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WO2002033051A1 - Gene autophage de plante - Google Patents

Gene autophage de plante Download PDF

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Publication number
WO2002033051A1
WO2002033051A1 PCT/US2001/031489 US0131489W WO0233051A1 WO 2002033051 A1 WO2002033051 A1 WO 2002033051A1 US 0131489 W US0131489 W US 0131489W WO 0233051 A1 WO0233051 A1 WO 0233051A1
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WIPO (PCT)
Prior art keywords
sequence
plant
seq
aut1
dna
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PCT/US2001/031489
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English (en)
Inventor
C. Jaycn Baker
Elizabeth Orlandi
Kenneth Dealh
Frank J. Turano
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The United States Of America, As Represented By The Secretary Of Agriculture
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Application filed by The United States Of America, As Represented By The Secretary Of Agriculture filed Critical The United States Of America, As Represented By The Secretary Of Agriculture
Priority to AU2002211540A priority Critical patent/AU2002211540A1/en
Publication of WO2002033051A1 publication Critical patent/WO2002033051A1/fr

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to autophagy in plants.
  • it provides a nucleic acid molecule which encodes an autophagy 1 (AUT 1 ) protein in plants. It further provides methods for using the nucleic acid molecules and proteins of the invention.
  • Autophagy is a process by which eukaryotic cells recycle parts of their own cytoplasmic components; degrading macromolecules or structures to allow synthesis of new components. A balance between the breakdown and synthesis must be reached to avoid cellular chaos during times of normal metabolism, however, this balance can be rapidly shifted in times of stress. In the animal kingdom, nutrient deprivation will trigger autophagy turning on bulk degradation of cellular proteins (Mortimore et al. 1996. Autophagy. In Subcellular Biochemistry, Vol. 27: Biology of the Lysosome, Lloyd et al. (Eds.). Plenum Press, New York, NY).
  • Autophagy has been implicated in several important cellular processes such as embryogenesis, disease development, apoptosis, and senescence. The process is crucial for survival during starvation and/or upon exposure to biotic or abiotic stressors. In plants, autophagy may contribute to many aspects of cellular, tissue and organ growth and differentiation. Autophagy could be integral to apoptotic events including: the differentiation of xylem elements, autolysis of leaf petal cells after fertilization, autolysis of embryo sac cells during embryo formation, senescence of leaves, and hypersensitive cell death which protect plants from invading pathogens (Nooden, L.D. 1988. The Phenomena of Senescence and Aging.
  • autophagy appears to play a role in many aspects of plant development, including flower longevity, nutrient status, plant disease resistance, and the increased cellular breakdown involved in senescence and in ripening.
  • Autophagy has been well studied in yeast and mammalian cells and has been recently described in plant cells undergoing nutrient deprivation; however, the previous lack of relevant genes in plants has led to slow progress in elucidating the control and involvement of autophagy in plant physiology.
  • this invention provides for a strategy for manipulating a gene involved in autophagy and thus is an invaluable tool for further research of cellular stress or developmental processes.
  • manipulation of the gene of the invention can provide quantitative information on the role of autophagic processes on metabolic fluxes, nutrient utilization and storage, cellular differentiation, growth, and senescence.
  • Such manipulation also provides a method for increasing crop productivity through enhancing crop resistance to stress.
  • Crop quality and yield is improved by increasing tolerance to a variety of environmental stresses, including disease. Both stress and disease cause a decrease in photosynthetic efficiency of crop plants resulting in less carbohydrate available for cells.
  • This deprivation triggers the autophagic degradation of cytosolic and membrane-bound proteins and lipids leading to cellular breakdown. Gaining control of this process through the use of gene promoters or overexpression, activation-tagging (Weigel etal. 2000. Plant Physiol. 122: 1003-1014), co-suppression (Elmayan etal. 1998.
  • nucleic acid molecule which encodes a protein involved in autophagy in plants, wherein the protein is AUT1 or an AUT1-like protein.
  • the preferred nucleic acid molecule encodes the plant autophagy protein (AUT1).
  • an antisense nucleic acid molecule having a nucleotide sequence that is complementary to the nucleotide sequences which encode a plant autophagy protein or to portions thereof having autophagy activity.
  • the antisense nucleic acid molecule includes a nucleotide sequence complementary to an RNA sequence, preferably a mRNA sequence, transcribed from the sequences described herein.
  • a plant transformed with a gene that encodes the plant autophagy 1 protein, AUT1 is particularly provided.
  • the invention further includes transgenic plant parts, including seeds, as well as tissue culture or protoplasts produced with nucleic acids or vectors of the invention.
  • a plant in which the AUT1 gene is repressed or overexpressed or the AUT1 protein is not active are particularly provided.
  • a method includes introducing into a host cell a nucleotide sequence which encodes a plant AUT1 peptide described herein and culturing under conditions to achieve expression of the AUT1 peptide.
  • Another object of the invention is to provide methods of transforming a plant with the nucleic acid molecules of the invention.
  • a method includes introducing into a plant cell a nucleic acid molecule encoding a plant protein described herein.
  • the method includes introducing into a plant cell an antisense nucleic acid molecule having a nucleotide sequence that is complementary to the nucleotide sequences which encode a plant autophagy protein or to portions thereof having autophagy activity.
  • the antisense nucleic acid molecule includes a nucleotide sequence complementary to an RNA sequence, preferably a mRNA sequence, transcribed from the sequences described herein. The antisense nucleotide sequence hybridizes to nucleic acid, including either the template strand or the RNA transcript, of the plant to reduce formation of a plant protein described herein.
  • Another object of the invention is to provide methods of regulating plant development by reduction of the formation of a plant protein or of a RNA transcript using one or more of the following technologies 1) co-suppression, utilizing sense DNA or RNA constructs, 2) DNA-insertional mutagenesis or "knockouts", or 3) chemical mutagenesis, to alter the resulting protein.
  • Plant transformation vectors comprising the gene which encodes the autophagy protein of the invention are also provided, as are plant cells transformed by these vectors, and plants and their progeny containing the genes. It is an object of the invention to provide plasmids containing DNA sequences, which when inserted into the genome of a plant bring about changes in its carbohydrate concentration and in the carbohydrate composition in plants regenerated from this material.
  • a further object is to provide plant cells and plants that contain these DNA sequences located in the plasmids.
  • a host cell containing the DNA of the invention, ⁇ wherein said host cell is a bacterial cell, in particular, an Agrobacterium tumefaciens or E. co/ cell, or eukaryotic cells such as yeast or fungi, mammalian or other vertebrate and/or insect, or other invertebrate cell lines.
  • FIGURES Figure 1 depicts the alignment of the deduced amino acid sequence of the yeast (SEQ ID NO:3) and potato (SEQ ID NO:2) AUT1 gene products or AUT1 peptides.
  • Figure 2 depicts the predicted transmembrane domains for the potato AUT ⁇ gene product.
  • the X axis reflects amino acid position and the negative Y axis, hydrophobicity.
  • This invention provides an isolated nucleic acid molecule that encodes a plant AUT1 or a plant AUT1-like protein encoded by said nucleic acid molecule.
  • An isolated nucleic acid molecule (AUT1) that encodes for a plant protein that is required for autophagy, which has been designated the plant autophagy 1 (AUT1) protein encoded by said nucleic acid molecule is particularly provided.
  • the invention further provides a transgenic plant transformed with nucleic acid molecules of the invention.
  • the invention provides a method of making a recombinant transgenic plant comprising transforming plant cells with a nucleic acid encoding a plant autophagy gene, AUT ⁇ .
  • the invention particularly provides transforming plants with constructs comprising a promoter that allows for overexpression of the A (/PI gene.
  • An advantage of plants thus transformed is that such plants can be programmed for localized cell death in the face of pathogen spread for example.
  • the invention further provides that said nucleic acid can be used in plants to repress autophagy, e.g., to maintain nutritive or nutrient value in time of stress.
  • the nucleic acid encoding plant AUT1 or AUT1-like protein which is used to make the recombinant transgenic plant is selected from the group consisting of: (a) an isolated DNA encoding an AUT1 protein; (b) an isolated DNA which hybridizes to isolated DNA of (a) above and which encodes an AUT1- like protein or a peptide having AUT1 , AUT1-like, or autophagy biological activity; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encodes a plant AUT1 or AUT1-like protein or a peptide having plant AUT1 biological activity.
  • DNA which hybridizes to isolated DNA refers to DNA sequences that can be identified in a Southern hybridization experiment under moderately stringent conditions as is known in the art. See, for example, Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • an isolated nucleic acid molecule has at least about 50% identity, preferably at least about 60% identity, further preferably at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity to a nucleic acid sequence set forth in SEQ ID NO:1.
  • the nucleic acid encoding a plant autophagy protein is the nucleic acid molecule having the sequence identified by SEQ ID NO:1 or a portion thereof, wherein the portion encodes a polypeptide having plant AUT1 biological activity.
  • said DNA is a recombinant chimeric gene construct comprising a promoter operable in a plant cell and a DNA encoding the plant AUT1 or AUT1-like protein, described above.
  • the chimeric gene construct additionally encodes at least one selectable marker and/or further comprises a heterologous coding sequence, wherein the heterologous coding sequence is an isolated DNA encoding a polypeptide sequence having a property which is advantageous to the plant and which is different from the autophagy protein.
  • Genes encoding polypeptides having properties advantageous to the plant and anti- phytopathogenic polypeptides are well known in the art. Examples include genes which encode proteins which protect plants against pathogens, herbicides, fungicides, insecticides, or disadvantageous environmental influences, wherein the disadvantageous environmental influences comprise heat, cold, wind, unfavorable soil conditions, moisture and dryness.
  • said recombinant chimeric gene or fusion gene construct further comprises DNA encoding a 5' untranslated region containing a translational enhancer and DNA encoding a 3' untranslated region containing a functional polyadenylation signal or parts of these regulatory elements.
  • the DNA sequence encoding the protein or peptide having autophagy activity or the DNA sequence comprising the heterologous coding sequence is derived from a plant gene or a microorganism gene or is a synthetic gene.
  • the DNA is contained in a vector under the control of a promoter allowing its expression in said transgenic plant.
  • Further embodiments of the invention include plant cells transformed by these vectors, plant parts, and plants and their progeny containing the chimeric or fusion genes.
  • said vector is one that can be expressed in bacteria (i.e., pBluescript), plant (pB101), fungal, vertebrate, or invertebrate cells.
  • a host cell containing the DNA of the invention is a bacterial cell, in particular, an Agrobacterium tumefaciens or E. co// ' cell, or eukaryotic cells such as yeast or fungi, mammalian or other vertebrate and/or insect, or other invertebrate cell lines.
  • the protein encoded by said DNA sequence is a plant AUT1 or AUT1-like protein.
  • said plant AUT1 protein is identified by SEQ ID NO:2.
  • isolated nucleic acid is intended to refer to nucleic acid which is not in its native environment.
  • the nucleic acid is separated from other contaminants that naturally accompany it, such as proteins, lipids, and other nucleic acid sequences.
  • the term includes nucleic acid which has been removed or purified from its naturally occurring environment or clone library, and further includes recombinant or cloned nucleic acid isolates and chemically synthesized nucleic acid.
  • nucleotide sequence is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, including deoxyribonucleic acid and ribonucleic acid, and derivatives thereof.
  • recombinant refers to a nucleic acid molecule which has been obtained by manipulation of genetic material using restriction enzymes, ligases, topoisomerases, DNA or RNA polymerases, thermostable DNA or RNA polymerases (i.e., Tag or Tag-like activity) and/or similar recombinant techniques as described by, for example, Sambrook et al., supra.
  • Recombinant does not refer to naturally occurring genetic recombinations.
  • the term “express”, “expressed”, or “expression” is defined to mean transcription alone.
  • encoding and “coding” refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional polypeptide, such as, for example, an active enzyme or other protein that has a specific function.
  • a DNA sequence in the normal (non-inverted) coding orientation in a plasmid is arranged such that the 3' end of the DNA sequence, as defined by the open reading frame, is linked to the 5' end of the plasmid, and the 5' end of the DNA is linked to the 3' end of the plasmid. The DNA may then be read correctly, transcribed, and translated.
  • inverted orientation is used herein to denote that a DNA sequence in a plasmid is arranged such that the 3' end of the DNA sequence, as defined by the open reading frame, is linked to the 3' end of the plasmid, and the 5' end of the DNA is linked to the 5' end of the plasmid.
  • inverted construction is transcribed, for example in a host plant, the resulting mRNA is "anti-sense" with respect to the normal "sense" mRNA.
  • a DNA sequence in inverted orientation may be regarded as equivalent to the non-coding strand of that DNA sequence.
  • AUT1 may have an amino acid sequence that is different by one or more amino acid "substitutions”.
  • the variant may have "conservative substitutions", wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product.
  • a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
  • Similar minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.
  • biological activity refers to AUT1 having structural, regulatory or biochemical functions of the naturally occurring AUT1 gene orAUTI peptide. Whether a given substitution will affect the functionality of the protein can be determined without undue experimentation using synthetic techniques and screening assays known in the art.
  • nucleic acid derivative refers to the chemical modification of a nucleic acid sequence encoding AUT1 or the encoded AUT1 wherein the subject nucleic acid or polypeptide has one or more residues chemically derivatized by reaction of a functional side group. Examples of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group; however, replacements are not limited to these groups.
  • a nucleic acid derivative would encode a polypeptide which retains essential biological characteristics of natural AUT1. Also included are those peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids, e.g., 5-hydroxylysine orornithine may be substituted for lysine.
  • nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the encoded polypeptide molecule would also not generally be expected to alter the activity of the polypeptide. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide sequence encoding the proteins described herein.
  • gene product is intended to refer to a natural or synthetic linear and sequential array of amino acids, i.e., peptide or protein, either full-length or fragmented portions.
  • peptide refers to a molecular chain of amino acids with a biological activity and does not refer to a specific length of the product.
  • proteins, oligopeptides, polypeptides and fusion proteins as well as fusion peptides are included.
  • chimeric or “fusion” refers to two or more DNA molecules which are derived from different sources, strains, or species, which do not recombine under natural conditions, or to two or more DNA molecules from the same species, which are linked in a manner that does not occur in the native genome.
  • present constructs and vectors permit the augmentation of plant genomes with a limited number of preselected genes.
  • the regulatory elements are operably linked to the coding sequence of the autophagy gene such that the regulatory element is capable of controlling expression of the autophagy gene.
  • Heterologous coding sequence refers to coding sequences which encode peptides or proteins, unrelated to, or, other than, the autophagy protein provided above and which are not intrinsically found in the position provided in the chimeric or fusion gene construct, i.e., DNA coding sequences which are derived from different sources, strains, or species, which do not recombine under natural conditions, or to two or more DNA coding sequences from the same species, which are linked in a manner that does not occur in the native genome.
  • Genes encoding an autophagy protein e.g., plant AUT1 or AUT1-like protein can be cloned using a variety of techniques according to the invention.
  • the simplest procedure for the cloning ofAUTI genes requires the cloning of genomic DNA or synthesized cDNA from an organism identified as producing AUT1 protein, and the transfer of the cloned DNA on a suitable plasmid or vector to a host organism which does not produce AUT1 protein, followed by the identification of transformed host colonies to which the ability to produce AUT1 protein has been conferred.
  • the transforming autophagy- conferring DNA can be cleaved into smaller fragments and the smallest which maintains the autophagy-conferring ability can be further characterized.
  • AUT1 or AUT1-like genes which are required for the synthesis of AUT1 or AUT1-like proteins and which are similar to known compounds may be identified or cloned by virtue of their sequence identity or similarity to the biosynthetic genes of the known compounds.
  • Techniques suitable for cloning by homology include standard library screening by DNA hybridization.
  • the term "substantial similarity" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence has a sequence sufficiently similar to a reference nucleotide sequence that it will hybridize therewith under moderately stringent conditions. This method of determining similarity is well known in the art to which the invention pertains. Briefly, moderately stringent conditions are defined in Sambrook etal.
  • 5X SSC sodium chloride/sodium citrate solution
  • SDS sodium dodecyl sulfate
  • EDTA 1.0 mM ethylene diamineteraacetic acid
  • nucleotide sequences having selected percent identities to specified regions of the nucleotide sequence set forth in SEQ ID NO:1 are provided.
  • nucleotide sequences are provided that have at least about 50% identity, preferably at least about 60% identity, more preferably at least about 70% identity, and most preferably at least about 80 or 90% identity, to a nucleotide sequence of substantial length within the nucleotide sequence set forth in SEQ ID NO:1.
  • such length may be no more than about 25, 50, 100, 200, 300, 800, 900, or 1317 nucleotides or may be the entire sequence.
  • nucleotide sequence set forth in SEQ ID NO:1 or portions thereof encode a protein that functions as described herein, i.e., one which regulates autophagy in plant cells.
  • Preferred nucleic acid sequences include the sequence spanning nucleotides 1 to 1317, 180 to 1050, and 240 to 960 of SEQ ID NO:1.
  • the percent identity may be determined, for example, by comparing sequence information using the MacVector computer program, version 6.0.1 (Oxford Molecular Group, Inc., Beaverton, OR). Briefly, the MacVector program defines identity as the number of identical aligned symbols, i.e., nucleotides or amino acids, divided by the total number of symbols in the shorter of the two sequences.
  • nucleotide sequences encoding the proteins described herein are provided.
  • a nucleotide sequence is provided that encodes a protein having an amino acid sequence having at least about 60% identity, preferably at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to the amino acid sequence set forth in SEQ ID NO-2.
  • a suitable DNA sequence may be obtained by cloning techniques using cDNA or genomic libraries of potato (Solanum tuberosum) or other plant species such as Arabidopsis thaliana, maize (lea maize), etc. which are available commercially or which may be constructed using standard methods known in the art.
  • Suitable nucleotide sequences may be isolated from DNA obtained from a wide variety of species by means of nucleic acid hybridization or polymerase chain reaction (PCR) procedures, using as probes or primers nucleotide sequences selected in accordance with the invention, such as those set forth in SEQ ID NO:1 , nucleotide sequences having substantial similarity thereto, or portions thereof or complements thereof.
  • the nucleotide sequences provided herein are cDNA sequences.
  • nucleic acid sequences encoding a plant protein described herein may be constructed by recombinant DNA technology, for example, by cutting and splicing nucleic acids using restriction enzymes and DNA ligase or topoisomerases.
  • nucleic acid sequences may be constructed using chemical synthesis, such as solid- phase phosphoramidate technology, or PCR. PCR may also be used to increase the quantity of nucleic acid produced.
  • the particular nucleic acid sequence is of a length which makes chemical synthesis of the entire length impractical, the sequence may be broken up into smaller segments which may be synthesized and ligated together to form the entire desired sequence by methods known in the art.
  • the DNA sequences of the invention can be used to prepare recombinant DNA molecules by cloning in any suitable vector.
  • a variety of vector-host combinations may be employed in practicing the present invention.
  • Host cells may be either prokaryotic or eukaryotic, and, when the host cells are bacterial cells, they may be either gram-negative or gram-positive bacteria.
  • examples of hosts suitable for use herein are prokaryotic and eukaryotic hosts such as E. coli K12 and related bacteria, Saccharomyces cerevisiae, Sf9 or Sf21 insect cells (Spodoptera frugiperda), Chinese hamster ovary cells, and plant cells in culture.
  • other hosts may also be utilized.
  • Vectors used in practicing the present invention are selected to be operable as cloning vectors or expression vectors in the selected host cell. Numerous vectors are known to those of skill in the art, and selection of an appropriate vector and host cell is a matter of choice.
  • the vectors may, for example, be bacteriophage, plasmids, viruses, or hybrids thereof.
  • various plasmid and phage vectors are known that are ideally suited for use in the invention, e.g., ⁇ Zap and pBluescript.
  • the vector may be a T-DNA vector. Representative T-DNA vector systems are discussed in the following publications: An et al. 1986. EMBO J. 4: 277; Herrera-Estrella etal. 1983. EMBO J. 2: 987; Herrera-Estrella et al. In Plant Genetic Engineering. Cambridge University Press, New York, NY, p. 63).
  • the vectors may be non-fusion vectors (i.e., those producing the protein of the invention not fused to any heterologous polypeptide), or alternatively, fusion vector (i.e., those producing the protein fused to a vector encoded polypeptide).
  • the fusion proteins would of course vary with the particular vector chosen.
  • Suitable non-fusion plasmid vectors for use with E. coli include but are not limited to pTrc99 for use with E. coli JM 105, or pANK-12, pANH-1 or pPL2 for use with E. coli MZ 1.
  • suitable fusion plasmid vectors include pGEX and pMC1871 for use with E.
  • Non-E. coli expression systems which may also be employed include pAc360 or pBluescript for use with SP2 or High 5 insect cells, pYesHis with the yeast C. cerevisiae INVSd or INVSc2, pLS405 with Salmonella dublin SL598, and pYUB12 with Mycobacterium smegmatis or M. bovis.
  • Still other suitable vector-host combinations that may be used in practicing the instant invention are described, for example, in U.S. Pat. No. 5,122,471, the contents of which are incorporated by reference herein.
  • the desired recombinant vector may be constructed by ligating DNA linker sequences to the 5' and 3' ends of the desired nucleotide insert, cleaving the insert with a restriction enzyme that specifically recognizes sequences present in the linker sequences and the desired vector, cleaving the vector with the same restriction enzyme, mixing the cleaved vector with the cleaved insert and using DNA ligase to incorporate the insert into the vector as known in the art.
  • the particular site chosen for insertion of the selected DNA fragment into the vector to form a recombinant vector is determined by a variety of factors. These include size and structure of the polypeptide to be expressed, susceptibility of the desired polypeptide to enzymatic degradation by the host cell components and contamination by its proteins, expression characteristics such as the location of start and stop codons, and other factors recognized by those of skill in the art. None of these factors alone absolutely controls the choice of insertion site for a particular polypeptide. Rather, the site chosen reflects a balance of these factors, and not all sites may be equally effective for a given protein.
  • the vectors may include other nucleotide sequences, such as those encoding selectable markers, including those for antibiotic resistance or color selection.
  • the vectors also preferably include a promoter nucleotide sequence.
  • the desired nucleic acid insert is preferably operably linked to the promoter.
  • a nucleic acid is "operably linked" to another nucleic acid sequence, such as a promoter sequence, when it is placed in a specific functional relationship with the other nucleic acid sequence.
  • the functional relationship between a promoter and a desired nucleic acid insert typically involves the nucleic acid and the promoter sequences being contiguous such that transcription of the nucleic acid sequence will be facilitated.
  • Two nucleic acid sequences are further said to be operably linked if the nature of the linkage between the two sequences does not (1) result in the introduction of a frame-shift mutation; (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region.
  • the promoter element is generally upstream (i.e., at the 5' end) of the nucleic acid insert coding sequence.
  • the vector should be selected so as to have a promoter operable in the host cell into which the vector is to be inserted (that is, the promoter should be recognized by the RNA polymerase of the host cell).
  • the vector should have a region which codes for a ribosome binding site positioned between the promoter and the site at which the DNA sequence is inserted so as to be operatively associated with the DNA sequence of the invention once inserted (in correct translational reading frame therewith).
  • the vector should be selected to provide a region which codes for a ribosomal binding site recognized by the ribosomes of the host cell into which the vector is to be inserted.
  • This invention encompasses a hybrid vector, that comprises a vector capable of replication, transcription and expression of DNA segments operably linked thereto; and a DNA segment encoding a polypeptide of this invention comprising the peptide disclosed herein operatively linked thereto, wherein when the vector is placed in an appropriate host it can express the polypeptide encoded by the DNA segment.
  • hybrid vector that comprises a vector capable of replication, transcription and expression of DNA segments operably linked thereto; and a DNA segment encoding a polypeptide of this invention comprising the peptide disclosed herein operatively linked thereto, wherein when the vector is placed in an appropriate host it can express the polypeptide encoded by the DNA segment.
  • Examples of such vectors are pGex (Pharmacia), baculovirus, pET-9d (Novagen) or pRSET T7 (Invitrogen).
  • the vector may be a eukaryotic or a prokaryotic vector depending on the host selected for transfection and in which the gene product is going
  • Still part of this invention is another hybrid vector, that comprises a vector capable of replication, transcription and expression of DNA segments operably coupled thereto; and a DNA segment comprising a DNA fragment encoding at least one of the polypeptides of the invention and a second unrelated DNA segment, both sequences being operably linked to one another and to the vector.
  • the preparation of the hybrid vector described above is known in the art and need not be further described herein (Smith, D. et al. 1988. Gene 67: 31 ; Studier, F. W. et al. 1990. Meth. Enzymol. 185:60-89).
  • DNA segments can be introduced into plasmids and coupled with promoters and terminators such that the coding part when read leads to the formation of an antisense RNA which under-expressesthe AUT1 protein.
  • the coding sequences contain information for the formation of an antisense RNA to the corresponding genes. Whether a translatable mRNA or an antisense nucleic acid is formed depends on the orientation of the coding sequence in relation to the promoter. If the 3' end of the coding sequence is fused to the 3' end of the promoter, an antisense RNA results, while fusion of the 5' end of the coding segment to the 3' end of the promoter produces a translatable RNA. The former leads to a reduction of AUT1 activity in the cell; the latter leads to an increase in biological activity associated with plant AUT1 protein.
  • Still another embodiment of this invention is the method of making a recombinant transgenic plant, said method comprising: providing a plant cell capable of regeneration; transforming said plant cell with a DNA segment encoding a plant AUT1 protein, where said DNA segment is selected from the group consisting of: (a) an isolated DNA encoding a plant AUT1 protein; (b) an isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a plant AUT1-like protein or a peptide having plant AUT1-like biological activity; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encodes a plant AUT1 or AUT1-like protein or a peptide having plant AUT1 biological activity; and then regenerating a recombinant plant from said transformed plant cell.
  • Yet another embodiment of this invention is the method of making a recombinant plant, said method comprising: providing a plant cell capable of regeneration; transforming said plant cell with the chimeric gene construct comprising a promoter operable in said plant cell, and a DNA segment encoding a plant AUT1 protein, where said DNA segment is selected from the group consisting of: (a) an isolated DNA encoding a plant AUT1 protein; (b) an isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a plant AUT1 -like protein or a peptide having plant AUT1 -like biological activity; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encodes a plant AUT1 or AUT1-like protein or a peptide having plant AUT1 biological activity; and then regenerating a recombinant plant from said transformed plant cell.
  • Yet a further embodiment of this invention is the method of making a recombinant plant, said method comprising: providing a plant cell capable of regeneration; transforming said plant cell with the chimeric gene construct comprising a promoter operable in said plant cell, and a DNA segment encoding a plant AUT1 -like protein, where said DNA segment is selected from the group consisting of: (a) an isolated DNA encoding a plant AUT1-like protein; (b) an isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a plant AUT1-like protein or a peptide having plant AUT1 , AUT1-like or autophagy biological activity; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a plant AUT1-like protein or a peptide having plant AUT1 , AUT1-like or autophagy biological activity; and then regenerating a
  • an inventive method includes introducing into a plant cell an antisense nucleotide sequence having a nucleotide sequence which is complementary in sequence, according to the well-known rules for nucleotide base-pairing, to the nucleotide sequences provided herein, such as one that is complementary to a nucleotide sequence having at least about 50% identity, preferably at least about 60% identity, more preferably at least about 70% identity, most preferably at least about 80% or 90% identity to a length of nucleotides within the nucleotide sequence set forth in SEQ ID NO:1 , preferably from, nucleotide residue 1 to nucleotide residue 1317.
  • An antisense nucleic acid molecule is capable of binding or hybridizing to a target nucleic acid molecule, either over a portion or over its whole length.
  • the antisense nucleic acid molecule typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 20 consecutive nucleotides of the sense sequence of SEQ ID NO:1.
  • the antisense molecule is at least about 20 nucleotides in length, and up to and including, the entire coding sequence and/or the entire cDNA sequence of SEQ ID NO:1.
  • the antisense nucleotide sequence may have a length of about 20 to about 100 nucleotides, about 20 to about 400 nucleotides, about 20 to about 800 nucleotides, and about 20 to about 1317 nucleotides. In more preferred embodiments, the antisense nucleotide sequence is the entire length of the nucleotide sequence set forth in SEQ ID NO:1. The antisense nucleotide sequence may hybridize to the template strand, which serves as the strand from which RNA is produced, so that transcription will be reduced.
  • the antisense nucleotide sequence may be complementary to, and therefore hybridize to, the sequences described herein, so that translation of the mRNA sequence to express the encoded protein, AUT1 , will be reduced.
  • the antisense nucleotide sequence may be either DNA or RNA. Additionally, inhibition can be via DNA:DNA:RNA triplex formation, which inhibits transcriptional expression of AUT1 mRNA. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to specifically hybridize.
  • an antisense compound specifically hybridizes when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • Such antisense sequences may be produced as described above for the nucleotide sequences and by further methods known in the art. Oligonucleotides can be chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications, which alter the chemistry of the backbone, sugars or heterocyclic bases, have been described in the literature.
  • One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.
  • the genes for autophagy of this invention can be expressed in transgenic plants thus causing increased biosynthesis, activation and/or overexpression of AUT1 protein in the transgenic plants. In this way transgenic plants which overexpress AUT1 protein are generated.
  • antisense constructs can be expressed resulting in repression of the AUT gene.
  • the autophagy genes and adjacent sequences may require modification and optimization.
  • genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from autophagy genes having codons which are not preferred in plants. It is known in the art and summarized, for example, in U.S. Patent 5,817,502 herein incorporated by reference, that all organisms have specific preferences for codon usage, and the autophagy gene codons can be changed to conform with plant preferences, while maintaining the amino acids encoded. Furthermore, high expression in plants is best achieved from coding sequences which have at least 35% GC content, and preferably more than 45%.
  • Microbial genes which have low GC contents may express poorly in plants due to the existence of ATTTA motifs which may destabilize messages, and AATAAA motifs which may cause inappropriate polyadenylation.
  • the AUT1 genes can be screened for the existence of illegitimate splice sites which may cause message truncation. All changes required to be made within the autophagy protein coding sequence such as those described above can be made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using methods well known in the art.
  • the preferred autophagy genes may be unmodified genes, should these be expressed at high levels in target transgenic plant species, or alternatively may be genes modified by the removal of destabilization and inappropriate polyadenylation motifs and illegitimate splice sites, and further modified by the incorporation of plant preferred codons, and further with a GC content preferred for expression in plants.
  • preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. 1989. Nucl. Acid Res. 17: 477-498).
  • sequences adjacent to the initiating methionine may require modification.
  • the sequences cognate to the selected autophagy genes may initiate translation efficiently in plants, or alternatively may do so inefficiently. In the case that they do so inefficiently, they can be modified by the inclusion of sequences known to be effective in plants.
  • Joshi (1987. Nucl. Acid Res.15: 6643-6653) has suggested an appropriate consensus for plants and Clontech suggests a further consensus translation initiator (1993/1994 catalog, page 210). These consensuses are suitable for use with the autophagy genes of this invention.
  • sequences are incorporated into the autophagy gene construction, up to and including the ATG (whilst leaving the second amino acid of the autophagy gene unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibility of modifying the second amino acid of the transgene).
  • Transgenic plants can be transformed with a DNA segment encoding a plant autophagy protein in the absence of an exogenously provided promoter.
  • chimeric gene constructs comprising a promoter operable in said plant cell and a DNA segment encoding a plant autophagy protein are utilized for the transformation, optimal expression of the plant autophagy protein results.
  • the expression of plant A/JTi gene(s) in transgenic plants is behind a promoter shown to be functional in plants. The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target tissue or species.
  • promoters are known in the art, including cell-specific promoters, inducible promoters, and constitutive promoters. Any promoter that directs transcription in plant cells may be used.
  • the promoters may be of viral, bacterial, or eukaryotic origin, including those from plants and plant viruses.
  • the expression of plant AUT ⁇ genes in transgenic plants can be under the regulation of promoters which are constitutive or promoters which are regulated.
  • Such promoters are well known in the art and described, for example, in U.S. Patent 5,589, 625; examples are: cauliflower mosaic virus 35S-promoter, rice actin promoter, rbc S promoter from different species, Agrobacter TR2' promoter, phaseolin gene promoter or the NOS promoter.
  • the promoter is of viral origin, including the cauliflower mosaic virus 35S-promoter (CaMV), such as CaMV 35S or 19S, a figwort mosaic virus promoter (FMV 35S), or the coat protein promoter of tobacco mosaic virus (TMV).
  • the promoter may further be, for example, a promoter for the small subunit of ribulose-1 ,3-diphosphate carboxylase. Promoters of bacterial origin include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids as discussed in Herrera-Estrell etal. (1983. Nature 303: 209-213).
  • the expression of the plant AUT genes of this invention can also be controlled, i.e., under the regulation of promoters which are regulated.
  • this transformation method can be developed to control disease in particular crops.
  • An advantage of controlled expression of the chimeric gene construct is that plant AUT1 protein is expressed only at the appropriate time and/or to the appropriate extent or level and/or only in particular parts of the plant.
  • a further advantage is that parts of plants that are inaccessible to conventional protective measures, can be protected using this method either through constitutive expression of the nucleic acid in all tissues or through tissue-specific expression of the nucleic acid as controlled by tissue or stage-specific promoters.
  • the promoter may further be one that responds to various forms of environmental stresses or other stimuli.
  • the promoter may be one induced by abiotic stresses such as wounding, cold, dessication, ultraviolet-B (van Der Krol et al. 1999. Plant Physiol. 121 : 1153-1162), heat shock (Shinmyo et al. 1998. Biotechnol. Bioeng. 58: 329-332) or other heat stress, drought stress or water stress.
  • the promoter may further be one induced by biotic stresses including pathogen stress, such as stress induced by a virus (Sohal etal.
  • the promoters may further be selected such that they require activation by elements known in the art, so that production of the protein encoded by the nucleic acid sequence insert may be regulated as desired.
  • Preferred promoters are foreign promoters.
  • a "foreign promoter" is defined herein to mean a promoter other than the native, or natural, promoter which promotes transcription of a length of DNA.
  • tissue specific expression patterns as controlled by tissue or stage-specific promoters include fiber specific, green tissue specific, root specific, stem specific, and flower specific.
  • tissue specific expression patterns include fiber specific, green tissue specific, root specific, stem specific, and flower specific.
  • expression in leaves is preferred;
  • inflorescences e.g. spikes, panicles, cobs etc.
  • protection of plants against root pathogens expression in roots is preferred;
  • seedlings against soil-borne pathogens expression in roots and/or seedlings is preferred.
  • protection against more than one type of phytopathogen will be sought, and thus expression in multiple tissues will be desirable.
  • promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.
  • promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons.
  • a preferred promoter is the maize PEPC promoter from the phosphenol carboxylase gene (Hudspeth et al. 1989. Plant Molec.
  • a preferred promoter for root specific expression is that described by de Framond (1991. FEBS 290: 103-106) or by Hudspeth et al. (1996. Plant Molec. Biol. 31 : 701-705).
  • a preferred stem specific promoter is that described in patent application WO 93/07278 (to Ciba-Geigy) and which drives expression of the maize trpA gene.
  • constructions for plant AUT1 protein expression in plants require an appropriate transcription terminator to be attached downstream of the plant A (/PI gene.
  • Several such terminators are available and known in the art (e.g. tml from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of this invention.
  • the vectors may further include other regulatory elements, such as enhancer sequences, which cooperate with the promoter to achieve transcription of the nucleic acid insert coding sequence.
  • enhancer sequences nucleotide sequence elements which can stimulate promoter activity in a cell, such as a plant host cell. Examples include sequences which have been shown to enhance expression such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV and AMV).
  • the vectors may include 3' regulatory sequence elements known in the art, such as those, for example, that increase the stability of the RNA transcribed.
  • the vectors may include another nucleotide sequence insert that encodes a peptide or polypeptide used as a tag to aid in purification of the desired protein encoded by the desired nucleotide sequence.
  • the additional nucleotide sequence is poistioned in the vector such that a fusion, or chimeric, protein is obtained.
  • a protein described herein may be produced having at its C-terminal end, linker amino acids, as known in the art, joined to the other protein that acts as a tag. After purification procedures known to the skilled artisan, the additional amino acid sequence is cleaved with an appropriate enzyme. The protein may then be isolated from the other proteins, or fragments thereof, by methods known in the art.
  • Plants may be used to express foreign gene products or to overexpress endogenous gene products via introduction of a genetically engineered DNA sequence encoding the foreign or endogenous gene into the genetic material of a suitable plant through the use of various biotechnological methods.
  • Methods of transforming a plant include introducing into a plant cell a nucleic acid molecule having a nucleotide sequence as described herein, such as one, for example, that encodes a protein having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • introduction refers to a method that is capable of introducing the genetically engineered DNA sequence into the genetic material of a plant cell.
  • Methods of transforming a plant are well known in the art. Examples of such biotechnological methods are Agrobacterium-medi ' ate ⁇ transfer, plant virus mediated-transfer, microinjection, microprojectile bombardment, electroporation of various plant parts, organs, tissues or cells, PEG-mediated transformation and transformation of plant protoplasts with virus-based stable vectors, all methods well known and practiced in the art. These techniques and others are found in references such as: Sambrook et al. (supra), Current Protocols in Molecular Biology (1993. Ausubel et al. (Eds.).
  • a host cell that includes the inventive recombinant vectors described above.
  • host cells may be used in the invention, including prokaryotic and eukaryotic host cells.
  • Preferred host cells are eukaryotic and are further preferably plant cells.
  • the transgenic plant is derived from plant cells of monocotyledons, such as duckweed, corn, turf (including rye grass, Bermuda grass, Blue grass, Fescue), dicotyledons, including lettuce, cereals such as wheat, crucifers (such as rapeseed, radishes and cabbage), solanaceae (including green peppers, potatoes, and tomatoes), and legumes such as soybeans and bush beans.
  • a plant AUT1 or AUT1-like protein or peptide having plant AUT1 biological activity is expressed in any plant cell or crop plant especially potato and/or model plant systems such as Arabidopsis thaliana or ice plant (Mesembryanthemum crystallinum).
  • binary vectors or vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be preferred.
  • direct gene transfer transformation with a single DNA species or co-transformation can be used (Schocher efa/. 1986. Biotechnology 4: 1093-1096).
  • transformation is usually (but not necessarily) undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methatrexate) or a herbicide (sulfonylurea, imidazolinone, or basta).
  • the marker should permit the selection of transformed cells rather than cells that do not contain the inserted DNA.
  • the choice of selectable marker is not, however, critical to the invention.
  • a method may include in vivo mutagenesis of the gene present in the plant genome encoding the plant AUT1 protein described herein in order to alter its activity to provide the desired results.
  • a plant may be mutated by methods known to the skilled artisan, including chemical methods and DNA insertion mutagenesis.
  • the invention includes transgenic plants produced with nucleic acids or vectors of the invention.
  • Transgenic plants containing the constructs of this invention can also be regenerated from plant tissues, plant parts, or protoplasts by methods known to those of skill in the art.
  • Plant part is meant to include any portion of a plant capable of producing a regenerated plant.
  • this invention encompasses cells, tissue (especially meristematic and/or embryonic tissue), protoplasts, epicotyls, hypocotyls, cotyledons, cotyledonary nodes, pollen, ovules, stems, roots, leaves, seed, and the like.
  • Plants can also be regenerated from explants. Methods will vary according to the plant species.
  • Seed can be obtained from the regenerated plant or from a cross between the regenerated plant and a suitable plant of the same species.
  • the plant can be vegetatively propagated by culturing plant parts under conditions suitable for the regeneration of such plant parts.
  • descendants, asexual or sexual, of a transgenic plant, which descendants express the nucleic acid of the invention are provided.
  • nucleotide sequences described above, and preferably portions thereof may be used as probes to locate other, similar nucleotide sequences that may encode other AUT1 proteins.
  • General methods for screening for selected nucleotide sequences in a DNA or RNA sample are known to the art. For example, DNA may be isolated from selected plants, treated with various restriction enzymes and analyzed by Southern blotting techniques utilizing a radioactively or fluorescently-labeled probe of interest. RNA fragments may be similarly analyzed by northern blotting techniques. Alternatively, commercially available cDNA or genomic libraries may be screened. Other techniques, such as oligonucleotide ligation assays, in situ hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use.
  • the nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding AUT1 , for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of AUT1.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 20 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NO:1.
  • a probe nucleic acid molecule has a nucleotide sequence having a least about 50% identity to a nucleotide sequence having a length of about 20 to about 100 nucleotides, preferably 20 to about 400, and further preferably about 20 to about 800, and about 20 to about 1317 nucleotides within the nucleotide sequence set forth in SEQ ID NO:1, preferably from nucleotide 1 to nucleotide 1317 in SEQ ID NO:1.
  • the probe has a nucleotide sequence having at least about 60% identity, preferably at least about 70% identity, more preferably at least about 80% identity, most preferably at least about 90% identity, to the length of nucleotides indicated directly above.
  • the probe may be radioactively labeled at its 5' end, for example, with polynucleotide kinase and 32 P and hybridized to the islolated nucleic acid fragments.
  • Hybridization utilizing a nucleotide sequence of the invention requires that hybridization be performed under relatively stringent conditions such that non-specific hybridization is minimized.
  • Appropriate hybridization conditions can be determined empirically, or can be estimated based, for example, on the relative G+C content of the probe and the number of mismatches between the probe and target sequence, if known.
  • Hybridization conditions can be adjusted as desired by varying, for example, the temperature of hybridizing or the salt concentration (Sambrook, supra, 1989).
  • a nucleotide sequence that hybridizes under relatively stringent conditions to a nucleic acid molecule is a single-stranded nucleic acid sequence that can range in size from about 10 nucleotides to the full-length of a gene or a cDNA.
  • Such a nucleotide sequence can be chemically synthesized, using routine methods or can be purchased from a commercial source.
  • such nucleotide sequences can be obtained by enzymatic methods such as random priming methods, the polymerase chain reaction (PCR) or by standard restriction endonuclease digestion, followed by denaturation.
  • Hybridization and wash conditions are well known and exemplified in Sambrook, etal., 1989, particularly Chapter 11 therein, the disclosure of which is hereby incorporated in its entirety by reference.
  • An example of stringent hybridization conditions is washing the filters in 0.1. X SSC and 0.5% SDS and incubating at 68°C for 2 hr with gentle agitation. Change the buffer and continue incubating for a further 30 min. (Sambrook et al. 1982. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, page 388).
  • the invention comprises a method for specifically identifying the plant AUT1 protein which comprises amplifying a subject mRNA by the RT-PCR method with the use of such DNA primers and thus assaying the expression of the plant A/JP1 gene.
  • the manner in which one probes cells for the presence of particular nucleotide sequences, as genomic DNA or RNA, is well established in the literature and does not require elaboration here.
  • a biological specimen is used as a source of mRNA.
  • the mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences.
  • a method includes providing a plant having an introduced nucleic acid molecule described herein, such as one having at least about 60% identity to the sequence set forth in SEQ ID NO:1 , that encodes a protein described herein.
  • the introduced nucleic acid molecule may include a promoter, preferably a foreign promoter, operably linked to a terminal 5' end of the nucleotide sequence so that the sequence is expressed, typically in plants during autophagy.
  • Such treating of the plant is expected to stimulate growth of the plant, as well as to provide other beneficial results, including reducing the effects of plant stress.
  • purified plant proteins expected to function as regulators or controllers of autophagy in plants such as AUT1 proteins, and therefore having the ability to regulate autophagy are provided.
  • the AUT1 proteins are substantially pure (i.e., the proteins are essentially free, e.g., at least about 95% free, from other proteins with which they naturally occur).
  • the amino acid sequence of a protein expected to function as a regulator or controller of autophagy in plants, originally found in potato is set forth in SEQ ID NO:2.
  • a variant of the proteins described herein is expected to be functionally similar to that set forth in SEQ ID NO:2, for example, if it includes amino acids which are conserved among a variety of plant species or if it includes non-conserved amino acids which exist at a given location in another plant species that expresses the proteins described herein.
  • inventive amino acid sequences similar to the amino acid sequences set forth herein that have at least about 60% identity thereto that preferably function in regulating cellular autophagy.
  • inventive amino acid sequences have at least about 70% identity, further preferably at least about 80% identity, and most preferably at least about 90% identity to these sequences and to portions of these sequences.
  • Particularly preferred portions include the segment of amino acid 1 to amino acid 314, the segment of amino acid 60 to amino acid 350, and the segment of amino acid 80 to amino acid 320 of SEQ ID NO:2.
  • percent identity may be determined, for example, by comparing sequence information using the MacVector computer program, version 6.0.1.
  • the program may be used to determine percent identity over the entire length of the proteins being compared.
  • the invention also encompasses fragments of the amino acid sequences set forth herein, said fragments having a biological function in regulating cellular autophagy or an immunological function wherein said fragment is immunogenic.
  • An immunogenic peptide refers to a peptide of at least six amino acid residues to which an antibody can be generated and/or to which the antibody is specifically immunoreactive.
  • the specific immunoreactive sites within the antigen are known as epitopes or antigenic determinants.
  • These epitopes can be a linear array of monomers in a polymeric composition-such as amino acids in a protein-or consist of or comprise a more complex secondary or tertiary structure.
  • the method includes providing a nucleotide sequence described above, or variants thereof, that encodes a protein described herein, and introducing the nucleotide sequence into a host cell, as described above.
  • the desired nucleotide sequence may be advantageously incorporated into a vector to form a recombinant vector.
  • the recombinant vector may then be introduced into a host cell according to known procedures in the art. Such host cells are then cultured under conditions, well known to the skilled artisan, effective to achieve expression of the plant protein.
  • the protein may then be purified using conventional techniques.
  • the cDNA clone of the invention isolated from potato, and sequenced, encodes a plant autophagy protein.
  • the deduced amino acid sequence has 32.6% identity and 43.4% similarity with the deduced amino acid sequence of the protein encoded by the Aufl gene from Saccharomyces cerevisiae, a gene that is essential for autophagy in yeast.
  • Percentage identity was determined utilizing the MacVector computer program, described supra. The percent similarity may be determined, for example, by comparing sequence information using the MacVector computer program, version 6.0.1 (Oxford Molecular Group, Inc., Beaverton, OR).
  • the MacVector program defines identity as the number of identical and similar, i.e., conserved substitutions (defined supra), among aligned amino acid sequences at any particular position, divided by the total number of amino acid residues in the shorter of the two sequences.
  • An additional embodiment of the invention relates to peptides which have plant AUT1 activity which can be used to generate antibodies. Such antibodies can be used to detect the presence a peptide having plant AUT1 biological activity in biological samples.
  • the surface of two 100 mM petri plates containing Kings B agar were inoculated with P. fluorescens. Plates were incubated for 20 hours at 27°C. Bacteria were washed off the surface of the plates using 10ml deionized water per plate. The bacterial suspension was added to individual centrifuge tubes (50 ml) and filled to three quarters full using PBS (NaCI 8.7gm; NaP04 1M pH 7 10ml; deionized water 990ml; final pH of 7). Tubes were centrifuged at ⁇ OOOrpm for 3 minutes and decanted. The pellet was suspended in 5ml of PBS for each tube.
  • Bacterial suspensions from all tubes were pooled, vortexed for 15 minutes, and centrifuged for 20 minutes at 10,000rpm. The supernatant was carefully separated from the pellet, placed in a glass tube, covered with parafilm, and heated to 100°C for 3 minutes.
  • Example 3 Determination of Nucleic Acid Sequence Encoding Plant AUT1 Autophagy Protein
  • the original clone that encoded for the potato AUT1 cDNA contained a DNA insert of approximately 1 ,300 nucleotides, as estimated by gel electrophoresis.
  • the DNA insert was sequenced using the Thermus aquaticus (Taq) DyeDeoxy terminator cycle sequence (PE Applied Biosystems, Foster City, CA, USA) method at the Center for Agricultural Biotechnology, University of Maryland, College Park, MD.
  • the deduced amino acid sequence has 32.6% identity and 43.4% similarity with the deduced amino acid sequence of the protein encoded by the Autl gene from Saccharomyces cerevisiae, a gene essential for autophagy in yeast (Fig. 1).

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Abstract

L'invention concerne des protéines recombinantes de plante qui fonctionnent en tant que régulateurs de l'autophagie, telles que des protéines AUT1, ainsi que des séquences nucléotidiques codant pour ces protéines. Elle concerne aussi des vecteurs de recombinaison, des cellules hôtes, des plantes transgéniques, ainsi que des procédés d'utilisation des molécules d'acides nucléiques et des protéines de l'invention.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011106684A3 (fr) * 2010-02-25 2012-02-02 San Diego State University Foundation Compositions et procédés pour moduler l'autophagie
WO2012057640A1 (fr) 2010-10-27 2012-05-03 Instytut Biochemii I Biofizyki Pan Homologue végétale de la protéine autophage p62

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 29 June 1999 (1999-06-29), ALCALA ET AL.: "Generation of ESTs from tomato carpel tissue", XP002906487, Database accession no. AI487832 *
DATABASE GENBANK [online] 29 June 1999 (1999-06-29), ALCALA ET AL.: "Generation of ESTs from tomato carpel tissue", XP002906488, Database accession no. AI488627 *
DATABASE GENBANK [online] 29 June 1999 (1999-06-29), ALCALA ET AL.: "Generation of ESTs from tomato carpel tissue", XP002906489, Database accession no. AI771633 *
DATABASE GENBANK [online] 29 June 1999 (1999-06-29), D'ASCENZO ET AL.: "Generation of ESTs from pseudomonas susceptible tomato", XP002906485, Database accession no. AI778748 *
DATABASE GENBANK [online] 29 June 1999 (1999-06-29), D'ASCENZO ET AL.: "Generation of ESTs from pseudomonas susceptible tomato", XP002906486, Database accession no. AI778747 *

Cited By (3)

* Cited by examiner, † Cited by third party
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WO2011106684A3 (fr) * 2010-02-25 2012-02-02 San Diego State University Foundation Compositions et procédés pour moduler l'autophagie
WO2012057640A1 (fr) 2010-10-27 2012-05-03 Instytut Biochemii I Biofizyki Pan Homologue végétale de la protéine autophage p62
US9534229B2 (en) 2010-10-27 2017-01-03 Instytut Biochemii I Biofizyki Pan Plant homolog to autophagy protein P62

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