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WO2014113605A1 - Protéines impliquées dans une réaction de stress végétale - Google Patents

Protéines impliquées dans une réaction de stress végétale Download PDF

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Publication number
WO2014113605A1
WO2014113605A1 PCT/US2014/011924 US2014011924W WO2014113605A1 WO 2014113605 A1 WO2014113605 A1 WO 2014113605A1 US 2014011924 W US2014011924 W US 2014011924W WO 2014113605 A1 WO2014113605 A1 WO 2014113605A1
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Prior art keywords
plant
seq
promoter
nos
tolerance
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Inventor
Pamela Ronald
David DE VLEESSCHAUWER
Harkamal WALIA
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • a major limitation of crop production is imposed by a suite of abiotic and biotic stresses resulting in 30%-60% yield losses globally each year.
  • Recently genes involved in stress response have been determined.
  • SUBIA and SUBIC are involved in response to the abiotic stress of submergance.
  • Such submergence tolerance genes may be involved in triggering changes to gene transcription in response to hypoxic conditions or waterlogged roots.
  • These abiotic stress response genes may also be involved in response to other types of abiotic stress such as drought, high heat, cold, frost, salinity, poor nutrient conditions, wounding, and the like.
  • the pattern recognition gene XA21 is involved in triggering changes to gene transcription in response to the presence of Gram-negative bacterial pathogens.
  • Such biotic stress response genes may also be involved in a variety of responses to pathogenic organisms. However, much remains to be learned about the signaling pathways controlled by these pivotal stress response proteins.
  • the present invention provides a plant comprising a modulated expression of a component involved in plant stress response compared to a control plant, wherein the component comprises a polypeptide that is substantially identical to a protein selected from the group consisting of SEQ ID NOS: 1-89 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS: 1-89, and wherein the plant has enhanced stress tolerance relative to the control plant.
  • the component comprises a polypeptide that is substantially identical to a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64.
  • the plant has increased expression of the component compared to the control plant.
  • the plant comprises a heterologous expression cassette, and the expression cassette comprises a promoter operably linked to a polynucleotide encoding the component.
  • the promoter can be constitutive, tissue-specific or inducible, stress-inducible, hypoxia inducible, drought inducible, root or foliage specific, and/or selected from the group consisting of the promoters of SEQ ID NOS: 1-89, or an ortholog thereof.
  • the promoter is a promoter of SEQ ID NO:38, 63, or 64.
  • the plant has decreased expression of the component compared to the control plant. In other aspects, the plant has increased expression of the component compared to the control plant. In some cases, the decreased expression is stress induced. For example, the decreased expression is stress induced relative to a control plant, such as a control plant that is not under stress or a control plant that is under stress. In other cases the increased expression is stress induced. For example, the increased expression is stress induced relative to a control plant, such as a control plant that is not under stress or a control plant that is under stress. [0007]
  • the plant can comprise a heterologous expression cassette comprising a promoter operably linked to a polynucleotide that reduces expression of the component.
  • the promoter can be constitutive, tissue-specific or inducible, stress- inducible, hypoxia inducible, drought inducible, root or foliage specific, and/or selected from the group consisting of the promoters of SEQ ID NOS: 1-89, or an ortholog thereof.
  • the promoter is a promoter of SEQ ID NO:38, 63, or 64.
  • the polynucleotide is an antisense polynucleotide, a co- suppression polynucleotide, a micro RNA, or an siRNA.
  • the component can interact with both XA21 and SUB 1 A.
  • the enhanced stress tolerance comprises abiotic stress tolerance.
  • the abiotic stress tolerance can be submergence tolerance, drought tolerance, heat tolerance, hypoxia tolerance, cold tolerance, frost tolerance, salinity tolerance, wounding tolerance, increased ABA sensitivity, or decreased ABA sensitivity.
  • the enhanced stress tolerance comprises enhanced biotic stress tolerance, such as enhanced resistance or tolerance to a bacterial, viral, or fungal pathogen.
  • the plant comprises a modulated SUB1A interacting gene, an ortholog, or a
  • the plant comprises a modulated SUB1C interacting gene, an ortholog, or a
  • the plant comprises an inactivated, modulated expression, or altered stability of a protein selected from the group consisting of SEQ ID NOS: 1-89, wherein the protein binds XA21, Xb2, OsWRKY62, Xbl l, Xbl2, Xb22, PBZ1, NPR1, NRR, rTGA2.1, GR L1, SUB1A, SUB1C, a protein substantially identical, or a functional ortholog thereof.
  • the protein binds TOR, RAPTOR, or SnR l, a protein substantially identical, a domain, or a functional ortholog thereof.
  • the plant comprises an inactivated, modulated expression, or altered stability of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64, wherein the protein binds TOR, RAPTOR, or SnRKl, a protein substantially identical, a domain, or a functional ortholog thereof.
  • the invention provides a plant comprising an inactivated component involved in plant stress response, wherein the component comprises a polypeptide that is substantially identical to a protein selected from the group consisting of SEQ ID NOS: 1-89 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS: 1-89, wherein the plant has enhanced stress tolerance relative to the control plant.
  • the invention provides a plant comprising an inactivated component involved in plant stress response, wherein the component comprises a polypeptide that is substantially identical to a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS
  • the invention provides a plant comprising a component involved in plant stress response comprising modulated stability compared to a control plant, wherein the component comprises a polypeptide that is
  • the invention provides a plant comprising a component involved in plant stress response comprising modulated stability compared to a control plant, wherein the component comprises a polypeptide that is
  • the plant comprising a polypeptide that is substantially identical to a protein selected from the group consisting of SEQ ID NOS: 1-89 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS: 1-89 has an altered ubiquitination, sumoylation, glycosylation, or thermal stability relative to a control plant.
  • the plant comprising a polypeptide that is substantially identical to a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64 has an altered ubiquitination, sumoylation, glycosylation, or thermal stability relative to a control plant.
  • the invention provides an expression cassette comprising a promoter operably linked to a component involved in plant stress response, wherein the component comprises a polypeptide that is substantially identical to a protein selected from the group consisting of SEQ ID NOS: 1-89 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS: 1-89, wherein introduction of the expression cassette into a plant results in a plant having enhanced stress tolerance compared to a control plant in which the expression cassette is not expressed.
  • the polypeptide is substantially identical to a protein selected from SEQ ID NOS:38, 63, and 64 or is substantially identical to a functional ortholog of a protein selected from the group consisting of SEQ ID NOS:38, 63, and 64.
  • the promoter can be constitutive, tissue-specific or inducible, stress- inducible, hypoxia inducible, drought inducible, root or foliage specific, and/or selected from the group consisting of the promoters of SEQ ID NOS: 1-89, or an ortholog thereof. In some cases, the promoter is selected from the group consisting of the promoters of SEQ ID NOS:38, 63, and 64, or an ortholog thereof.
  • the expression cassette comprises a SUB1A interacting gene, a SUB1C interacting gene, or an ortholog, or conservatively modified variant thereof.
  • the expression cassette comprises TOR, RAPTOR, SnRKl, or an ortholog, a domain, or a conservatively modified variant thereof.
  • the expression cassette encodes a protein selected from SEQ ID NOS: 1-89, wherein the protein binds XA21, Xb2, OsWRKY62, Xbl 1, Xbl 2, Xb22, PBZ1, NPR1, NRR, rTGA2.1, GRNL1, SUB1A, SUB1C, or a protein substantially identical, or an ortholog thereof.
  • the expression cassette encodes a protein selected from SEQ ID NOS:38, 63, and 64.
  • the expression cassette encodes a protein that binds TOR, RAPTOR, or SnRKl, or a protein substantially identical, or an ortholog thereof.
  • the present invention provides a method of engineering a plant having enhanced stress tolerance, the method comprising:
  • the present invention provides a method of cultivating a plant having an enhanced stress tolerance comprising :providing seeds or seedlings of a plant of any of the plants provided herein; sowing said seeds or seedlings; and cultivating said seeds or seedlings under conditions suitable for growth of a plant; wherein said plant has enhanced stress tolerance relative to a wild-type plant.
  • the present invention provides a method for identifying plant genetic variants with enhanced stress tolerance comprising: mutating one or more genes involved in plant stress response, wherein the genes are selected from SEQ ID NOS: l-89, a protein substantially identical to one of SEQ ID NOS: l- 89, or a functional ortholog thereof; and selecting for plants comprising enhanced stress tolerance; wherein the selected plant comprises enhanced stress tolerance relative to a wild-type plant.
  • the present invention provides a method for identifying plant genetic variants with enhanced stress tolerance comprising: mutating one or more genes involved in plant stress response, wherein the genes are selected from SEQ ID NOS:38, 63, and 64, a protein substantially identical to one of SEQ ID NOS:38, 63, and 64, or a functional ortholog thereof; and selecting for plants comprising enhanced stress tolerance; wherein the selected plant comprises enhanced stress tolerance relative to a wild-type plant.
  • the present invention provides an expression cassette comprising a promoter operably linked a polynucleotide sequence encoding a gene, an antisense nucleotide, an shR A, or an siR A, wherein the promoter is selected from the group consisting of the promoters of SEQ ID NOS: 1-89.
  • the present invention provides an expression cassette comprising a promoter operably linked a polynucleotide sequence encoding a gene, an antisense nucleotide, an shRNA, or an siRNA, wherein the promoter is selected from the group consisting of the promoters of SEQ ID NOS:38, 63, and 64.
  • the invention provides stress-induced silencing of any one of SEQ ID NOS: l-89 (e.g., SEQ ID NO:38, 63, or 64, more preferably SEQ ID NO:63) or stress-induced upregulation of any one of SEQ ID NOS: l-89 (e.g., SEQ ID NO:38, 63, or 64, more preferably SEQ ID NO:63).
  • the stress induced silencing or stress induced upregulation provides enhanced stress response.
  • the stress induced silencing or stress induced upregulation provies enhanced resistance to abiotic stress.
  • the enhanced resistance to abiotic stress can be, for example, submergence tolerance, drought tolerance, heat tolerance, hypoxia tolerance, cold tolerance, frost tolerance, salinity tolerance, wounding tolerance, increased ABA sensitivity, or decreased ABA sensitivity.
  • the upregulation provides enhanced resistance to biotic stress.
  • the enhanced resistance to biotic stress can be, for example, enhanced resistance or tolerance to a bacterial, viral, or fungal pathogen.
  • Figure 1 depicts the effects of modulating TOR and Raptor expression in rice on growth and development.
  • Figure 2 depicts the pathways involved in TOR signaling in plants as determined by microarray analysis of rapamycin treated plant cells.
  • Figure 3 depicts the effect of modulating TOR and Raptor expression in rice on resistance to bacterial leaf blight disease.
  • Figure 4 depicts the inverse correlation between Xanthomonas oryzae pv. oryzae (Xoo) resistance and plant growth.
  • Figure 5 depicts the antagonizing effect of TOR and Raptor overexpression in rice on resistance to the necrotrophic pathogen Rhizoctonia solani.
  • stress includes biotic and abiotic stress.
  • Abiotic stress includes but is not limited to hypoxia, drought, high heat, cold, frost, salinity, poor nutrient conditions, high wind load, lodging, and wounding.
  • Biotic stress is stress induced by disease or pathogenic organisms, including for example Gram-negative bacteria.
  • biotic stress includes stress induced by the presence of
  • stress tolerance refers to the ability of an organism to tolerate biotic or abiotic stress or to recover once the stress conditions have passed.
  • enhanced stress tolerance refers to a phenotype in which an organism, such as a plant, has greater growth, multiplication, fertility, or yield during a stress condition or after a stress condition has passed than an organism that does not have enhanced stress tolerance.
  • a control plant could be a non-transgenic plant from the same plant line.
  • the enhancement can be an increase of 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.
  • Subjectgence tolerance refers to the ability of a plant survive hypoxia or low oxygen conditions, such as hypoxic conditions caused by flooding, for a period of time and to recover once the low oxygen conditions have passed.
  • Increased tolerance refers to an ability of plant to survive low oxygen conditions for a longer period of time, or to recover more quickly, than a control plant.
  • SUB1 nucleic acids refer to nucleic acids that encode a SUB1 polypeptide.
  • SUB1 polypeptides include SUB1A and SUB1C, comprise an ERF domain ⁇ see, e.g., Liu, 1998; Sakuma, 2002) and confer submergence tolerance when expressed in a plant.
  • SUB1A and SUB1C are ethylene response transcription factors that regulate response to prolonged submergence.
  • XA21 nucleic acids refer to nucleic acids that encode XA21 polypeptide.
  • XA21 polypeptide is a pattern recognition protein encoding a receptor kinase that confers resistance to strains of Gram-negative bacterium that contain the microbe associated molecular pattern AvrXA21.
  • the intracellular non-RD kinase domain of XA21 possesses intrinsic kinase activity.
  • XA21 and like proteins are also known as pathogen associated molecular pattern receptors, which typically recognize a broad range of pathogens, so long as the pathogen presents the recognized molecular pattern.
  • Enhanced (or improved or increased) disease resistance refers to an increase in the ability of a plant to prevent pathogen infection or pathogen-induced symptoms. Enhanced resistance can be specific for a particular pathogen species or genus, or can be increased resistance to all pathogens (e.g., systemic acquired resistance). Typically, enhancement is determined relative to a control, e.g., a wild type plant, or otherwise non-resistant plant.
  • Phenogens include, but are not limited to, viruses, bacteria, nematodes, fungi and insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA (1988)).
  • interactome refers to the group of genes and gene products that directly or indirectly interact or are capable of interacting.
  • stress-response interactome refers to the group of genes and gene products that respond to biotic or abiotic stress or affect stress tolerance or recovery, and the genes or gene products with which they interact.
  • a polypeptide is “capable of interacting" with another polypeptide in a number of different ways.
  • This interaction can, for instance, be a direct protein-protein interaction.
  • Typical bonds formed in a protein-protein interaction include hydrogen, ionic, van der Waals and covalent bonds.
  • the interaction may be indirect.
  • a third polypeptide may bind to both polypeptides, thereby keeping all three polypeptides in proximity to one another.
  • Protein interactions can be measured by a number of different methods that are known to those of ordinary skill in the art. Examples of systems to measure such interactions include, inter alia, the yeast two-hybrid system (see, e.g., Fields, Nature 340(6230):245-6 (1989) and Finley, R. L. JR & Brent R.
  • plant includes whole plants, shoot vegetative organs/structures ⁇ e.g. leaves, stems and tubers), roots, flowers and floral organs/structures ⁇ e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue ⁇ e.g. vascular tissue, ground tissue, and the like) and cells ⁇ e.g. guard cells, egg cells, trichomes and the like), and progeny of same.
  • shoot vegetative organs/structures ⁇ e.g. leaves, stems and tubers
  • roots flowers and floral organs/structures ⁇ e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules
  • seed including embryo, endosperm, and seed coat
  • fruit the mature ovary
  • plant tissue ⁇ e.g. vascular tissue, ground tissue, and the like
  • cells
  • the class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous. Specific embodiments of plants useful for the methods and compositions of the present invention include but are not limited to commercial food crops, and energy crops.
  • the invention has use over broad range of plants, including species from the genera Arabidopsis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,
  • Lycopersicon Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.
  • the plant is selected from the group consisting of rice, maize, wheat, soybeans, cotton, canola, turfgrass, alfalfa, poplar, eucalyptus, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, barley, tobacco, hemp, poppy, bamboo, canola, rape, sunflower, willow, or Brachypodium.
  • the plant is an ornamental plant.
  • the plant is a vegetable- or fruit-producing plant.
  • the plant is a monocot.
  • the plant is a dicot.
  • An "expression cassette” refers to a nucleic acid construct, which when introduced into a host cell ⁇ e.g., a plant cell), results in transcription and/or translation of a R A or polypeptide, respectively.
  • An expression cassette typically includes a sequence to be expressed, and sequences necessary for expression of the sequence to be expressed.
  • the sequence to be expressed can be a coding sequence or a non- coding sequence ⁇ e.g., an inhibitory sequence).
  • an expression cassette is inserted into an expression vector to be introduced into a host cell.
  • the expression vector can be viral or non- viral.
  • a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation ⁇ e.g., by methods described in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)).
  • a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide.
  • polynucleotides can be manipulated in many ways and are not limited to the examples above.
  • a recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).
  • a polynucleotide sequence is "heterologous to" an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form.
  • a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally-occurring allelic variants.
  • exogenous in reference to a polypeptide or polynucleotide, refers to polypeptide or polynucleotide which is introduced into a cell or organism (e.g., plant) by any means other than by a sexual cross.
  • transgenic e.g. , a transgenic plant or plant tissue, refers to a recombinantly modified organism with at least one introduced genetic element. The term is typically used in a positive sense, so that the specified gene is expressed in the transgenic organism. However, a transgenic organism can be transgenic for an inhibitory nucleic acid, i.e., a sequence encoding an inhibitory nucleic acid is introduced.
  • the introduced polynucleotide can be from the same species or a different species, can be endogenous or exogenous to the organism, can include a non-native or mutant sequence, or can include a non-coding sequence.
  • promoter refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of R A polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells.
  • a plant promoter used in the present invention may originally derive from the same species or variety of plant into which it is introduced, .e.g., methods and compositions using a canola promoter in a canola plant.
  • a plant promoter used in the present invention may originally derive from a different plant, e.g., methods using methods and compositions using a petunia promoter in a canola plant.
  • the plant promoters of the present invention may not derive from a plant, e.g. a bacterial or fungal promoter in a plant that is capable of initiating transcription in plant cells.
  • a "constitutive promoter” in the context of this invention refers to a promoter that is capable of initiating transcription in nearly all cell types, whereas a "cell type-specific promoter” or “tissue-specific promoter” initiates transcription only in one or a few particular cell types or groups of cells forming a tissue.
  • a promoter is tissue -specific if the transcription levels initiated by the promoter in a specific cell-type or tissue are at least 2-fold, 3 -fold, 4-fold, 5 -fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold higher or more as compared to the transcription levels initiated by the promoter in non-specific tissues.
  • the promoter is vessel-specific, root- specific, flower- specific, shoot-specific, or meristem-specific.
  • an "inducible promoter” refers to a promoter which can respond to a signal to increase or decrease transcription.
  • an inducible promoter may be silent, i.e., does not substantially initiate transcription, in the absence of a signal and active, i.e., initiates transcription, in the presence of the signal.
  • inducible promoters include promoters are provided herein. In some cases inducible promoters may initiate transcription in response to biotic stress or abiotic stress (i.e., stress- inducible promoters), temperature (e.g.
  • tissue specific promoters are inducible.
  • a promoter is inducible if the transcription levels initiated by the promoter under inducing conditions is at least 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold higher or more as compared to the transcription levels initiated by the promoter in a non-induced state.
  • the term "inactivate,” with reference to a particular gene, refers to methods or compositions in which one or more genes are rendered partially, substantially, or completely unable to perform their function. For example, a gene may be inhibited, mutated, knocked-out, or modulated such that it no longer effectively performs its function.
  • modulate as in to "modulate a gene” or “modulate expression” of a gene refers to increasing or decreasing the expression, activity, or stability of a gene. For example, a gene may be modulated by increasing or decreasing the amount of RNA that is transcribed from the gene or altering the rate of such transcription.
  • Decreased expression may include expression that is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 50%, 75%, 80%, 90%, 95%, 99% or more, such as 100%.
  • Increased expression includes expression that is increased by 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%), 90%), 100%), 200%), or more.
  • expression may be increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100- fold, 500-fold, 1000-fold higher. Expression may be modulated in a tissue specific or inducible manner as provided herein.
  • knockdown or “knockout,” with reference to a particular gene, describes an organism that is genetically modified to delete the gene, reduce expression of the gene (e.g., to less than 1 , 5, 10, or 20% of wild type expression), or to express a non-functional gene product.
  • gene knockdown is used synonymously with gene knockout or gene deficient.
  • polynucleotide “interfering polynucleotide,” and “interfering nucleic acid” are used generally herein to refer to RNA targeting strategies for reducing gene expression.
  • the antisense sequence is identical to the targeted sequence (or a fragment thereof), but this is not necessary for effective reduction of expression.
  • the antisense sequence can have 85, 90, 95, 98, or 99% identity to the complement of a target RNA or fragment thereof.
  • the targeted fragment can be about 10, 20, 30, 40, 50, 10-50, 20- 40, 20-100, 40-200 or more nucleotides in length.
  • RNAi refers to RNA interference strategies of reducing expression of a targeted gene.
  • RNAi technique employs genetic constructs within which sense and anti-sense sequences are placed in regions flanking an intron sequence in proper splicing orientation with donor and acceptor splicing sites.
  • spacer sequences of various lengths can be employed to separate self- complementary regions of sequence in the construct.
  • intron sequences are spliced-out, allowing sense and anti-sense sequences, as well as splice junction sequences, to bind forming double-stranded RNA.
  • Select ribonucleases then bind to and cleave the double-stranded RNA, thereby initiating the cascade of events leading to degradation of specific mRNA gene sequences, and silencing specific genes.
  • RNA interference The phenomenon of RNA interference is described and discussed in Bass, Nature 411 : 428-29 (2001); Elbahir et ah, Nature 411 : 494-98 (2001); and Fire et al, Nature 391 : 806-11 (1998); and WO 01/75164, where methods of making interfering RNA also are discussed.
  • siRNA refers to small interfering RNAs, that are capable of causing interference with gene expression and can cause post-transcriptional silencing of specific genes in cells, e.g., in plant cells.
  • the siRNAs based upon the sequences and nucleic acids encoding the gene products disclosed herein typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches.
  • Typical siRNAs have up to 40 bps, 35 bps, 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integer thereabout or therebetween.
  • Tools for designing optimal inhibitory siRNAs include that available from
  • a "short hairpin RNA” or “small hairpin RNA” is a ribonucleotide sequence forming a hairpin turn which can be used to silence gene expression. After processing by cellular factors the short hairpin RNA interacts with a complementary RNA thereby interfering with the expression of the complementary RNA.
  • Codon refers to the introduction of nucleic acid configured in the sense orientation to block the transcription of target genes.
  • this method to modulate expression of endogenous genes.
  • nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
  • the terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • a conservative substitution is given a score between zero and 1.
  • the scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity.
  • percent identity can be any integer from at least 25% to 100% ⁇ e.g., at least 25%, 26%, 27%, 28%, ... ,70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%), preferably calculated with BLAST using standard parameters, as described below.
  • amino acid sequences for these purposes normally means sequence identity of at least 40%.
  • Preferred percent identity of polypeptides can be any integer from at least 40% to 100% (e.g., at least 40%,41 %, 42%, 43%, . ..
  • the present invention provides polypeptides (and polynucleotides encoding such polypeptides) substantially identical to the sequences exemplified herein (e.g., any of SEQ ID NOS: l-89).
  • Polypeptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Unless other wise indicated, the comparison window extends the entire length of a reference sequence. Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.
  • BLAST algorithm is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive -valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • HSPs high scoring sequence pairs
  • nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • ortholog refers to genes from different species, that retain the same function in the course of evolution.
  • orthologs are conservatively modified variants of a sequence; e.g. , a conservatively modified variant of one of SEQ ID NOS: l-89.
  • Orthologs of a gene of interest can be identified by finding the closest ⁇ i.e., most conservatively modified) homolog of the gene of interest in the genome of another species.
  • orthologs of a gene of interest can be identified by determining which of a class of homologs has the most similar promoter as the gene of interest.
  • orthologs may be identified functionally. For example the ortholog of a transcription factor whose overexpression leads to submergence tolerance can be identified as a homologous transcription factor that also increases submergence tolerance when overexpressed.
  • 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. One of skill will recognize that each codon in a nucleic acid (except
  • AUG which is ordinarily the only codon for methionine
  • 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 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.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • the present invention provides polynucleotides that selectively hybridize to SEQ ID NOS:90-178.
  • sequenceselectively (or specifically) hybridizes to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 time background hybridization.
  • Polynucleotides that selectively hybridize to SEQ ID NO: l can be of any length, e.g., at least 10, 15, 20, 25, 30, 50, 100, 200 500 or more nucleotides or having fewer than 500, 200, 100, or 50 nucleotides, etc.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • genomic DNA or cDNA comprising nucleic acids of the invention can often be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here.
  • suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and at least one wash in 0.2X SSC at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions.
  • a positive hybridization is at least twice background.
  • a further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.
  • TOR e.g., SEQ ID NO:63
  • Tor is a phosphatidylinositol 3 kinase.
  • TOR is encoded by LOC_Os05gl4550 (RAP ID: Os05g0235300).
  • Rice TOR contains the following domains: Heat 1 , encoded by amino acids 184-221 ; Heat 2, encoded by amino acids 271-308, Heat 3, encoded by amino acids 348-389; Heat 4, encoded by amino acids 549-587; Heat 5, encoded by amino acids 588-625; Heat 6, encoded by amino acids 717-755; Heat 7, encoded by amino acids 761-799; Heat 8, encoded by amino acids 888-926; Heat 9, encoded by amino acids 981-1018; Heat 10, encoded by amino acids 1022-1059; Heat 1 1 , encoded by amino acids 1061-1098; Focal Adhesion Target Domain (FAT), encoded by amino acids 1297-1877; Rapamycin Binding Domain (FRB), encoded by amino acids 1910-2009; PI3 Kinase Domain (PI3K), encoded by amino acids 2077-2465; and C-terminal Focal Adhesion Target Domain (FATC), encoded by amino acids 2433-2465.
  • FAT Focal Adhesion Target Domain
  • FB Rapamycin
  • TOR also refers to orthologs and homologs of rice TOR, including genes or gene products that are at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to rice TOR.
  • expression of TOR, or one or more domains of TOR, or a homolog or ortholog thereof is modulated in a plant.
  • the modulation of expression protects a plant against abiotic or biotic stress.
  • the modulation of expression increases plant growth, crop yield, or resistance to drought, submergence, or pathogens.
  • SnRKl refers to a gene or gene product known as SNF1 -related kinase 1 (SnRKl).
  • SnRKl is a serine/threonine kinase.
  • SnRKl is encoded by LOC_Os05g45420.
  • Rice SnRKl contains the following domains: serine/threonine dual specificity protein kinase catalytic domain, encoded by amino acids 1-265; ubiquitin associated/translation elongation factor EFIB domain, encoded by amino acids 285-321 ; and C-terminal kinase associated domain 1 (KA1), encoded by amino acids 321-502.
  • SnRKl also refers to orthologs and homologs of rice SnRKl , including genes or gene products that are at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to rice SnRKl .
  • expression of SnRKl , or one or more domains of SnRKl , or a homolog or ortholog thereof, is modulated in a plant.
  • the modulation of expression protects a plant against abiotic or biotic stress.
  • the modulation of expression increases plant growth, crop yield, or resistance to drought, submergence, or pathogens.
  • RAPTOR refers to a gene or gene product known as Regulatory- Associated Protein of TOR (RAPTOR).
  • RAPTOR is an adaptor protein that binds to TOR.
  • RAPTOR is encoded by LOC_Osl2g01922.
  • Rice RAPTOR contains the following domains: Heat domain, encoded by amino acids 644-668; and WD40/YVTN repeat like containing domain, encoded by amino acids 1042-1347.
  • RAPTOR also refers to orthologs and homologs of rice RAPTOR, including genes or gene products that are at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to rice RAPTOR.
  • expression of RAPTOR, or one or more domains of RAPTOR, or a homolog or ortholog thereof, is modulated in a plant.
  • the modulation of expression protects a plant against abiotic or biotic stress.
  • the modulation of expression increases plant growth, crop yield, or resistance to drought, submergence, or pathogens.
  • the inventors have discovered a number of components, genes, or gene products (SEQ ID NOS: l-89) that are involved in plant stress response and are believed to regulate and/or control stress tolerance in rice. In some cases, these gene products are constituents of the stress response interactome of rice. In view of this discovery, it is believed that modulation (i.e., an increase or decrease) of expression, activity, and/or stability of one or more of these gene products in rice will enhance stress tolerance in rice. Moreover, it is believed that modulation of the same gene products, substantially similar gene products and/or orthologs in other plant species will enhance stress tolerance in other plant species.
  • methods of enhancing tolerance to biotic and abiotic stress in plants are provided by introducing a polynucleotide encoding one or more of the gene products into plants, altering the endogenous polynucleotides in plants, modulating the expression of one or more of the gene products involved in plant stress response, inactivating a gene or gene product, altering the activity of a gene product, or altering the stability of one or more gene products involved in plant stress response, a gene product substantially identical to a gene product involved in plant stress response, or an ortholog thereof.
  • stress tolerance is enhanced by increasing expression or activity of one of more of the above-mentioned gene products (SEQ ID NOS: l-89 or a gene product substantially identical or an ortholog thereof) in a plant.
  • Any of a number of methods can be used to increase activity of polypeptides or polynucleotides of the invention in plants.
  • Enhanced expression is useful, for example, to enhance systemic tolerance to abiotic or biotic stress.
  • Any organ can be targeted, such as shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g.
  • heterologous polypeptides or polynucleotides of the invention may be expressed in a transgenic plant under the control of an inducible promoter such as a stress-inducible promoter or a tissue specific stress-inducible promoter.
  • polypeptide activity of a gene of the present invention may be increased by inhibiting or inactivating a gene that suppresses the polypeptide.
  • a negative regulator of a stress-response component may be suppressed to provide a greater tolerance to stress.
  • the polypeptide activity of a gene of the present invention may be increased by altering the stability of the gene product. For example, one or more ubiquitination or sumoylation sites can be removed. Alternatively, the amino-terminus of a component may be altered to increase resistance to N-end rule proteolysis. Additionally, glycosylation sites may be added to enhance stability. In some cases increased glycosylation may inhibit ubiquitination, sumoylation, or proteolysis. i. Increasing gene expression
  • Isolated sequences prepared as described herein can be used to introduce expression of a particular nucleic acid to increase gene expression using methods well known to those of skill in the art. Preparation of suitable constructs and means for introducing them into plants are described below.
  • genes of the invention like other proteins, have different domains that perform different functions.
  • gene sequences of the invention need not be full length, so long as the desired functional domain of the protein is expressed.
  • Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described in detail below.
  • the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
  • mutations are introduced into a gene product as described herein (SEQ ID NOS: l-89 or a gene product substantially identical or an ortholog thereof) such that activity of the encoded polypeptide is either increased or decreased.
  • Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are known. For instance, seeds or other plant material can be treated with a mutagenic chemical substance, according to standard techniques. Such chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, ethyl methanesulfonate and N-nitroso-N-ethylurea.
  • ionizing radiation from sources such as, X-rays, fast neutrons or gamma rays can be used.
  • homologous recombination can be used to induce targeted gene modifications by specifically targeting gene of the invention in vivo ⁇ see, generally, Grewal and Klar, Genetics 146: 1221-1238 (1997) and Xu et al., Genes Dev. 10: 2411-2422 (1996)). Homologous recombination has been demonstrated in plants (Puchta et al, Experientia 50: 277-284 (1994), Swoboda et al, EMBO J. 13: 484-489 (1994); Offringa et al, Proc. Natl Acad. Sci. USA 90: 7346-7350 (1993); and Kempin et al. Nature 389:802-803 (1997)).
  • oligonucleotides composed of a contiguous stretch of R A and DNA residues in a duplex conformation with double hairpin caps on the ends can be used.
  • the RNA/DNA sequence is designed to align with the sequence of the target gene and to contain the desired nucleotide change.
  • Introduction of the chimeric oligonucleotide on an extrachromosomal T-DNA plasmid results in efficient and specific gene conversion directed by chimeric molecules in a small number of transformed plant cells. This method is described in Cole-Strauss et al, Science 273: 1386-1389 (1996) and Yoon et al. Proc. Natl. Acad. Sci. USA 93: 2071-2076 (1996).
  • Modified endogenous genes of the present invention as provided herein may exhibit increased or decreased expression or activity as compared to the wild-type gene, and thereby provide enhanced stress tolerance. iii. Other methods for increasing activity of polynucleotides and polypeptides of the invention
  • One method to increase expression of genes of the invention is to use
  • activation mutagenesis (see, e.g. Hiyashi et al. Science 258: 1350-1353 (1992)).
  • an endogenous gene of the invention can be modified to be expressed constitutively, ectopically, or excessively by insertion of T-DNA sequences that contain strong/constitutive promoters upstream of the endogenous gene.
  • preparation of transgenic plants overexpressing a gene of the invention can also be used to increase expression of that gene.
  • Activation mutagenesis of the endogenous gene of the invention will give the same effect as overexpression of a transgenic nucleic acid of the invention in transgenic plants.
  • an endogenous gene encoding an enhancer of activity or expression of an endogenous gene of the invention can be modified to be expressed by insertion of T-DNA sequences in a similar manner and activity of genes or polypeptides of the invention can be increased.
  • Another strategy to increase gene expression can be the use of dominant hyperactive mutants of a gene of the invention by expressing modified transgenes.
  • expression of a variant of one of SEQ ID NOS: 1-89 with a defective domain that is important for interaction with a negative regulator can be used to generate dominant hyperactive proteins.
  • expression of truncated proteins which have only a domain that interacts with a negative regulator can titrate the negative regulator and thereby increase endogenous activity.
  • Use of dominant mutants to hyperactivate target genes is described in Mizukami et al. Plant Cell 8:831-845 (1996).
  • stress tolerance is enhanced by decreasing expression or activity of one of more of the above-mentioned gene products (SEQ ID NOS: 1-89 or a gene product substantially identical or an ortholog thereof) in a plant.
  • Activity of polynucleotides or polypeptides of the invention can play a role in modulating, directly or indirectly, the expression of a number of components of the stress-response interactome through interaction with the genes' promoters as well as with other proteins ⁇ e.g., RNA polymerase, transcription factors and the like).
  • modulation of such gene expression activity can be used, to increase stress tolerance in plants. i. Inhibition of gene expression
  • stress tolerance is enhanced by decreasing expression of one of more of the above-mentioned gene products (SEQ ID NOS: 1-89 or a gene product substantially identical or an ortholog thereof) in a plant.
  • the nucleic acid sequences disclosed here can be used to design nucleic acids useful in a number of methods to inhibit expression of genes of the invention in plants.
  • antisense technology can be conveniently used. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The construct is then transformed into plants and the antisense strand of RNA is produced.
  • the nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous gene or genes (e.g., any one or more of SEQ ID NOS:90-178) to be repressed.
  • the sequence need not be perfectly identical to inhibit expression.
  • the vectors of the present invention can be designed such that the inhibitory effect applies to other genes within a family of genes exhibiting identity or substantial identity to the target gene.
  • the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher identity 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 identity of non-coding segments may be equally effective.
  • a sequence of between about 30 or 40 nucleotides to about the full length of a nucleotide 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 about 500 to about 3500 nucleotides is especially preferred.
  • a number of gene regions can be targeted to suppress expression of genes of the invention.
  • the targets can include, for instance, the coding regions, introns, sequences from exon/intron junctions, 5' or 3' untranslated regions, and the like.
  • Co-suppression may also be utilized in the methods of the present invention to decrease the expression of one or more of the genes or gene products provided herein.
  • Introduction of nucleic acid configured in the sense orientation has been recently shown to be an effective means by which to block the transcription of target genes.
  • the suppressive effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence.
  • the introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity is most preferred. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting identity or substantial identity.
  • the introduced sequence needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants that over-express the introduced sequence. A higher identity in a sequence shorter than full-length compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective.
  • Oligonucleotide -based triple-helix formation can also be used to disrupt expression of genes of the invention.
  • Triplex DNA can inhibit DNA transcription and replication, generate site-specific mutations, cleave DNA, and induce homologous recombination (see, e.g., Havre and Glazer J. Virology 67:7324-7331 (1993); Scanlon et al. FASEB J. 9: 1288-1296 (1995); Giovannangeli et al. Biochemistry 35: 10539- 10548 (1996); Chan and Glazer J. Mol. Medicine (Berlin) 75: 267-282 (1997)).
  • Triple helix DNAs can be used to target the same sequences identified for antisense regulation.
  • RNA molecules or ribozymes can also be used to inhibit expression of genes. 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. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. Thus, ribozymes can be used to target the same sequences identified for antisense regulation.
  • RNAs A number of classes of ribozymes have been identified.
  • One class of ribozymes is derived from a number of small circular RNAs that are capable of self- cleavage and replication in plants.
  • the RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs).
  • RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus.
  • the design and use of target RNA- specific ribozymes is described in Zhao and Pick, Nature 365:448-451 (1993); Eastham and Ahlering, J.
  • Methods for introducing genetic mutations described above can also be used to select for plants with decreased expression of one or more genes of the invention. iii. Other means for inhibiting polynucleotide or polypeptide
  • Activity of polynucleotides of the invention may be modulated by eliminating the proteins that are required for cell-specific expression of such polynucleotides.
  • expression of regulatory proteins and/or the sequences that control gene e.g., SEQ ID Nos: 1-89
  • expression can be modulated using the methods described here.
  • Another strategy is to inhibit the ability of a protein of the invention to interact with itself or with other proteins. This can be achieved, for instance, using antibodies specific to a polypeptide of the invention.
  • expression of antibodies specific for a polypeptide of the invention is used to inactivate functional domains through antibody :antigen recognition (see, Hupp et ah, Cell 83:237-245 (1995)). Interference with activity of protein(s) that interact with polypeptides of the invention can be applied in a similar fashion.
  • dominant negative alleles i.e. dominant gain of function mutants
  • of the genes of the invention can be prepared by expressing a transgene that encodes a truncated polypeptide. Use of dominant negative mutants to inactivate target genes in transgenic plants is described in
  • nucleic acids of the invention may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library.
  • genomic libraries large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector.
  • cDNA library mRNA is isolated from the desired organ, such as leaves, and a cDNA library that contains a gene transcript of the invention is prepared from the mRNA.
  • cDNA may be prepared from mRNA extracted from other tissues in which genes of the invention or homologs are expressed.
  • the cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned gene of the invention as disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against a polypeptide of the invention can be used to screen an mRNA expression library.
  • the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques.
  • PCR polymerase chain reaction
  • PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • PCR Protocols A Guide to Methods and Applications .
  • Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et ah, Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. d. Preparation of recombinant vectors
  • DNA sequence coding for the desired polypeptide for example a cDNA sequence encoding a full length protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.
  • a plant promoter fragment may be employed which will direct expression of the gene in all, or substantially all, tissues of a regenerated plant.
  • Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation.
  • constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the ⁇ - or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill.
  • Constitutive promoters and regulatory elements can also be isolated from genes that are expressed constitutively or at least expressed in most if not all tissues of a plant.
  • Such genes include, for example, ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33: 125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al, Mol. Gen. Genet. 251 : 196- 203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104: 1167-1176 (1994)), GPcl from maize (GenBank No. X15596, Martinez et al. J. Mol.
  • the plant promoter may direct expression of a nucleic acid of the invention in a specific tissue, organ or cell type ⁇ i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control ⁇ i.e. inducible promoters).
  • tissue-specific promoters examples include anaerobic conditions, elevated temperature, the presence of light, or sprayed with chemicals/hormones. Tissue- specific promoters can be inducible.
  • tissue-specific promoters may only promote transcription within a certain time frame of developmental stage within that tissue. Other tissue specific promoters may be active throughout the life cycle of a particular tissue.
  • tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.
  • a number of tissue-specific promoters can also be used in the invention. For instance, promoters that direct expression of nucleic acids in leaves, roots or flowers are useful for enhancing resistance to pathogens that infect those organs.
  • photosynthetic organ-specific promoters such as the RBCS promoter (Khoudi, et al, Gene 197:343, 1997), can be used.
  • Root-specific expression of polynucleotides of the invention can be achieved under the control of the root-specific ANRI promoter (Zhang & Forde, Science, 279:407, 1998).
  • Any strong, constitutive promoters such as the CaMV 35S promoter, can be used for the expression of polynucleotides of the invention throughout the plant.
  • Stress-inducible promoters of the present invention include promoters of the rice SUB1A, SUB1C, XA21, RAB17, HVA22, HSP17.5, HSP22, HSP16.9, PDC, ADH, SI, psL2, PDC2, PDC4, P5CS, OsDREB, NCED, PP2C, LP2, genes, or a promoter of a stress-inducible ortholog thereof.
  • stress-indubible promoters include one or more promoters of SEQ ID NOS: 1-89 or an ortholog thereof. Stress-inducible promoters may also include one or more promoters found in U.S. Patent Application Publication 2009/0229014 or an ortholog thereof.
  • the present invention provides constitutive, tissue-specific, cell-specific, and inducible promoters.
  • the promoters of the present invention include any promoter or set of promoters selected from the native promoters of the genes that encode SEQ ID NOS: 1-89, or an ortholog thereof.
  • the promoters of the present invention include a promoter that is substantially identical to a native promoter of one of SEQ ID NOS: 1-89.
  • polyadenylation region at the 3 '-end of the coding region should be included.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the vector comprising the sequences ⁇ e.g., promoters or coding regions) from genes of the invention may comprise a marker gene that 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 chlorosulfuron or Basta. e. Production of transgenic plants
  • DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques.
  • the DNA 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 DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
  • 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 82:5824 (1985).
  • Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).
  • the DNA constructs may be combined with one or more suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • the virulence functions of the Agrobacterium 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.
  • Agrobacterium tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. Science 233:496- 498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer to Plants, Potrykus, ed. (Springer- Verlag, Berlin 1995).
  • transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased stress-tolerance.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences.
  • Plant regeneration from cultured protoplasts is described in Evans et al, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 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).
  • nucleic acids of the invention can be used to confer desired traits on essentially any plant.
  • the invention has use over a broad range of plants, including, but not limited to, species from the genera Anacardium, Arabidopsis, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum,
  • 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.
  • the present invention provides for method of enhancing stress tolerance in plants by modulating the expression and/or altering the activity of polynucleotides and/or polypeptides of the invention.
  • tolerance can be enhanced, for instance, by increased expression of positive regulators of stress response-related genes.
  • polynucleotides or polypeptides of the invention can be modified to enhance tolerance, e.g., by increasing or decreasing the polypeptides' interactions with other components important in plant stress tolerance.
  • one possible mechanism by which the polypeptides of the invention modulate tolerance is, for example, by acting as components of a signal cascade between initiation of stress and the development of the stress response.
  • polypeptides of the invention may lead directly to increased transcription of stress response transcripts, thereby enhancing tolerance to stress.
  • polypeptides of the invention may interact with promoters of other genes as well as with other regulatory factors, thereby modulating expression of stress response genes or other genes involved in stress tolerance. For example, after a plant component (e.g., a plant disease resistance polypeptide) is activated by the presence of a pathogen (e.g.
  • the plant component provides a signal (e.g., via protein-protein interactions, phosphorylation/dephosphorylation, oxidative burst or the like) directly or indirectly, to the polypeptides of the invention.
  • a signal e.g., via protein-protein interactions, phosphorylation/dephosphorylation, oxidative burst or the like
  • some polypeptides of the invention may act as negative regulator of a stress response.
  • Negative regulators act to prevent a stress response by various mechanisms (e.g., via protein-protein interactions,
  • polypeptides of the invention can act as transcriptional repressors, thereby allowing for the expression of stress response genes. Such mechanisms may be altered when a plant is subject to stress, allowing a stress response to develop. i. Selecting for plants with enhanced stress tolerance
  • Plants with enhanced stress tolerance can be selected in many ways. One of ordinary skill in the art will recognize that the following methods are but a few of the possibilities. One method of selecting plants with enhanced stress tolerance is to determine tolerance of a plant to a specific plant stress such as a biotic or abiotic stress.
  • the stress is an abiotic stress.
  • abiotic stressors include but are not limited to: submergence, drought, heat, hypoxia, cold, frost, salinity, wounding, lodging or other physical insult, and changes in plant growth medium (e.g., soil) nutrient levels.
  • plant growth medium e.g., soil
  • enhanced tolerance is measured by the reduction or elimination of stress symptoms when compared to a control plant. In some cases, enhanced tolerance is measured by increased multiplication, fertility, or yield. In some cases, enhanced tolerance is measured by faster or more robust recovery after the stress condition is removed or mitigated.
  • the stress is the presence or activity of a specific pathogen.
  • pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi and insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA) (1988)).
  • pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi and insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA) (1988)).
  • HR hypersensitive response
  • Plants with enhanced tolerance can produce an enhanced hypersensitive response relative to control plants.
  • Enhanced tolerance can also be determined by measuring the increased expression of a gene operably linked a promoter of one or more genes provided herein, such as a gene involved in plant stress response or a component of the stress response interactome. Measurement of such expression can be measured by quantitating the accumulation of RNA or subsequent protein product ⁇ e.g., using northern or western blot techniques, respectively (see, e.g., Sambrook et al. and Ausubel et al).
  • a possible alternate strategy for measuring stress response promoter expression involves operably linking a reporter gene to the promoter of a gene involved in plant stress response. Reporter gene constructs allow for ease of measurement of expression from the promoter of interest. Examples of reporter genes include: ⁇ -gal, GUS (see, e.g., Jefferson, R. A., et al, EMBO J6: 3901-3907 (1987), green fluorescent protein, luciferase, and others.
  • Y2H yeast two-hybrid screening strategy
  • SUBl A and SUB1C were cloned into a Y2H screening vector.
  • Bait constructs were transformed into yeast strains, plated on selective medium, and screened.
  • BiFC bimolecular fluorescence complementation
  • the stress response interactome includes 5 candidate transcription factors that interact with both SUBl A and the pattern recognition receptor XA21. This result supports growing evidence that there is a set of proteins that link plant biotic and abiotic stress responses and suggests that the knowledge gained through the course of the proposed studies can also be used for future work in dissecting the abiotic stress tolerance response of plants of commercial, agronomic, and scientific interest. [0144] Interactome members were further prioritized for in-depth characterization based on SUB1A mediated transcriptome analysis, novelty of predicted member, and minimal redundancy in the rice genome.
  • RiceNet was then used to investigate the grass abiotic stress responses. By querying the network with genes encoding proteins present in the submergence tolerance stress response interactome and key AP2/ERF TFs, genes highly predicted to control the abiotic stress response were provided. Thus, the exploration of the rice abiotic stress response network has provided a rich resource of genes for selection, for refined engineering of stress tolerance.
  • Gene products involved in rice stress response include gene products encoded by genes at the following loci: LOC_Os03g46770, LOC_Os04g56400,
  • LOC Os01g64730 LOC Os04g52090, LOC Os07g47790, LOC_Os06g44010,
  • LOC Os01g40094 LOC Os03g63940, LOC Os03g20340, LOC_Os05g45420,
  • LOC Os03g08220 LOC Os04g50990, LOC Os08g44380, LOC_Os01g54870,
  • LOC Os02g28810 LOC Os07g42950, LOC Os01g01060.
  • Representative amino acid sequences of the gene products involved in plant stress response are provided herein as SEQ ID Nos: 1-89, and corresponding cDNA sequences as SEQ ID Nos: 90-178.
  • One of skill in the art can determine the nucleic acid sequence or amino-acid sequence for any of the loci provided herein, or an ortholog thereof, using appropriate databases including, for example: GenBank, GreenPhyl (www.greenphyl.org), the floral genome project (fgp.huck.psu.edu), PlantPan (plantpan.mbc.nctu.edu.tw), the rice genome annotation project
  • This example clarifies and illuminates the role of SnRKl and TOR during the onset and maintenance of plant innate immunity and assesses whether these master growth regulators are targets for pathogen manipulation.
  • the example also identifies and functionally characterizes upstream regulators and downstream effector molecules of SnRKl and TOR, and provides insight into the regulation and molecular intricacies of the SnRKl -TOR signal interaction. Finally, this example evaluates the importance of such interplay in determining disease outcomes.
  • Seeds were surface sterilized with 2% sodium hypochlorite solution for 2 min, rinsed three times in sterile distilled water, and germinated on a wet sterile filter paper in sealed Petri dishes (> 92% relative humidity) at 28 °C.
  • Five days later, germinated seeds were grown in commercial potting soil (Universal; Snebbout, Kaprijke, Belgium) under non-sterile greenhouse conditions (30 ⁇ 4 °C; 16 h light/8 h dark regime). Plants were watered daily and fertilized with 5g/m 2 (NFLi) 2 SC)4 and 10g/m 2 FeS0 4 .7H 2 0 on day 8, 15, 22 and 29 after sowing.
  • plants were propagated in the greenhouse and fertilized with 0.5% ammonium sulphate every two weeks until flowering.
  • Xanthomonas oryzae pv. oryzae strain PX099 was routinely grown on Sucrose Peptone Agar (SPA) medium at 28 °C.
  • SPA Sucrose Peptone Agar
  • single colonies were transferred to liquid SP medium and grown for 48h at 28 °C. Plants were inoculated when 6 weeks olds by clipping the fifth and sixth stage leaves with scissors dipped in a solution of Xoo cells in water (1 x 10 9
  • Inoculated plants were kept in a dew chamber (> 92% relative humidity; 28 ⁇ 2 °C) for 24 h and thereafter transferred to greenhouse conditions for disease development. Fourteen days after inoculation, disease severity was assessed by measuring the length of the water-soaked lesions
  • SnRKl sucrose non- fermenting-related kinasel
  • TOR rapamycin
  • TOR central protein kinase
  • TOR RNAi plants developed lesion mimics when aging and, in most cases, failed to set seeds. Similar yet less pronounced phenotypes were observed for SnRKl OX lines, uncovering SnRKl as a plant growth-repressive protein.
  • SnRKl RNAi lines were hyper- susceptible to Rs and Xoo infection, tagging SnRKl as a positive regulator of basal immunity.
  • TOR/Raptor activity can be rapidly and transiently induced or repressed using dexamethasone (Dex).
  • Dex dexamethasone

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Abstract

L'invention concerne des procédés et des compositions pour améliorer la tolérance végétale au stress.
PCT/US2014/011924 2013-01-16 2014-01-16 Protéines impliquées dans une réaction de stress végétale Ceased WO2014113605A1 (fr)

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CN113150097A (zh) * 2021-05-11 2021-07-23 黑龙江八一农垦大学 一种与植物耐逆性相关的蛋白OsERF096及其编码基因与应用
US11203765B2 (en) * 2016-05-12 2021-12-21 University Of Florida Research Foundation, Incorporated Drought tolerant plants
CN116064570A (zh) * 2022-07-29 2023-05-05 浙江大学 Tor基因在提高作物抗旱性和氮利用效率中的应用
CN116396947A (zh) * 2023-02-14 2023-07-07 中国农业大学 SnRK1的β亚基在水稻抗稻瘟病菌和白叶枯菌中的应用
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CN119530244A (zh) * 2024-12-10 2025-02-28 扬州大学 一种调控水稻抗病性基因OsSK8及其编码蛋白和应用
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11203765B2 (en) * 2016-05-12 2021-12-21 University Of Florida Research Foundation, Incorporated Drought tolerant plants
US20180163221A1 (en) * 2016-12-12 2018-06-14 Academia Sinica Transgenic plants expressing type 2c protein phosphatase abscisic acid (pp2caba) proteins and uses thereof
US11299744B2 (en) * 2016-12-12 2022-04-12 Academia Sinica Transgenic plants expressing type 2C protein phosphatase abscisic acid (PP2CABA) proteins and uses thereof
CN113150097A (zh) * 2021-05-11 2021-07-23 黑龙江八一农垦大学 一种与植物耐逆性相关的蛋白OsERF096及其编码基因与应用
CN113150097B (zh) * 2021-05-11 2023-09-15 黑龙江八一农垦大学 一种与植物耐逆性相关的蛋白OsERF096及其编码基因与应用
CN116064570A (zh) * 2022-07-29 2023-05-05 浙江大学 Tor基因在提高作物抗旱性和氮利用效率中的应用
CN116396947A (zh) * 2023-02-14 2023-07-07 中国农业大学 SnRK1的β亚基在水稻抗稻瘟病菌和白叶枯菌中的应用
WO2025080749A1 (fr) * 2023-10-11 2025-04-17 Benbarra Llc Méthodes d'atténuation des effets d'une maladie chez les plantes
CN118085050A (zh) * 2024-04-08 2024-05-28 四川农业大学 水稻转录因子erf及其编码基因在调控植物抗病性中的应用
CN118308410A (zh) * 2024-04-30 2024-07-09 中国农业科学院生物技术研究所 蛋白drw1或调控其表达的物质在调控水稻热胁迫抗性中的应用
CN119530244A (zh) * 2024-12-10 2025-02-28 扬州大学 一种调控水稻抗病性基因OsSK8及其编码蛋白和应用

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