subtilisin family proteins e.g., subtilisin family proteins; colicin family proteins; ribonuclease family proteins
  • regions that are not well conserved in all proteins e.g., subtilisin family proteins; colicin family proteins; ribonuclease family proteins
  • regions of the protein that are good candidates for separation into separate peptides. Such regions retain unaltered principal secondary structural features, such as alpha helices and beta-sheets.
  • a number of possible replacement and coding sequence variants can be tested using an assay either for protein function in vitro, or for function, or for in vivo lethality.
  • Exemplary polypeptides of the present invention include ribonucleases such as barnase (Mauguen et al., Nature 297: 162-64 (1982)), binase (Pavlovsky et al., FEBS Lett. 162: 167-70 (1983)), Ribonuclease Tl (Fujii et al, Biosci. Biotechnol. Biochem. 59: 1869-1874 (1995)), nucleases such as colicin E9 (Wallis et al, Eur. J.
  • subtilisin BPN' Error et al, J. Mol. Biol. 233:293-304
  • these polypeptides are used to yield male sterility when co-expressed in tapetal tissue.
  • Other polypeptides for creating cell toxicity or inhibition include those which produce toxic substances, disrupt cell function, suppress genes required by the cell (such as by using anti-sense, sense suppression, or ribozymes), and disruption of mitochondrial function.
  • the polypeptide is derived from a separate subsequence of a ribonuclease such as barnase.
  • a ribonuclease such as barnase.
  • barnase the minimal length of each polypeptide is at least 20 amino acids.
  • the extent of overlap of barnase polypeptides will be no more than 5 amino acids.
  • barnase is a well-studied cell lethality function that has already been shown to be cell-autonomous, independent of other cellular functions, and very sensitive. barnase expression has been shown to inhibit cell growth and development in specific plant tissues.
  • the mature barnase protein consists of a 110 amino acid polypeptide. It has been shown in in vitro studies that amino acid 37 of the mature protein can be converted from a valine to a methionine with good retention of ribonuclease activity. Sancho & Fersht, J. Mol.Biol 224:741-47 (1992).
  • cyanogen bromide treatment cleaves the protein into a 36 amino acid peptide and a 74 amino acid peptide and that neither peptide retains any activity. Further, at least 30% of normal activity is reconstituted when the two peptides are mixed in vitro. Sancho & Fersht, supra.
  • a particularly preferred embodiment is to produce the following two partial barnase coding subsequences via PCR amplification using a barnase gene as template and 1) primers designed to introduce a methionine codon at position 1 of the mature protein coding sequence and a stop codon after position 36 of the mature protein sequence, and 2) primers designed to introduce a methionine codon at position 37 of the mature protein coding sequence while leaving the end of the mature protein coding sequence intact.
  • the two partial coding sequences can then be manipulated further to produce expression cassettes, using, for example, a promoter, a 5' untranslated region, a 3' untranslated region, and a polyadenylation signal.
  • the two expression cassettes can be designed to create two-component lethality systems that could be used to create a range of useful traits.
  • Another exemplary two-component polypeptide system is the use of the Avr9 elicitor polypeptide from Cladosporium fulvum and the corresponding resistance gene, Cf9 from Lycopersicon esculentum. A hypersensitive response is elicited in cells expressing both Avr9 and Cf9 resulting in cell death. Means to assay for eukaryotic cell cytoxicity or inhibition produced by two peptide fragments of a single protein are well known in the art.
  • enzymatic activity can be assayed directly on cell extracts containing the expressed peptides or in purified preparations of the peptides. Further, eukaryotic cell cytotoxicity or inhibition can be assayed using a range of indicators for cell function.
  • the expression cassettes can be introduced to cells along with an expression cassette that produces an easily assayed function, such as the beta-glucuronidase protein (Jefferson et al, EMBO J. 6:3901-3907 (1987)) or firefly luciferase (De Wet et al, Mol. Cell. Biol.
  • the peptides of the invention may also be modified according to standard methodology to produce polypeptides with, either separately or in combination, e.g. , enhanced thermal stability, enhanced subunit association, enhanced activity at lower concentrations and the like. These peptide can also be modified to produce conservatively modified variants.
  • the modification of the polypeptides can be achieved, e.g. , by techniques known to those skilled in the art such as random or site-specific mutagenesis of the nucleic acids that encode the polypeptides. Using such methods of mutagenesis, genetically modified peptides are then assayed for reconstitution of activity in vivo. Activity at lower concentrations or at higher temperatures is measured by comparing the genetically modified and the original peptides.
  • the two barnase components described above are modified separately in a way that has been shown to enhance stability of the intact barnase protein (see Example 9).
  • the modifications selected are based on the work of Serrano et al , J. Mol Biol. 233:305-12 (1993), who compared the thermodynamic stability of barnase with a related protein, binase, obtained from Bacillus intermedius. Binase has the same amino acid sequence as barnase at all but 18 of the 110 amino acid positions.
  • Serrano et al. replaced each of the barnase amino acids with a different amino acid at positions in which the proteins differed, and measured the change in thermodynamic stability induced by the amino acid change.
  • Enzyme activity was also measured for a subset of the mutagenized barnase forms. In this way, specific amino acid changes that enhanced stability, but had little effect on enzyme activity were identified.
  • the glutamine at position 15 of the mature barnase protein was replaced by isoleucine, and the threonine at position 16 was replaced by arginine;
  • the carboxy-terminal peptide the glycine at position 65 was replaced with a serine, and the lysine at position 108 was replaced with an arginine.
  • the transcription products of number of DNA constructs can be used to suppress expression of endogenous plant genes and yield a beneficial or lethal result to the cell.
  • Anti-sense RNA inhibition of gene expression has been shown; see, e.g. , Sheehy et al, Proc. Nat. Acad. Sci. USA 85:8805-8809 (1988), and Hiatt et al, U.S. Patent No. 4,801,340.
  • sense suppression to modulate expression of endogenous genes see, Napoli et al, The Plant Cell 2:279-289 (1990), and U.S. Patent No. 5,034,323.
  • RNA molecules or ribozymes can also be used to inhibit gene expression.
  • a beneficial or lethal ribozyme can be transcribed upon induction by a polypeptide expressed from a second expression cassette (e.g. , tet repressor/ VP 16 activator fusion polypeptide). It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme.
  • ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
  • a general design and use of target RNA- specific ribozymes is described in Haseloff et al Nature, 334:585-591 (1988).
  • the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about 2000 nucleotides should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 500 nucleotides is especially preferred.
  • Transformed plant cells which are derived by any number of transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired expression cassette.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the polynucleotide encoding a desired polypeptide.
  • Plant regeneration from cultured protoplasts is described in Evans et al , Protoplasts ' Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73 (1985). Regeneration can also be obtained from plant callus, explants, organs, or parts thereof.
  • Such regeneration techniques are described generally in Klee et al, Ann. Rev. of Plant Phys. 38:467-486 (1987).
  • the expression cassettes encoding each component of the two or more component system are either introduced into a single cell by cotransfo ⁇ nation of cells with each of the two expression cassettes, or by sequential transformation of cells with the two expression cassettes.
  • two promoters with overlapping specificity are used, cell inhibition or lethality will result in only the target tissue in which both promoters are sufficiently active.
  • the expression cassettes are introduced into different cells by transformation. Whole organisms are regenerated from the separated transformed cells, and then a hybrid organism is produced by crossing the individual organisms. In this way, the original whole organisms, each carrying a single expression cassette show no cell inhibition or lethality. However, the hybrid organism resulting from the cross will have both expression cassettes in the same cell, and will express cell inhibitory function or lethality in a manner dependent upon the expression patterns of the chosen promoters.
  • One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • inbred lines are made, each comprising one of the two expression cassettes whose joint expression leads to inhibition or death of the cell.
  • each expression cassette can express a non-lethal polypeptide from a seed-specific promoter.
  • each of the two polynucleotides is operably linked to a promoter functional in tapetal cells or pollen cells, either using the same promoter with each polynucleotide or different promoters.
  • Inbred lines are maintained, each comprising one of the polynucleotides in homozygous condition. When the inbred lines are crossed, the resulting hybrid carries both partial genes and is male-sterile. If the two inbreds are the same line, except for the partial barnase polypeptide, then the resulting "hybrid" is a male-sterile inbred that can be used in a hybrid breeding strategy.
  • each of the polynucleotide sequences is operably linked with a promoter expressed in stigmatic tissues, tissues of the transmitting tract, ovule tissues, or other tissues essential for female fertility.
  • Inbred lines are maintained, each comprising one of the two polynucleotides in homozygous condition. When the inbred lines are crossed, the resulting hybrid carries both polynucleotides and is female-sterile. If the two inbreds are the same line, except for the partial barnase gene, then the resulting "hybrid" is a female-sterile inbred that can be used in a hybrid breeding strategy.
  • Disease resistance in plants can be mediated by a hypersensitive response in which cells infected by a pathogen are killed to prevent further spread of the pathogen.
  • a synthetic hypersensitive response can be created.
  • tolerance to root knot or cyst nematodes can be mediated by eliminating the giant cells or specialized feeder cells these pests require for continued growth and multiplication in plant roots.
  • promoters induced in the giant cells or specialized feeder cells in combination with a two-component system of the present invention these specialized root cells can be eliminated.
  • Hybrid seeds comprising both expression cassettes of the multi-component system are typically produced in a maintainer field using sub-lines Al and A 2 .
  • These sublines comprise one or the other of the two expression cassettes of the two-component system which when functional in the same cell lead to cell inhibition or cell death.
  • Both sublines A ! and A 2 are fertile since each carries one expression cassette of the two- component system of the invention.
  • cell death or inhibition is initiated in the cells where both expression cassettes are functional.
  • one subline will comprise an expression cassette comprising a polynucleotide from the Cladosporium fulvum Avr9 avirulence gene operably linked to a tapetal-specific promoter such as pMon.
  • the tomato Cf9 gene (the corresponding tomato resistance gene) is operably linked to a second tapetal-specific promoter, TA29.
  • Both sublines are male fertile since the Avr9 and Cf9 polypeptides individually do not confer cell death. However, when these sublines are crossed they yield a line in which a hypersensitive response is initiated in the tapetum resulting in cell death. Hominid-Kosack et al, PNAS 91: 10445-10449 (1994); Jones et al, Science 266:789-793 (1994). Tapetal cell death will confer male-sterility without adversely affecting other organs. In the hybrid seed production field, the male sterile line A can be crossed to any line B to produce hybrid seeds.
  • one subline has a dominant male sterile gene (Ms) with an artificial promoter comprising at least one tet operator and a TATA-box.
  • Ms dominant male sterile gene
  • the corresponding subline has a tapetal-specific promoter driving the expression of a chimeric transcriptional activator.
  • this chimeric transcriptional activator will be Act. Weinmann et al. , The Plant Journal 5(4):559-569 (1994).
  • the line produced from crossing the sublines will be male sterile since it contains both the transcriptional activator (Act), which is expressed specifically in the tapetum, and the tet operator-Ms gene.
  • Ms genes such as ribonucleases (e.g. , Barnase), or premature expression of 6-1,3 glucanases in the tapetum, have been shown to produce male sterility.
  • the male sterile line A can be crossed to any line B to produce hybrid seeds.
  • the present invention further provides packageable DNA or RNA (nucleic acid) constructs for a multi-component lethality or inhibitory system as described more fully above.
  • the various constructs employed in gene therapy methods can be had by reference to the compositions and methods described earlier.
  • the constructs of the invention can be used to target specific mammalian cells.
  • the packageable nucleic acid constructs allow for the transfection of eukaryotic cells in vivo or ex vivo.
  • the eukaryotic cells are mammalian hosts, such as mice, rodents, primates, and humans.
  • the packageable nucleic acids of the invention can be inserted into any of a number of well known vectors for the transfection of target cells and organisms as described below.
  • Cells are transfected with a expression cassette comprising a poynucleotide operably linked to a promoter functional in the cell or in a cell of a later developmental stage.
  • Cells transfected all of the functional expression cassettes of the multi-component lethality system will inhibit cell growth (i.e. , the cell doesn't enter the cell cycle) or kill the cell.
  • the multi-component system of the present invention can be modified to provide a therapeutic effect upon transfection with all functional expression cassettes of the multi-component system; such embodiments can employ any polypeptide which produces a desirable effect.
  • Preferred embodiments of the present invention is targeted killing of cancerous cells.
  • a cell capable of causing cancerous growth in a mammal is altered in multiple ways via mutation from a mammalian cell that is not capable of causing cancerous growth. It is known in the art that defects occur in the control of cell growth control networks, in the control of telomere length and in the control of contact-mediated cell growth inhibition. Although there are common themes and mechanisms underlying causation of specific cancers, a wide range of genes have been identified as oncogenic when their expression is altered or their function is altered. Certain genes are often expressed in cancerous cells which are normally not expressed in the mature cells of an intact organism.
  • the promoters of such genes can be used in the instant invention to create a two-component system that will form a lethal function in a cancerous cell, but not in a cell under normal controls for the cell cycle, contact inhibition and telomere formation.
  • Typical promoters would be selected from a group that includes, but is not limited to, a telomerase promoter, or a promoter under the control of myc genes.
  • the polynucleotides of the two-component system can be delivered to cancerous cells via disarmed human viruses, liposome fusion, or other method.
  • transfected includes reference to the introduction of a nucleic acid into a eukaryotic cell where the nucleic acid can be incorporated into the genome of the cell (i.e., chromosome, plasmid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g. , transfected mRNA).
  • the nucleic acids are transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell. Vectors which target distinct cell types are known in the art.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof. See, e.g., Buchscher et al. , J. Virol. 66(5): 2731-2739 (1992); Johann et al , J. Virol 66(5): 1635-
  • AAV-based vectors are also used to transduce cells with target nucleic acids, e.g. , in the in vitro production of nucleic acids and polypeptides, and in vivo and ex vivo gene therapy procedures.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with an expression cassette of the present invention (gene or cDNA), and re- infused back into the subject organism (e.g., patient).
  • an expression cassette of the present invention gene or cDNA
  • Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al, Culture of Animal Cells, A Manual of Basic Technique, third edition (1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients) .
  • stem cells are used in ex-vivo procedures for cell transfection and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34 + cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN- ⁇ and TNF- ⁇ are known (see, Inaba et al , J. Exp. Med. 176: 1693-1702 (1992)).
  • stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4 + and CD8 + (T cells), CD45 + (panB cells), GR-1 (granulocytes), and Ia d (differentiated antigen presenting cells).
  • unwanted cells such as CD4 + and CD8 + (T cells), CD45 + (panB cells), GR-1 (granulocytes), and Ia d (differentiated antigen presenting cells).
  • T cells CD4 + and CD8 +
  • CD45 + panB cells
  • GR-1 granulocytes
  • Ia d differentiate antigen presenting cells
  • hematopoietic progenitor and stem cells are characterized by the presence of a CD34 surface membrane antigen. This antigen is used for purification, e.g. , on affinity columns which bind CD34. See, Ho et al. ' , Stem Cells 13 (suppl. 3): 100-105 (1995). See also, Brenner, Journal of Hematotherapy 2:7-17 (1993).
  • hematopoietic stem cells are isolated from fetal cord blood. Yu et al , Proc. Natl Acad. Sci. USA 92:699-703 (1995) describe a preferred method of transducing CD34 + cells from human fetal cord blood using retro viral vectors.
  • Vectors e.g., retroviruses, adeno viruses, liposomes, etc.
  • therapeutic nucleic acids can be administered directly to the organism for transduction of cells in vivo. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells.
  • the packaged nucleic acids are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such packaged nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • compositions of the present invention are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • the packaged nucleic acids can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the packaged nucleic acid with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • Parenteral administration and intravenous administration are the preferred methods of administration.
  • the formulations of packaged nucleic acid can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Cells transduced by the packaged nucleic acid as described above in the context of ex vivo therapy can also be administered intravenously or parenterally as described above.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient.
  • the physician evaluates circulating plasma levels of the vector, vector toxicities, progression of the disease, and the production of anti-vector antibodies.
  • the dose equivalent of a naked nucleic acid from a vector is from about 1 ⁇ g to 100 ⁇ g for a typical 70 kilogram patient, and doses of vectors which include a retroviral particle are calculated to yield an equivalent amount of therapeutic nucleic acid.
  • inhibitors and transduced cells of the present invention can be administered at a rate determined by the LD-50 of the transduced cell type, and the side-effects of the cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
  • Transduced cells are prepared for reinfusion according to established methods. See, Abrahamsen et al., J. Clin. Apheresis, 6:48-53 (1991); Carter et al, J.
  • the cells should number between lxlO 8 and lxlO 12 .
  • the growth characteristics of cells vary from patient to patient and from cell type to cell type. About 72 hours prior to reinfusion of the transduced cells, an aliquot is taken for analysis of phenotype, and percentage of cells expressing the therapeutic agent.
  • the two or more component lethality system of the present invention is preferably employed with multicellular eukaryotic organisms such as a mammal or plant, it is also applicable to any organism, as cell inhibitory or lethality functions (e.g., ribonucleases, deoxyribonucleases, proteases, toxins) are known that effect all types of cells.
  • cell inhibitory or lethality functions e.g., ribonucleases, deoxyribonucleases, proteases, toxins
  • the principles of protein structure and protein folding apply universally in living cells, and thus multi-component systems will function in cells of all types.
  • a range of promoters with various expression patterns are known in the art for prokaryotic and non-plant eukaryotic cells. The combination of specifically expressed promoters with a two-component system is therefore general.
  • a two-component lethality system can also be applied to prokaryotic organisms.
  • a two or more component system can be delivered to the bacterial cells specifically using specialized bacteriophage.
  • a single promoter that is highly expressed in the target bacterial cell but without activity in the host organism or other beneficial bacterial cells can be used with each partial coding sequence, and the two genes delivered to the target bacterial cells in the genome of a specialized bacteriophage.
  • the system can be expanded to utilize greater than two components.
  • Example 1 describes the synthesis of partial barnase genes using a constitutive promoter.
  • PCR amplifications were done according to manufacturer's recommended conditions, using Taq DNA polymerase, each pair of primers separately, and chromosomal DNA isolated from Bacillus amyloliquifaciens strain ATCC #23842. Bacterial DNA was isolated using standard methods.
  • the resulting fragments comprising the 5' partial coding sequence (bn5) and the 3' partial coding sequence (bn3), synthesized to have an Ncol site at the 5' end and an Xbal site at the 3' end of each fragment were gel purified.
  • each fragment was cloned into plasmid pNG5104, with the coding sequence of the ⁇ -glucuronidase (uidA) gene removed by Ncol -Xbal digestion and gel purification of the resulting large fragment.
  • the resulting genes have each partial coding sequence operably linked to the SpMas promoter (Gelving et al, 1995) and to the 3' polyadenylation region of the octopine synthase gene.
  • the size of the inserted fragments was verified by gel electrophoresis, and at least two clones for each of the 5' genes (SpMas-bn5) and the 3' genes (SpMas-bn3) was selected for sequence analysis. The sequences were confirmed for each of the genes, and the resulting plasmids were designated pER4013 (SpMas-bn5) and pER4022 (SpMas-bn3).
  • the bn5 coding sequence encodes a peptide with a methionine added prior to the amino-terminal alanine of the mature barnase protein, and with a carboxy-terminal tryptophan (mature protein position 35).
  • the bn3 coding sequence encodes a peptide with the initiation methionine in place of the valine of the barnase mature protein position 36 and with the correct carboxy-terminal arginine (mature protein position 110).
  • Example 2 describes biollistics assay for the reconstitution of a lethal function in plant cells.
  • Reconstitution of cell lethality was assayed by bombarding plant leaves with a mixture of pER4013 and pER4022 phagemid DNAs, along with DNA of phagemid pLVC320, which comprises a SpMas-LUC chimeric gene.
  • pER4013 and pLVC320 were delivered biollistically, and pER4022 and pLVC320 were delivered biollistically.
  • the plasmid DNA's were prepared as single stranded, circular molecules using the M 13 origin of replications in these phagemids.
  • each plasmid was adjusted to 400 ⁇ g/ ⁇ l in 10 mM Tris-1 mM EDTA, pH 8.0.
  • the plasmid DNA's were mixed according to standard methods and coated onto tungsten particles by mixing 80 ⁇ l of each DNA mixture with 40 ⁇ l of M17 tungsten particles (100 mg/ml in sterile water), 40 ⁇ l of 100 mM spermidine, and 100 ⁇ l of 2.5 M CaC12 in rapid succession. After mixing by vortexing, the particles were allowed to remain on ice for 5 min, after which the particles were collected by centrifugation in a microcentrifuge for 20 sec. The supernate was removed, and the particles suspended in 100 ⁇ l of 70% ethanol.
  • the luciferase assay results in Table 1 are reported as cpm/leaf disk.
  • the combination of SpMas-bn5 with SpMas-bn3 results in a 84% reduction in luciferase activity compared with SpMas-bn5 alone and a reduction of 71 % compared with SpMas-bn3 alone. This indicates reconstitution of cell lethal function by expression of the separate bn5 and bn3 peptides in the same cells. Similar results are reported in TAbles 2- 4.
  • Example 3 describes tobacco lines expressing an amino-terminal barnase partial peptide and a carboxy-terminal barnase partial peptide under control of a strong constitutive promoter.
  • the SpMas-bn5 gene in pER4013 is recloned as a PvuII fragment into the
  • T-DNA vector pWTT2200 which has an nptll gene as the selectable marker to confer resistance to kanamycin or G418.
  • the SpMas-bn3 gene in pER4022 is recloned as a PvuII fragment into the Smal site of T-DNA vector pNG5185, which has an ALS gene as the selectable marker to confer resistance to chlorsulfuron.
  • Each construct, along with vector only controls, is introduced separately into Nicotiana tobaccum cultivar petite Havana using standard leaf disk transformation methods.
  • Several independent transgenic individuals carrying the SpMas-bn5 and SpMas-bn3 transgenes are assayed for expression by northern analysis.
  • Three independent transgenic individuals from each class are selected as high expressers, and three of each control transformant class are also selected.
  • Crosses are done between 1) SpMas-bn5 transgenic plants and pNG5185 transgenic plants, 2) SpMas-bn3 transgenic plants and pWTT2200 transgenic plants, and 3) SpMas-bn5 and SpMas-bn3 transgenic plants. Only in the third set of crosses would a functional cell lethal function be expressed in those cells which carry both the SpMas-bn5 and SpMas-bn3 genes. This represents one-fourth of the progeny population.
  • Example 4 describes tobacco lines expressing an amino-terminal barnase partial peptide and a carboxy-terminal barnase partial peptide under control of a promoter expressed in seed coats.
  • chimeric genes are prepared in which bn5 and bn3 coding sequences are each operably linked to a seed coat promoter isolated from watermelon, and known as pWM403. A 2 kbp fragment of the region preceeding the start of transcription of the WM403 gene is used for each chimeric gene.
  • the individual bn5 and bn3 chimeric genes are cloned into plant binary vectors pWTT2200 and pNG5185 respectively, as described in example 3 above.
  • transgenic individuals from each class are selected as high expressers, and three transgenic individuals of each control transformant class (vector only) are also selected.
  • Crosses are made between 20 1) pWM403-bn5 transgenic plants and pNG5185 transgenic plants, 20 2) pWM403-bn3 transgenic plants and pWTT2200 transgenic plants, and 3) pW M403-bn5 and pWM403-bn3 transgenic plants.
  • Progeny plants resistant to both chlorsulfuron and kanamycin are selected and grown to maturity for evaluation of seed and seed coat development.
  • Example 5 describes tobacco lines expressing an amino-terminal barnase partial peptide and a carboxy-terminal barnase partial peptide under control of two different promoters expressed in seed coats.
  • chimeric genes are prepared in which bn5 and bn3 coding sequences are each operably linked to a seed coat promoter isolated from tobacco, and known as pT218 (Fobert et al, The Plant Journal 6:567-77 (1994)). A 2 kbp fragment of the T218 promoter is used for each chimeric gene.
  • bn5 and bn3 chimeric genes are cloned into the plant binary vectors pWTT2200 and pNG5185 respectively. High expressing transgenic individuals are identified as described in example 4.
  • Crosses are made between 1) pT218-bn5 transgenic plants and ⁇ NG5185 transgenic plants, 2) pT218-bh3 transgenic plants and pWTT2200 transgenic plants, 3) pT218-bn5 and pWM403-bn3 transgenic plants, and 4) pWM403-bn5 and pT218-bn3 transgenic plants.
  • Progeny plants resistant to both chlorsulfuron and kanamycin are selected and grown to maturity for evaluation of seed and seed coat development.
  • Example 6 describes tobacco lines expressing an amino-terminal barnase partial peptide and a carboxy-terminal barnase partial peptide under control of a promoter expressed in tapetal cells.
  • chimeric genes are prepared in which bn5 and bn3 coding sequences are each operably linked to a tapetal-specific promoter isolated from tobacco, and known as pTA29 (Mariani et al, Nature 347:737-41, (1990)).
  • pTA29 Median et al, Nature 347:737-41, (1990)
  • bn5 and bn3 chimeric genes are cloned into the plant binary vectors pWTT2200 and pNG5185, respectively. High expressing transgenic individuals are identified as described in example 4.
  • Crosses are made between 1) pTA29-bn5 transgenic plants and pNG5185 transgenic plants, 2) pTA29-bn3 transgenic plants and pWTT2200 transgenic plants, and 3) pTA29-bn5 and pTA29-bn3 transgenic plants.
  • Progeny plants resistant to both chlorsulfuron and kanamycin are selected and grown to maturity for evaluation of pollen development and male fertility.
  • Example 7 describes development of two-component system based on colicin E7 nuclease function.
  • a region of approximately 15,000 daltons of the carboxy terminal end of the colicins comprises a non-specific nuclease function known to be involved in the cell death of cells into which the nuclease is targeted via the amino terminal segment. Alignment of these sequences reveals a region of approximately 30 amino acids located 65 - 35 amino acids from the carboxy terminus which is highly variable. Within this region of colicin is a methionine residue 55 amino acid residues from the carboxy terminus of the protein.
  • the 5' coding sequence is created, through polymerase chain reaction, by incorporating a methionine codon in place of the leucine codon located at position 135 residues from t he carboxy terminus, with a stop codon inserted in place of the methionine codon at residue 55 from the carboxy terminus.
  • the 3' coding sequence is created, through polymerase chain reaction, using the methionine codon at residue 55 from the carboxy terminus as the initiation codon and using the normal stop codon of the colicin E7 message.
  • Each pair of coding sequences is cloned, as described in example 1 , into a vector with the SpMas promoter to create genes that are constitutively expressed in plant cells.
  • the pair of candidate genes is then introduced biollistically into plant leaf disks either separately or in combination as shown in example 2.
  • Example 8 describes a two-component system for the reconstitution of a lethal function in plant cells.
  • Example 8A describes the subcloning of two partial barnase genes (bn3 and bn5) using the 35S promoter, and the subcloning of a barstar gene using a enhanced 35S promoter.
  • Plasmids pER4013 (SpMAS-bn5, encoding the barnase gene bn5) and pER4022 (SpMAS-bn3, encoding the barnase gene bn3) were digested with Ncol and Xbal, and the fragments corresponding to the bn5 and bn3 coding regions were gel purified.
  • Plasmid pEL5051 which contains a 35S-Gus synthetic gene, was digested with Ncol and Xbal, which excised the GUS coding region.
  • Ncol/Xbal fragment from pEL5051 containing the 35S promoter, was then gel purified.
  • the bn3 and bn5 fragments were then each separately cloned into this Ncol/Xbal fragment of pEL5051 to make pEL5152 and pEL5161, which respectively encode bn3 and bn5, each linked to the 35S promoter.
  • the barstar gene operably linked to the enhanced 35S promoter was subcloned in the following manner.
  • the complete barstar coding sequence from Bacillus amyloliquefaciens is available from GenBank under Accession No. XI 5545 (Hartley, J. Mol. Biol. 202:913-915 (1988)).
  • a complete coding sequence of barstar was obtained.
  • PCR primers corresponding to the ends of the barstar coding sequence were made. These primers contained additional restriction sites for BspHI at the 5' end and Xbal at the 3' end.
  • the primers were used for PCR amplifications using Bacillus amyloliquefaciens genomic DNA with TAQ DNA polymerase according to the manufacturer's recommendations.
  • Bacillus amyloliquefaciens strain ATCC No. 23842 chromosomal DNA was isolated according to standard methods.
  • the PCR fragments were digested with BspHI and Xbal and the fragment with the barstar coding sequence was gel purified and cloned into pUC120 digested with Ncol and Xbal.
  • Plasmid pNG5011 was used as a source of DNA for a second round of PCR amplification as described above, except that Vent DNA polymerase was used. The resulting fragments were gel purified and cloned into EcoRV- digested pBluescript plasmid. At least two plasmids identified by restriction digest as having the correct size fragment were sequenced and the correct barstar sequence was confirmed for at least one of these clones. This plasmid was designated pBH4004. Plasmid pBH4004 was digested with BspHI and Xbal and the fragment corresponding to barstar was gel purified.
  • Plasmid pKL3049 contains a synthetic gene comprised of a 35S promoter with two copies of an 35S enhancer region (this promoter is designated e35S or enhanced 35S) attached at an Ncol restriction site to a chitinase coding sequence, ChiA, which is joined to a NOS 3' region at a Xbal restriction site. Plasmid pKL3049 was digested with Ncol and Xbal and the fragment containing the e35S promoter was gel purified.
  • this promoter is designated e35S or enhanced 35S
  • Example 8B describes a biollistics assay for the reconstitution of a lethal function in plant cells. This example shows that the lethal function is due to the RNase activity of a reconstituted barnase. This reconstitution was demonstrated by the specific reduction of this lethal function with co-expression of barstar, the specific inhibitor of the RNase activity of barnase.
  • Reconstitution of cell lethality was assayed by bombarding plant leaves with a mixture of pEL5152 and pEL5161 plasmid DNA along with DNA of the plasmid pJJ3792, which comprises a 35S-LUC chimeric gene.
  • plant leaves were bombarded with a mixture of pEL5152, pEL5161 and pSG5351, along with pJJ3792.
  • pEL5152, pEL5161, and pSG5351 were delivered biollistically, separately, with pJJ3792.
  • the plasmid DNAs were prepared as double stranded circular molecules purified by two rounds of CsCl density gradient centrifugation. After determining the DNA concentration by spectrophotometer, the concentration of each plasmid was adjusted to 400 mg/ml in 10 mM tris 1 mM EDTA, pH 8.0. The plasmids were mixed according to standard methods as described in example 2, except that 40 ⁇ L of each DNA mixture was mixed with 20 ⁇ L of tungsten particles, 20 ⁇ L of 100 mM spermidine, and 50 ⁇ L of 2.5 M CaC12. After centrifugation, the particles were suspended in 50 ⁇ L of 70% ethanol.
  • the 40 ⁇ L DNA mixture contained 4 ⁇ L of pJJ3792 and 12 ⁇ L of ⁇ EL5152, pEL5161, or pSG5351 either separately or together.
  • a CsCl purified filler DNA was used to bring the volume for each sample to 40 ⁇ L.
  • Luciferase assays were performed as described in example 2 except that plant leaf extracts were prepared in 200 ⁇ L of Cell Lysis Buffer (Promega), the samples were centrifuged, and then 20 ⁇ L of the supernatant was diluted to 100 ⁇ L with Cell Lysis Buffer. The assay used 40 ⁇ L of assay solution and 5 ⁇ L of diluted plant extract. Typical luciferase assay results are shown in Table 5 and are reported as the average cpm for 10 leaf disk per treatment.
  • 35S-bn5 and 35S-bn3 resulted in an 86% reduction in luciferase activity as compared with the average of the three controls.
  • the addition of e35S-barstar to 35S-bn3 and 35S-bn5 resulted in a restoration of luciferase activity to 48% of the average of the controls. This result indicates that the cell lethal function reconstituted by the expression of bn5 and bn3 in the same cell is the RNase activity of barnase.
  • Example 9 describes improvement in function of the two-component system through site-specific mutagenesis.
  • Example 9A describes construction of chimeric genes that encode mutagenized peptides for enhanced stability.
  • bn5 For the bn5 gene, specific nucleotide changes were introduced so that the amino acid sequence was changed from MAQVINTFDGVADYLQIYHKLPDNYITKSEAQALGW to MAQVINTFDGVADYLIRYHKLPDNYITKSEAQALGW.
  • the glutamine residue at position 16 of bn5 (position 15 of mature barnase protein) was replaced by an isoleucine residue
  • the arginine residue at position 17 of bn5 was replaced by an arginine residue (changes underlined).
  • nucleotide changes were introduced by PCR, using the same general methods of example 1.
  • two PCR reactions were performed, using a barnase coding sequence-containing plasmid as template, one with primers NC207 and NC225, the other with NC224 and NC143.
  • NC207 5' GGCCATGGCACAGGTTATCAACACGTTTGACGGGGTTGC 3' NC225: 5' AGGAAGCTTATGATATCTGATAAGATAATCCGCAACCCCG 3 ' NC224: 5' CATAAGCTTCCTGATAATTACATTACAAAATC 3' NC143: 5' CGTCTAGATTACCAGCCGAGGGCTTGTGCTTC 3'
  • the resulting DNA fragments were gel purified and then mixed together prior to performing a PCR reaction with primer pair NC207 and NC143.
  • the resulting fragment was digested with Hindlll to confirm the fragment identity and then digested with Ncol and Xbal prior to cloning into pUC120.
  • the resulting plasmid, comprising the full, modified coding sequence of bn5 (bn5-2) was designated pAR4554.
  • the cloned fragment was sequenced.
  • NC144 5' GGCCATGGCATCAAAAGGGAACCTTGCAGA 3'
  • NC221 5' CC ATGTGCGGCCGCTTTTGCTCGGGAGTTTGCCTTC 3 '
  • NC222 5 ' AAAAGCGGCCGCACATGGCGTGAAGCGGATATTAA CTATGTATCAGGCTTCAG 3'
  • NC223 5' CGTCTAGAGTTATCTGATCCTTGTAAAGGTCTG 3 '
  • the resulting DNA fragments were gel purified and then mixed together prior to performing a PCR reaction with primer pair NC144 and NC223.
  • the resulting fragment was digested with NotI to confirm the fragment identity and then digested with Ncol and Xbal prior to cloning into pUC120.
  • the cloned fragment was sequenced.
  • plasmids pAR4554 and pAR4561 were digested with Ncol and Xbal, the suitable fragments were gel purified, and each was inserted into plasmid pEL5051, with the bn5-2 and bn3-2 coding sequences replacing the coding sequence of the beta-glucuronidase (uidA) gene removed by Ncol-Xbal digestion and gel purification of the resulting large fragment.
  • the resulting chimeric genes have each partial coding sequence operably linked to the CaMV 35S promoter and to the 3 ' polyadenylation region of the octopine synthase gene.
  • the size of the inserted fragments was verified by agarose gel electrophoresis.
  • the plasmid containing the chimeric bn5-2 gene was designated pEL5061, and the plasmid containing the chimeric bn3-2 gene was designated pEL5071.
  • Example 9B describes bioUistics assays to determine activity of the two modified peptides compared with the unmodified peptides in plant cells.
  • Reconstitution of ribonuclease activity is assayed as described in example 8, by the bioUistics delivery of the two partial genes together in comparison with the 2 partial genes individually. This is done in a single experiment, with leaf tissue of a young tobacco plant as the target for DNA delivery. The data from a typical experiment are shown in Table 6. When the modified barnase fragments were expressed in the same cell, the luciferase activity was reduced to 19% of the average of the four controls. When the unmodified barnase fragments were expressed in the same cell, the luciferase activity was reduced to less than 56% of the average of the four controls. Table 6. Luciferase activity recorded as cpm per tobacco leaf disk and as the average for 11 leaf disks per treatment. Data is reported as (measured cpm minus background) x .001.
  • Example 9C describes bioUistics assays to determine the relative activity of the 2 modified peptides compared with the unmodified peptides in plant cells.
  • a dilution experiment was performed, with decreasing amounts of the 2 modified partial genes used for DNA delivery. This was done in a single experiment, with leaf tissue of a young pepper plant as the target for DNA delivery as described in example 8.
  • the data from a typical experiment are shown in Table 8.
  • Experiment 9D describes bioUistics assays to determine activity of the 2 modified peptides compared with the unmodified peptides in pea pod tissue. Reconstitution of ribonuclease activity in pea pod tissue was assayed as described in example 8, by the bioUistics delivery of the 2 partial genes together in comparison with a luciferase with filler DNA only control. This was done in a single experiment, with tissue from the inner surface of immature pea pods as the target for DNA delivery. The data from a typical experiment are shown in Table 9. When the modified barnase fragments are expressed in the same cell, the luciferase activity is reduced to 2% of the Luc controls. Table 9. Luciferase activity recorded as cpm per pea pod disk and as the average for 12 disks per treatment. Data is reported as (measured cpm minus background) x .001.
  • Example 9E describes bioUistics assays to determine activity of the two modified peptides compared with the unmodified peptides in pea seed coat tissue.
  • Example 10 describes that when the two partial genes are introduced into tobacco cells by Agrobacterium, cell lethality results.
  • Plasmids carrying the bn5 and bn3 chimeric genes are digested to liberate the chimeric genes themselves.
  • the chimeric genes are each inserted at an appropriate restriction site in a T-DNA vector.
  • a control lethality gene which has a barnase coding sequence disrupted by an intron to prevent bacterial expression of barnase activity.
  • the st-ls ivs2 intron used by Vancanneyt et al. , Mol Gen. Genet. 220:245-50 (1990) is inserted into the valine codon at position 36 of the mature barnase protein (position 37 of the barnase gene used here, due to the addition of a methionine residue prior to the alanine residue of the mature barnase protein).
  • a chimeric gene is constructed using the barnase coding sequence with the intron operably linked to the CaMV 35S promoter and to the octopine synthase 3' untranslated region at the 3' end.
  • An appropriate T-DNA vector is prepared from this construct.
  • Young tobacco leaf plants are used for Agrobacterium-mediated DNA delivery, according to standard techniques, of 1) 35S-bn5 by itself, 2) 35S-bn3 by itself, 3) 35S-bn5/35S-bn3 together, or 4) 35S-barnaseINT.
  • An Agrobacterium colony containing the appropriate plasmid, each from a fresh plate is used to inoculate 5 ml of LB containing 10 ⁇ g per ml tetracycline.
  • Tobacco leaves 7 to 10 cm in length are nicked only on the underside and Agrobacterium bacteria is infiltrated into the leaf by filling a 1 ml syringe with Agrobacterium and placing the syringe tip (with no needle) tight against the nick. Pressure is applied to the syringe plunger and the sector in the leaf between the mid-rib and two lateral veins is infused with the Agrobacterium solution.
  • One can monitor the introduction of the Agrobacterium solution by the change in color of the leaf from green to dark green when the solution is introduced. After approximately 5 days of incubation at room temperature, cell death can be observed by chlorosis and browning followed by complete tissue collapse.
  • bn5 nor bn3 chimeric genes individually give rise to cell death.
  • bn5 and bn3 chimeric genes are introduced together or when intact barnaseINT is introduced, cell death is observed. Precise timing of cell death can vary.
  • Example 11 describes use of a repressor/activator fusion protein to induce expression of barnase in tapetal cells.
  • the method is diagramed in Figure 1.
  • Subline A has a dominant male sterile gene (Ms) with an artificial promoter consisting of one or more tet operators and a TATA -box. In this condition, the male sterile gene is not transcribed and subline A ] is male fertile.
  • Subline A 2 has a tapetal-specific promoter driving the expression of a chimeric transcriptional activator.
  • This transcriptional activator is made by fusing the tet repressor, which recognizes the tet operator, to a eukaryotic activation domain, the virion protein 16 (VP16) activation domain from Herpes simplex virus.
  • This tet repressor/VP16 activator fusion (which is abbreviated in the diagram as "Act") has been shown by Weinmann et al. (1994), supra, to activate transcription in plants from a minimal promoter plus 7 tet operators. Sublines A ! and A 2 are crossed to produce line A, which is male sterile since it contains both the transcriptional activator (Act), which is expressed specifically in the tapetum, and the tet operator-Ms gene.
  • the transcriptional activator binds to the tet operator inducing expression of the male sterile gene specifically in the tapetum.
  • Tapetal-specific expression of Ms genes such as ribonucleases (e.g. , Barnase), has been shown to produce male sterility.
  • line A which is male sterile, can then be crossed to any line B, to produce hybrid seeds. In many crops it is advantageous for the hybrids produced to be male fertile.
  • Figure 1 shows the use of the cre/lox system to create two alternative alleles at one locus, one allele (A ( ) consisting of the tet operator-Ms gene and the other allele (A 2 ) consisting of the tet repressor/VP16 activator driven off a tapetal-specific promoter.
  • the initial transformant has a tet operator-Ms gene inserted in opposite orientation between a tapetal-specific promoter and the tet repressor/VP16 activator. Lox sites are placed in the same orientation on both sides of the tet operator-Ms insert.
  • line A ⁇ The tet repressor/activator in this line is silent since an insert is present in between the tapetal-specific promoter and the tet repressor/activator.
  • the Ms gene is also silent, but is activated upon crossing to a line carrying a tet repressor/activator.
  • Line A 2 is created by crossing line Aj to a line carrying ere recombinase (from bacteriophage PI). Cre recombinase excises the tet operator-Ms gene, allowing the tet repressor/activator to be expressed in the tapetum. When made homozygous, this is used as line A 2 .
  • Example 12 describes use of the AVR9 elicitor polypeptide from Cladosporium fulvum and the corresponding resistance gene, Cf9 from Lycopersicon esculentum to specifically kill tapetal cells.
  • a method to produce hybrid seed using this embodiment is diagramed in Figure 2.
  • the Maintainer Field sub-lines A ⁇ and A 2 which are male fertile, are crossed to yield line A which is male sterile.
  • the Cladosporium fulvum Avr9 avirulence gene is expressed off of a tapetal-specific promoter (pl27a, described in U.S. Patent No. 5,254,801).
  • the AVR9 polypeptide is fused to a signal peptide from the tobacco Prla protein, as described in Hammond-Kosack et al (1994).
  • the tomato Cf9 gene (the corresponding tomato resistance gene) is expressed off of a second tapetal-specific promoter ("TA29" in this illustration).
  • Both sublines A and A 2 are male fertile since the AVR9 and CF9 polypeptides, when expressed separately, do not confer cell-death. However, when sublines A, and A 2 are crossed together to produce line A, a hypersensitive response (HR) is initiated in the tapetum resulting in cell death. Hammond-Kosack et al. (1994) and Jones at al. (1994) have shown that cells expressing both AVR9 and CF9 become necrotic and that Cf9 expression is cell-autonomous. Tapetal cell-death should therefore confer male-sterility without adversely affecting other organs.
  • HR hypersensitive response
  • line A which is male sterile
  • line B can then be crossed to any line B, to produce hybrid seeds.
  • the cre/lox system is used to create two alternative alleles at one locus, one allele (A,) carrying the pl27a-Avr9 gene and the other allele (A 2 ) carrying the TA29-Cf9 gene.
  • the initial transformant has a pl27a-Avr9 gene inserted in opposite orientation between a tapetal-specific promoter and Cf9.
  • Lox sites are placed in the same orientation on both sides of the pl27a-Avr9 insert. When made homozygous, this is used as line A, .
  • the TA29-Cf9 gene in this line is silent since an insert is present between the tapetal-specific promoter and the Cf9 gene. Only the pl27a-Avr9 gene will be expressed in this line.
  • Line A 2 is created by crossing line A, to a line carrying cre recombinase (from bacteriophage PI). Cre recombinase would excise the pl27a-Avr9 gene, allowing the TA29-Cf9 gene to be expressed. When made homozygous, this is used as line A 2 .
  • Example 13 describes use of the cre-lox system to insert two functional expression cassettes into a lox site previously introduced into a plant genome.
  • the method uses a combination of mutant lox sites as described by Albert et al. Plant J. 7:649-659 (1995) to increase efficiency of the insertion event as compared to excision.
  • a mutant lox site (lox 66 , as described by Albert et al.) is introduced into a desired plant genome using a recombinant expression cassette having a CaMV35S promoter linked to a structural gene encoding cre.
  • the lox 66 site is placed between the structural gene and the promoter.
  • Protoplasts from this plant are then transformed with a plasmid carrying a second mutant site (lox 71 ) linked to a selectable marker such as hygromycin phosphotransferase (hpt) plus a first functional expression cassette (e.g. , AVR9 under control of a tapetal-specific promoter) .
  • a selectable marker such as hygromycin phosphotransferase (hpt) plus a first functional expression cassette (e.g. , AVR9 under control of a tapetal-specific promoter) .
  • Insertion of the hpt at the lox site yields a 35S-lox wt -/zpt, which provides a selectable marker (hygromycin resistance).
  • the wild-type lox site is reconstructed from the cross-over event between the two mutant sites.
  • a second double mutant lox site is created, which prevents cross-over events at the insertion sites, thereby preventing excision of the inserted fragment.
  • insertion renders the 35S-cre expression cassette non- functional, thereby preventing continued expression of the cre recombinase, which interferes with stable integration.
  • the above process is repeated using the original plant line and a second plasmid carrying a second functional expression cassette (e.g. , CF9 under control of a tapetal-specific promoter).
  • a second functional expression cassette e.g. , CF9 under control of a tapetal-specific promoter.
  • An alternate means for using insertion to create alternate alleles is to transform one plant with the 35S-lox 66 -cre construct described above and a second plant with a construct comprising the following elements: lox 71 -/y?t-AVR9 under control of a tapetal-specific promoter-lox 71 .
  • the hpt- AVR9 sequence will be excised from its site of insertion and will integrate at the lox 66 site between the 35S promoter and the cre structural gene. Selection for hygromycin resistance allows selection of transformed cells.

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Abstract

La présente invention concerne des procédés permettant d'inhiber la croissance d'une cellule ou de tuer une cellule dans un organisme, en particulier chez les plantes. L'invention se rapporte également à des cellules génétiquement modifiées permettant de tuer des cellules spécifiques ou d'exercer un effet bénéfique sur des cellules spécifiques.
PCT/US1998/001315 1997-01-24 1998-01-23 Procedes et compositions a deux composants induisant la letalite cellulaire vegetale WO1998032325A1 (fr)

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AU62482/98A AU744675B2 (en) 1997-01-24 1998-01-23 Two component plant cell lethality methods and compositions
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US6791011B1 (en) 1997-02-21 2004-09-14 Gene Shears Pty. Limited Protein complementation in transgenic plants
AU784973B2 (en) * 1999-08-31 2006-08-10 Fertiseeds Ltd. Exogenic allelism
US20070107079A1 (en) * 2000-10-14 2007-05-10 Thomas Christopher J R Plant cell death system
EP2455476A1 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
DE102012003848A1 (de) * 2012-02-29 2013-08-29 Kws Saat Ag Pathogenresistente transgene Pflanze
US8742207B2 (en) 2007-02-01 2014-06-03 Enza Zaden Beheer B.V. Disease resistant plants
EP2781151A1 (fr) 2013-03-18 2014-09-24 Bayer CropScience AG Procédés de séparation de graines hybrides à partir d'un mélange d'agents de contrôle biologique de graines
US9970925B2 (en) 2010-09-30 2018-05-15 Rigel Pharmaceuticals, Inc. In vivo reporter system
WO2019042935A1 (fr) 2017-08-29 2019-03-07 Scienza Biotechnologies 3 B.V. Plants de soja résistant à phytophthora sojae
US10501754B2 (en) 2007-02-01 2019-12-10 Enza Zaden Beheer B.V. Disease resistant potato plants
US10597675B2 (en) 2013-07-22 2020-03-24 Scienza Biotechnologies 5 B.V. Downy mildew resistance providing genes in sunflower
US10787673B2 (en) 2007-02-01 2020-09-29 Enza Zaden Beheer B.V. Disease resistant Brassica plants
US11299746B2 (en) 2014-06-18 2022-04-12 Enza Zaden Beheer B.V. Disease resistant pepper plants
US11685926B2 (en) 2007-02-01 2023-06-27 Enza Zaden Beheer B.V. Disease resistant onion plants

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US6791011B1 (en) 1997-02-21 2004-09-14 Gene Shears Pty. Limited Protein complementation in transgenic plants
AU784973B2 (en) * 1999-08-31 2006-08-10 Fertiseeds Ltd. Exogenic allelism
AU784973C (en) * 1999-08-31 2007-05-03 Fertiseeds Ltd. Exogenic allelism
US20070107079A1 (en) * 2000-10-14 2007-05-10 Thomas Christopher J R Plant cell death system
US7282624B2 (en) 2000-10-14 2007-10-16 Advanced Technologies (Cambridge) Limited Plant cell death system
US9994861B2 (en) 2007-02-01 2018-06-12 Enza Zaden Beheer B.V. Disease resistant grape plants
EP2455476A1 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455480A2 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455475A1 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455483A2 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455479A2 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455474A1 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455481A2 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455477A2 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455473A1 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
EP2455482A2 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
US11685926B2 (en) 2007-02-01 2023-06-27 Enza Zaden Beheer B.V. Disease resistant onion plants
US8742207B2 (en) 2007-02-01 2014-06-03 Enza Zaden Beheer B.V. Disease resistant plants
US10787673B2 (en) 2007-02-01 2020-09-29 Enza Zaden Beheer B.V. Disease resistant Brassica plants
US10501754B2 (en) 2007-02-01 2019-12-10 Enza Zaden Beheer B.V. Disease resistant potato plants
US9121029B2 (en) 2007-02-01 2015-09-01 Enza Zaden Beheer B.V. Disease resistant plants
US9546373B2 (en) 2007-02-01 2017-01-17 Enza Zaden Beheer B.V. Disease resistant plants
US9932600B2 (en) 2007-02-01 2018-04-03 Enza Zaden Beheer B.V. Disease resistant tomato plants
EP2455478A2 (fr) 2007-02-01 2012-05-23 Enza Zaden Beheer B.V. Plantes résistantes aux maladies
US9970925B2 (en) 2010-09-30 2018-05-15 Rigel Pharmaceuticals, Inc. In vivo reporter system
EP2622102B1 (fr) * 2010-09-30 2019-03-27 Rigel Pharmaceuticals, Inc. Système rapporteur in vivo
EP2820137A1 (fr) * 2012-02-29 2015-01-07 KWS Saat AG Plante transgénique résistante aux pathogènes
DE102012003848A1 (de) * 2012-02-29 2013-08-29 Kws Saat Ag Pathogenresistente transgene Pflanze
EP2781151A1 (fr) 2013-03-18 2014-09-24 Bayer CropScience AG Procédés de séparation de graines hybrides à partir d'un mélange d'agents de contrôle biologique de graines
US10597675B2 (en) 2013-07-22 2020-03-24 Scienza Biotechnologies 5 B.V. Downy mildew resistance providing genes in sunflower
US11299746B2 (en) 2014-06-18 2022-04-12 Enza Zaden Beheer B.V. Disease resistant pepper plants
WO2019042935A1 (fr) 2017-08-29 2019-03-07 Scienza Biotechnologies 3 B.V. Plants de soja résistant à phytophthora sojae

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