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WO1998026045A1 - Plantes transgeniques a l'epreuve des agressions du milieu - Google Patents

Plantes transgeniques a l'epreuve des agressions du milieu Download PDF

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WO1998026045A1
WO1998026045A1 PCT/US1997/023019 US9723019W WO9826045A1 WO 1998026045 A1 WO1998026045 A1 WO 1998026045A1 US 9723019 W US9723019 W US 9723019W WO 9826045 A1 WO9826045 A1 WO 9826045A1
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gene
plant
transgenic plant
expression
cell
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Jen Sheen
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General Hospital Corp
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General Hospital Corp
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Priority to EP97954562A priority patent/EP1015556A4/fr
Priority to CA002274580A priority patent/CA2274580A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

  • This invention relates to the fields of plant genetic engineering and crop protection.
  • ABA abscisic acid
  • environmental stress conditions such as drought, cold, and salinity
  • ABA abscisic acid
  • the accumulation of these gene products is thought to protect plants from stress induced damage.
  • Many of these genes are also expressed at the late stage of embryogenesis during seed development and are thought to be important for seed desiccation and dormancy.
  • Several studies have identified cis-acting elements and trans-acting factors important for the regulation of these stress-inducible genes. Background information relating to the aforementioned topics is found in the following references: Skriver and Mundy, Plant Cell 2:503, 1990; Bray, Plant Physiol.
  • the invention features a method for protecting a plant against an environmental stress, the method including the steps of: (a) producing a transgenic plant cell including a recombinant protein kinase (PK) domain gene integrated into the genome of the transgenic plant cell and positioned for expression in the plant cell; and (b) growing a transgenic plant from the plant cell, wherein the PK domain gene is expressed in the transgenic plant.
  • the method of the invention involves the expression of a PK domain gene which is capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, or temperature stress.
  • the invention includes a PK domain gene which encodes a polypeptide that includes an amino acid sequence that is substantially identical to the amino acid sequence of ATCDPK1 or ATCDPKla.
  • the invention includes a gene encoding a polypeptide having a PK domain that includes an amino acid sequence that is substantially identical to the amino acid sequence of ATCDPK1 or ATCDPKla.
  • the invention features a method for protecting a plant against environmental stress, the method including the steps of: (a) producing a transgenic plant cell including a recombinant calcium-dependent protein kinase (CDPK) gene integrated into the genome of the transgenic plant cell and positioned for expression in the plant cell, the CDPK gene being capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, and temperature stresses; and (b) growing a transgenic plant from the plant cell, wherein the CDPK gene is expressed in the transgenic plant.
  • a recombinant calcium-dependent protein kinase (CDPK) gene integrated into the genome of the transgenic plant cell and positioned for expression in the plant cell, the CDPK gene being capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, and temperature stresses.
  • an environmental stress e.g., dehydration, salt, and temperature stresses
  • the invention features a method for protecting a plant against environmental stress, the method including the steps of: (a) producing a transgenic plant cell including a recombinant calcium/calmodulin- dependent (CaM-K) gene integrated into the genome of the transgenic plant cell and positioned for expression in the plant cell; and (b) growing a transgenic plant from the plant cell wherein the CaM-K gene is expressed in the transgenic plant.
  • the method of the invention includes the expression of a CaM-K gene (e.g., a mammalian CaM-KII gene) which is capable of increasing the level of tolerance to an environmental stress, e.g., against dehydration, salt, or temperature stress.
  • a CaM-K gene e.g., a mammalian CaM-KII gene
  • the invention features a method for protecting a plant against environmental stress, the method including the steps of: (a) producing a transgenic plant cell which includes a combination of at least two genes selected from the group consisting of a recombinant PK domain gene, a recombinant CDPK gene, and a CaM-K gene, each of the genes being integrated into the genome of the transgenic plant cell and positioned for expression in the plant cell; and (b) growing a transgenic plant from the plant cell, wherein each of the genes is expressed in the transgenic plant.
  • the method of the invention includes the expression of genes which are capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, or temperature stress.
  • the invention features a transgenic plant including a recombinant PK domain gene integrated into the genome of the transgenic plant and positioned for expression in the plant, the PK domain gene being capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, or temperature stress, on a transgenic plant expressing the recombinant PK domain.
  • the invention features a transgenic plant including a recombinant CDPK gene integrated into the genome of the transgenic plant and positioned for expression in the plant, the recombinant CDPK gene being capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, or temperature stress, on a transgenic plant expressing the recombinant CDPK.
  • the invention features a transgenic plant including a recombinant CaM-K gene integrated into the genome of the transgenic plant and positioned for expression in the plant, the recombinant CaM-K gene being capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, or temperature stress, on a transgenic plant which is expressing the recombinant CaM-K gene.
  • an environmental stress e.g., dehydration, salt, or temperature stress
  • the invention features a transgenic plant including a recombinant CDPK gene, PK domain gene, CaM-K gene, or any combination thereof integrated into the genome of the transgenic plant cell and positioned for expression in the plant cell, the CDPK, PK domain, and CaM-K genes being capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, or temperature stress, on a transgenic plant expressing the DNA.
  • a recombinant CDPK gene, PK domain gene, CaM-K gene, or any combination thereof integrated into the genome of the transgenic plant cell and positioned for expression in the plant cell, the CDPK, PK domain, and CaM-K genes being capable of increasing the level of tolerance to an environmental stress, e.g., dehydration, salt, or temperature stress, on a transgenic plant expressing the DNA.
  • an environmental stress e.g., dehydration, salt, or temperature stress
  • the invention includes seeds and cells from any of the aforementioned transgenic plants.
  • the invention features a substantially pure PK domain polypeptide capable of increasing the level of tolerance to an environmental stress, e.g., environmental stress in a transgenic plant.
  • the invention features a PK domain polypeptide including an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 5 (SEQ ID NO: 2).
  • the invention features substantially pure DNA encoding a PK domain polypeptide capable of conferring tolerance to an environmental stress in a transgenic plant.
  • This DNA may include a nucleic acid sequence substantially identical to the nucleic acid sequence shown in Fig. 5 (SEQ ID NO: 1).
  • Such DNA may be operably linked to an expression control region for the expression of the PK polypeptide; and the expression control region may include a promoter (for example, a constitutive or inducible promoter).
  • the invention features a cell (e.g., a plant cell) which includes the DNA of the invention.
  • polypeptide is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
  • substantially identical is meant a polypeptide or nucleic acid exhibiting at least 70%, preferably 80%, more preferably 85%, and most preferably 90%, or even 95% sequence identity to a reference sequence.
  • the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids.
  • the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, FastA, or PILEUP/PRETTYBOX programs).
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, FastA, or PILEUP/PRETTYBOX programs.
  • Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • a substantially pure polypeptide is meant a polypeptide (for example, the PK domain polypeptide shown in Fig. 5 (SEQ ID NO: 2)) that has been separated from components which naturally accompany it.
  • the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a substantially pure polypeptide.
  • a substantially pure PK domain polypeptide may be obtained, for example, by extraction from a natural source (for example, a plant cell); by expression of a recombinant nucleic acid encoding a PK domain polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • derived from is meant isolated from or having the sequence of a naturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic DNA, or combination thereof).
  • substantially pure DNA DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • transformed cell is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a regulator polypeptide (e.g., a CDPK, a PK domain, or a CaM-K polypeptide).
  • a regulator polypeptide e.g., a CDPK, a PK domain, or a CaM-K polypeptide
  • positioned for expression is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation, if appropriate, of the sequence (i.e., facilitates the production of, for example, a CDPK polypeptide, a PK domain polypeptide, a CaM-K polypeptide, a recombinant protein, or an RNA molecule).
  • reporter gene is meant a gene whose expression may be assayed; such genes include, without limitation, ⁇ -glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), and ⁇ -galactosidase.
  • expression control region is meant any minimal sequence sufficient to direct transcription.
  • promoter elements that are sufficient to render promoter- dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, stress-, or hormone-inducible elements or chemical inducers such as SA or INA); such elements may be located in the 5' or 3' regions of the native gene or engineered into a transgene construct.
  • operably linked is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).
  • Plant cell is meant any self-propagating cell bounded by a semi- permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired.
  • Plant cell includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • crucifer any plant that is classified within the Cruciferae family.
  • the Cruciferae include many agricultural crops, including, without limitation, rape (for example, Brassica campestris and Brassica napus), broccoli, cabbage, Bmssel sprouts, radish, kale, Chinese kale, kohlrabi, cauliflower, turnip, rutabaga, mustard, horseradish, and Arabidopsis.
  • transgene is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell.
  • Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
  • transgenic any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell.
  • the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into the nuclear or plastidic genome.
  • the level of tolerance in a transgenic plant of the invention is at least 5%, 10%, or 20% (and preferably 30% or 40%) greater than the tolerance to an environmental stress exhibited in a control plant.
  • the level of tolerance to an environmental stress is 50% greater, 60% greater, and more preferably even 75% or 90% greater than a control plant; with up to 100% above the level of tolerance as compared to a control plant being most preferred.
  • the level of tolerance is measured using conventional methods.
  • the level of stress tolerance to salinity may be determined by comparing physical features and characteristics (for example, plant height and weight) of transgenic plants and control plants.
  • the invention provides a number of important advances and advantages for the protection of plants against environmental stress, such as drought, salt, and temperature.
  • the invention facilitates an effective and economical means to improve agronomically important traits of plants for tolerating the effects of dehydration, salinity, cold, and heat.
  • the invention provides for increased production efficiency, as well as for improvements in quality and yield of crop plants and ornamentals.
  • the invention contributes to the production of high quality and high yield agricultural products: for example, fruits, ornamentals, vegetables, cereals, and field crops.
  • the invention further provides a means for mediating the expression of stress- related protective proteins that enable a plant to tolerate the effects of environmental stress.
  • transgenic plants constitutively producing a recombinant CDPK gene product, a PK domain, or CaM-K are capable of turning on a plant's stress signal transduction pathway by allowing the expression of multiple stress-related proteins, which in turn enhances the plant's tolerance to multiple stress conditions. Expression of these gene products therefore obviates the need to express individual stress-related genes as a means to promote plant defense mechanisms against adverse conditions.
  • FIG. 1 shows a series of photographs demonstrating stress signaling in maize leaf protoplasts as visualized by green-fluorescent (GFP) expression.
  • GFP green-fluorescent
  • Fig. 2 shows a series of photographs demonstrating that intracellular Ca 2+ elevation activates stress signaling.
  • Fig. 3 A shows a schematic illustration of the structural comparison between plant CDPKs and mammalian CaMKII.
  • Fig. 3B shows the sequence comparison among the kinase domains of four ATCDPKs. Identical amino acids are highlighted.
  • Fig. 3C shows the schematic illustrations of various PK constructs.
  • Fig. 3D shows a photograph of a gel illustrating the immunoprecipitation of eight PKs with anti-HA.
  • Fig. 3E shows a graph demonstrating that ATCDPK1 and ATCDPKla activate stress-inducible transcription.
  • Fig. 4A shows a photograph of a gel illustrating the immunoprecipitation of ATCDPK1 and ATCDPK1(K40M) mutant proteins.
  • Fig. 4B shows a series of photographs demonstrating that the ATCDPK1 (K40M) mutant does not activate stress signaling.
  • Fig. 4C shows a graph demonstrating that PP2C blocks the action of
  • Fig. 4D shows a schematic illustration of a model for stress signal transduction in plant cells.
  • Fig. 5 shows the nucleotide and amino acid sequences of the ATCDPKla PK domain, SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • CDPKl and CDPKl a are closely related Ca 2+ dependent protein kinases that activate a stress-inducible promoter, bypassing stress signals.
  • the effects of CDPKl and CDPKl a are specific since six distinct plant protein kinases, including two other CDPKs, failed to mimic stress signaling. The activation is abolished by a CDPKl mutation in the kinase domain, and diminished by a constitutively active protein phosphatase 2C capable of blocking the stress hormone ABA responses.
  • CDPKs including their PK domains
  • CDPKl and CDPKl a are therefore examples of positive regulators for controlling stress signal transduction in plants. Expression of such regulators in transgenic plants is useful for turning on the stress signal transduction pathway as a means for increasing plant tolerance to multiple stress conditions, including drought, salinity, and extreme temperature conditions.
  • GFP green- fluorescent protein
  • the barley HVA1 promoter was obtained by PCR using barley genomic DNA and two primers: 5TCCACCGAGATGCCGACGCA-3' (SEQ ID NO: 17) and 5'-GTTGGAGGCCATGGTCGTCTCACGAT-3' (SEQ ID NO: 18).
  • the CDPK HVA1 promoter and the SGFP were fused at the ATG Ncol site.
  • the CDPK HVA1 gene has been reported to be activated by multiple stress signals in vegetative tissues (Straub et al., Plant Mol. Biol. 26:617, 1994). Four clones were selected and tested for stress responses with identical results as is discussed below.
  • Maize leaf protoplasts were electroporated with the plasmid DNA carrying HVA1-SGFP and divided (10 5 cells/ml per sample) for various treatments: constant light (15 ⁇ Em "2 S " ') at 23 °C for sixteen hours (Control), 0°C for four hours followed by twelve hours at 23 °C (Cold), 0.2 M NaCl for three hours, washed, and incubated for thirteen hours (Salt), constant darkness for sixteen hours (Dark), and 100 ⁇ M ABA for sixteen hours (ABA) (Fig. 1).
  • the protocol for transient expression analysis using maize leaf protoplasts has been described by Sheen (EMBO J. 12: 3497, 1993) and Chiu et al. (Curr. Biol. 6:225, 1996).
  • HVA1-SGFP into maize leaf protoplasts
  • the expression of GFP was found to be enhanced by cold, high salt, dark, and ABA (Fig. 1).
  • These responses were specific to HVA1-SGFP because the expression of an internal control, generated by fusing the maize ubiquitin promoter (Christensen et al., Plant Mol. Biol. 18:675, 1992) and the ⁇ -glucuronidase gene (UBI-GUS) (Jefferson, Plant Mol. Biol. Rep. 5:387, 1987), was not affected (data not shown).
  • the GFP expression derived from UBI-SGFP was not changed by the same treatments (data not shown).
  • SGFP (UBI) were treated with 1 mM Ca 2+ , 1 mM Ca 2 7l 00 nM A23187, and 1 mM Ca 2+ /100 nM ionomycin, respectively designated "C,” "A,” and “I” in Fig.
  • UBI-SGFP was constructed by inserting the UBI promoter from pAHC27
  • Ca 2 7calmodulin-dependent KPII Ca 2 7calmodulin-dependent KPII (CaMKII) (Kapiloff et al, Proc. Natl. Acad. Sci. USA 88:3710, 1991).
  • this family of PKs carries a calmodulin-like domain at the C-terminus (Fig. 3A). This unique feature presumably allows CDPKs to respond to Ca 2+ signals directly without calmodulin.
  • Various other domains found in CaMKII and CDPK are shown in Fig. 3A, these include: (H) hinge, (N) NH 2 . terminal domain for some CDPKs (J) junction, (EF) EF-hand Ca 2+ -binding site (Harper et al.
  • CDPKs are the most prevalent serine/threonine PKs found in higher plants as the cloning of numerous CDPKs in a broad range of plant species has been reported (Harper et al. Science 252:951, 1991 ; Suen and Choi, Plant Mol. Biol. 17:581 , 1991; Roberts and Harmon, Ann. Rev. Plant Physiol. Plant Mol. Biol. 43:375, 1992; Estruch et al, Proc. Natl. Acad. Sci. USA 91 :8837, 1994; Urao et al. Plant Physiol. 105: 1461, 1994; Urao et al, Mol. Gen. Genet. 244:331, 1994; Harper et al, Biochem.
  • AKl/ATCDPK and ATCDPKla Two CDPKs (ATCDPKl and ATCDPKla) are closely related (96% amino acid and similarity) while the other two CDPKs (AKl/ATCDPK and ATCDPK2 have more divergent sequences (78% and 75% amino acid similarity respectively, to ATCDPKl) (Fig. 3B).
  • AKl/ATCDPK and ATCDPK2 possess calcium-dependent PK activity and the truncated AKl/ATCDPK has calcium-independent (constitutively active) PK activity by (Urao et al, Mol. Gen. Genet. 244:331 , 1994; and Harper et al, Bioch. 33:7267, 1994).
  • ATCDPKla The PK activity of ATCDPKl, however, has not been demonstrated in vitro because it does not phosphorylate common PK substrates (Urao et al, Mol. Gen. Genet. 244:331, 1994).
  • the ATCDPKla cDNA that has restriction enzyme digestion patterns distinct from those of ATCDPKl , was identified during the isolation of ATCDPKl by polymerase chain reaction (PCR) (Minet et al. Plant J. 2:417,1992).
  • PCR polymerase chain reaction
  • the nucleotide and amino acid sequences of the ATCDPKla PK are shown in Fig. 5 (SEQ ID NO: 1 and 2, respectively).
  • GCGGATCCATGGAGACGAAGCCAAACCCTA-3' (SEQ ID NO: 7) and 5*- GTCAAGGCCTTGCTTGTTCATCGACAATCC-3' (SEQ ID NO: 8 ); (ATPKa) 5'-CATGCCATGGCTCCGGCGACTAATTCACCG-3' (SEQ ID NO: 9) and
  • Truncated forms containing all eleven PK domains analogous to the construction of a constitutively active mutant of CaMKII in mammals (Kapiloff et al, Proc. Natl. Acad. Sci. 88, 7267, 1994) (Fig. 3 A and Fig. 3B), were inserted into the plant expression vector with a strong constitutive promoter 35SC4PPDK (Sheen, EMBO J 12:3497, 1993; Chiu et al. Curr. Biol. 6:225,1996). The putative regulatory domains of theses PKs were deleted (Fig. 3C).
  • PKs were fused in frame to a double hemagglutinin (HA) epitope tag (designated DHA in Fig. 3C) at the C-terminus and inserted into a plant expression vector (Sheen, EMBO J. 12:3497, 1993; and Chiu et al, Curr. Biol. 6:225, 1996).
  • HA hemagglutinin
  • PKs in transfected maize leaf protoplasts were demonstrated by immunoprecipitation of [ 35 S] methionine labeled proteins with the anti-HA monoclonal antibody (Fig. 3D).
  • Transfected protoplasts were incubated for four hours to allow mRNA accumulation and then labeled with 200 ⁇ Ci/ml of [ 35 S] methionine for twelve hours before harvest.
  • Immunoprecipitation was carried out based on a published protocol by Kapiloff et al. (Proc. Natl. Acad. Sci. USA 88:3710, 1991). The proteins were separated on a 12.5% SDS-PAGE gel and visualized by fluorography.
  • HVA1-LUC luciferase coding sequence
  • Maize leaf protoplasts were transfected with HVA 1 -LUC alone and incubated without (Fig. 3E, "C") or with 100 ⁇ M ABA (Fig. 3E, "A”). HVA1- LUC was also co-electroporated with the PK constructs (1-8) shown in Fig. 3C and Fig. 3D, and incubated without ABA (Fig. 3E, "1-8"). Relative LUC activities from duplicated samples are shown. About 2% of the cell lysates were used for LUC (Luehrsen et al, Meth. Enz. 216:397, 1992) and GUS assays (Sheen, EMBO J. 12:3497, 1993; and Jefferson, Plant Mol. Biol.
  • CDPKl Activates but PP2C Abolishes Stress Signaling
  • PK activity is important for the activation of the stress- inducible HVA1 promoter
  • a null mutation was made by site-directed mutagenesis to eliminate the ATP binding site (K40) in ATCDPKl (Urao et al, Mol. Gen. Genet. 244:331 , 1994; and Kapiloff et al, Proc. Natl. Acad. Sci. USA 88:3710, 1991) and analyzed as follows. Maize leaf protoplasts were electroporated with HVA1-SGFP alone, or with ATCDPKl (CDPKl) and the ATCDPKl (K40M) mutant (CDPKlmut). About 10 5 protoplasts from each treatment were observed using a fluorescence microscope (Chiu et al, Curr.
  • the kinase mutation did not affect the expression of the protein (Fig. 4A), but it could no longer activate the expression of HVA1-GFP (Fig. 4B).
  • the expression of UBI-SGFP was not affected by ATCDPKl or the ATCDPKl mutant (data not shown). This result indicates that the PK domain of ATCDPKl was required and sufficient to recognize specific protein substances mediating stress signal transduction.
  • the deleted regulatory domain was likely involved in PK activity control in response to stress signals (Harper et al. Science 252:951, 1991 ; Suen and Choi, Plant Mol. Biol. 17:581, 1991 ; Roberts and Harmon, Ann. Rev. Plant Physiol. Plant Mol. Biol.
  • ATCDPKl and ATCDPKl a are positive regulators in plant stress signal transduction
  • a specific and constitutively active Arabidopsis protein phosphatase 2C (PP2C) capable of abolishing ABA responses was examined as follows. Maize leaf protoplasts were electroporated with HVA 1 -LUC alone or with the effectors as indicated. PP2C null did not show PP2C activity (data not shown).
  • the CDPK sequences described herein may be used, together with conventional screening methods of nucleic acid hybridization screening.
  • hybridization techniques and screening procedures are well known to those skilled in the art and are described, for example, in Benton and Davis, Science 196: 180, 1977; Grunstein and Hogness, Proc. Natl. Acad. Sci, USA 72:3961, 1975; Ausubel et al. Current Protocols in Molecular Biology, Wiley Interscience, New York, and Berger and Kimmel, Guide to Molecular Cloning Techniques, 1987, Academic Press, New York.;; and Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • all or part of the CDPK may be used as a probe to screen a recombinant plant DNA library for genes having sequence identity to the CDPK gene or the PK domain.
  • Hybridizing sequences are detected by plaque or colony hybridization according to the methods described below.
  • using all or a portion of the amino acid sequence of the CDPK may be used as a probe to screen a recombinant plant DNA library for genes having sequence identity to the CDPK gene or the PK domain.
  • PK polypeptide SEQ ID NO: 2
  • PK degenerate oligonucleotide probes i.e., a mixture of all possible coding sequences for a given amino acid sequence.
  • oligonucleotides may be based upon the sequence of either DNA strand and any appropriate portion of the PK sequence.
  • General methods for designing and preparing such probes are provided, for example, in Ausubel et al, 1996, Current Protocols in Molecular Biology, Wiley Interscience, New York, and Berger and Kimmel, Guide to Molecular Cloning Techniques, 1987, Academic Press, New York.
  • oligonucleotides are useful for PK gene isolation, either through their use as probes capable of hybridizing to PK complementary sequences or as primers for various amplification techniques, for example, polymerase chain reaction (PCR) cloning strategies.
  • PCR polymerase chain reaction
  • a combination of different oligonucleotide probes may be used for the screening of a recombinant DNA library.
  • the oligonucleotides may be detectably- labeled using methods known in the art and used to probe filter replicas from a recombinant DNA library.
  • Recombinant DNA libraries are prepared according to methods well known in the art, for example, as described in Ausubel et al. (supra), or they may be obtained from commercial sources.
  • PK oligonucleotides may also be used as primers in amplification cloning strategies, for example, using PCR.
  • PCR methods are well known in the art and are described, for example, in PCR Technology, Erlich, ed, Stockton Press, London, 1989; PCR Protocols: A Guide to Methods and Applications, Innis et al, eds. Academic Press, Inc., New York, 1990; and Ausubel et al. (supra).
  • Primers are optionally designed to allow cloning of the amplified product into a suitable vector, for example, by including appropriate restriction sites at the 5' and 3' ends of the amplified fragment (as described herein).
  • PK sequences may be isolated using the PCR "RACE” technique, or Rapid Amplification of cDNA Ends (see, e.g., Innis et al. (supra)).
  • RACE Rapid Amplification of cDNA Ends
  • oligonucleotide primers based on an PK sequence are oriented in the 3' and 5' directions and are used to generate overlapping PCR fragments. These overlapping 3'- and 5 '-end RACE products are combined to produce an intact full-length cDNA. This method is described in Innis et al. (supra); and Frohman et al, Proc. Natl. Acad. Sci. USA 85:8998, 1988.
  • Confirmation of a sequence's relatedness to the PK polypeptide family may be accomplished by a variety of conventional methods including, but not limited to, sequence comparison of the gene and its expressed product.
  • sequence comparison of the gene and its expressed product may be accomplished by a variety of conventional methods including, but not limited to, sequence comparison of the gene and its expressed product.
  • activity of the gene product may be evaluated according to any of the techniques described.
  • a regulator of the stress response e.g., CDPK, PK, or CaM-K sequences
  • CDPK e.g., CDPK, PK, or CaM-K sequences
  • the regulators of the invention may be produced in a prokaryotic host, for example, E.
  • coli or in a eukaryotic host, for example, Saccharomyces cerevisiae, mammalian cells (for example, COS 1 or NIH 3T3 cells), or any of a number of plant hosts including, without limitation, algae, tree species, ornamental species, temperate fruit species, tropical fruit species, vegetable species, legume species, crucifer species, monocots, dicots, or in any plant of commercial or agricultural significance.
  • a eukaryotic host for example, Saccharomyces cerevisiae, mammalian cells (for example, COS 1 or NIH 3T3 cells), or any of a number of plant hosts including, without limitation, algae, tree species, ornamental species, temperate fruit species, tropical fruit species, vegetable species, legume species, crucifer species, monocots, dicots, or in any plant of commercial or agricultural significance.
  • suitable plant hosts include, but are not limited to, Conifers, Petunia, Tomato, Potato, Tobacco, Arabidopsis, Lettuce, Sunflower, Oilseed rape, Flax, Cotton, Sugarbeet, Celery, Soybean, Alfalfa, Medicago, Lotus, Vigna, Cucumber, Carrot, Eggplant, Cauliflower, Horseradish, Morning Glory, Poplar, Walnut, Apple, Grape, Asparagus, Rice, Maize, Millet, Onion, Barley, Orchard grass, Oat, Rye, and Wheat.
  • expression constructs may be expressed in a transgenic plant to turn on the stress signal transduction pathway to enhance plant tolerance to multiple stress conditions.
  • the method of transformation or transfection and the choice of vehicle for expression of the regulator polypeptide will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; Gelvin et al. Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990; Kindle, K, Proc. Natl. Acad. Sci, U.S.A 87:1228, 1990; Potrykus, I, Annu. Rev. Plant Physiol. Plant Mol.
  • Expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P.H. Pouwels et al, 1985, Supp. 1987); Gasser and Fraley (supra); Clontech Molecular Biology Catalog (Catalog 1992/93 Tools for the Molecular Biologist, Palo Alto, CA); and the references cited above.
  • Other expression constructs are described by Fraley et al. (U.S. Pat. No. 5,352,605).
  • a regulator polypeptide e.g. CDPK, PK domain, or
  • CaM-K is produced by a stably-transfected plant cell line, a transiently- transfected plant cell line, or by a transgenic plant.
  • a number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants are available to the public; such vectors are described in Pouwels et al. (supra), Weissbach and Weissbach (supra), and Gelvin et al. (supra). Methods for constructing such cell lines are described in, e.g., Weissbach and Weissbach (supra), and Gelvin et al. (supra).
  • plant expression vectors include (1) a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker.
  • plant expression vectors may also contain, if desired, a promoter regulatory region (for example, one conferring inducible or constitutive, pathogen- or wound-induced, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • nucleic acid sequence encoding a regulator polypeptide e.g., CDPK, PK, or CaM-K sequence
  • a regulator polypeptide e.g., CDPK, PK, or CaM-K sequence
  • the flanking regions may be subjected to mutagenesis.
  • the regulator DNA sequence of the invention may, if desired, be combined with other DNA sequences in a variety of ways.
  • the regulator DNA sequence of the invention may be employed with all or part of the gene sequences normally associated with itself.
  • a DNA sequence encoding a regulator polypeptide e.g., a CDPK, a PK domain, or a CaM-K
  • a DNA construct having a transcription initiation control region capable of promoting transcription and translation in a host cell is combined in a DNA construct having a transcription initiation control region capable of promoting transcription and translation in a host cell.
  • the constructs will involve regulatory regions functional in plants which provide for modified production of the regulator protein as discussed herein.
  • the open reading frame coding for the regulator protein or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region such as the sequence naturally found in the 5' upstream region of the regulator structural gene (e.g., CDPK, PK domain, or CaM-K). Numerous other transcription initiation regions are available which provide for constitutive or inducible regulation.
  • appropriate 5' upstream non-coding regions are obtained from other genes, for example, from genes regulated during meristem development, seed development, embryo development, or leaf development.
  • Transcript termination regions may be provided by the DNA sequence encoding the regulator protein (e.g., CDPK, PK domain, or CaM-K) or any convenient transcription termination region derived from a different gene source.
  • the transcript termination region will contain preferably at least 1-3 kb of sequence 3' to the structural gene from which the termination region is derived.
  • Plant expression constructs having, for example, CDPK as the DNA sequence of interest for expression may be employed with a wide variety of plant life. Such genetically-engineered plants are useful for a variety of industrial and agricultural applications as discussed herein.
  • a useful plant promoter is a caulimovirus promoter, for example, a cauliflower mosaic virus (CaMV) promoter.
  • CaMV cauliflower mosaic virus
  • These promoters confer high levels of expression in most plant tissues, and the activity of these promoters is not dependent on virally encoded proteins.
  • CaMV is a source for both the 35S and 19S promoters. In most tissues of transgenic plants, the CaMV 35S promoter is a strong promoter (see, e.g., Odell et al. Nature 313:810, 1985).
  • the CaMV promoter is also highly active in monocots (see, e.g., Dekeyser et al. Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet. 220:389, 1990). Moreover, activity of this promoter can be further increased (i.e., between 2-10 fold) by duplication of the CaMV 35S promoter (see e.g., Kay et al. Science 236: 1299, 1987; Ow et al, Proc. Natl. Acad. Sci., U.S.A. 84:4870, 1987; and Fang et al. Plant Cell 1 : 141 , 1989).
  • the regulator gene product e.g., CDPK, PK domain, or CaM-K
  • the regulator gene product e.g., CDPK, PK domain, or CaM-K
  • gene promoters each with its own distinct characteristics embodied in its regulatory sequences, shown to be regulated in response to the environment, hormones, and/or developmental cues. These include gene promoters that are responsible for heat-regulated gene expression (see, e.g., Callis et al. Plant Physiol. 88:965, 1988; Takahashi and Komeda, Mol. Gen. Genet. 219:365, 1989; and Takahashi et al, Plant J.
  • light-regulated gene expression e.g., the pea rbcS-3A described by Kuhlemeier et al. Plant Cell 1 :471 , 1989; the maize rbcS promoter described by Schaffner and Sheen, Plant Cell 3:997, 1991 ; or the cholorphyll a/b-binding protein gene found in pea described by Simpson et al, EMBO J. 4:2723, 1985
  • hormone- regulated gene expression for example, the abscisic acid (ABA) responsive sequences from the Em gene of wheat described by Marcotte et al.
  • Plant expression vectors may also optionally include RNA processing signals, e.g, introns, which have been shown to be important for efficient RNA synthesis and accumulation (Callis et al. Genes and Dev. 1 : 1183, 1987).
  • RNA processing signals e.g, introns
  • the location of the RNA splice sequences can dramatically influence the level of transgene expression in plants.
  • an intron may be positioned upstream or downstream of a CDPK, Cam-K, or PK domain polypeptide-encoding sequence in the transgene to modulate levels of gene expression.
  • the expression vectors may also include regulatory control regions which are generally present in the 3' regions of plant genes (Thomburg et al, Proc. Natl. Acad.
  • the 3' terminator region may be included in the expression vector to increase stability of the mRNA.
  • One such terminator region may be derived from the PI-II te ⁇ ninator region of potato.
  • other commonly used terminators are derived from the octopine or nopaline synthase signals.
  • the plant expression vector also typically contains a dominant selectable marker gene used to identify those cells that have become transformed.
  • Useful selectable genes for plant systems include genes encoding antibiotic resistance genes, for example, those encoding resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, or spectinomycin. Genes required for photosynthesis may also be used as selectable markers in photosynthetic-deficient strains. Finally, genes encoding herbicide resistance may be used as selectable markers; useful herbicide resistance genes include the bar gene encoding the enzyme phosphinothricin acetyltransferase and conferring resistance to the broad spectrum herbicide Basta® (Hoechst AG, Frankfurt, Germany).
  • Efficient use of selectable markers is facilitated by a determination of the susceptibility of a plant cell to a particular selectable agent and a determination of the concentration of this agent which effectively kills most, if not all, of the transformed cells.
  • Some useful concentrations of antibiotics for tobacco transformation include, e.g., 75-100 ⁇ g/mL (kanamycin), 20-50 ⁇ g/mL (hygromycin), or 5-10 ⁇ g/mL (bleomycin).
  • a useful strategy for selection of transformants for herbicide resistance is described, e.g., by Vasil et al, supra.
  • Plant Transformation Upon construction of the plant expression vector, several standard methods are available for introduction of the vector into a plant host, thereby generating a transgenic plant. These methods include (1) Agrobacterium- mediated transformation (A. tumefaciens or A. rhizogenes) (see, e.g., Lichtenstein and Fuller, In: Genetic Engineering, vol 6, PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C.P, and Draper, J,. In: DNA Cloning, Vol II, D.M. Glover, ed, Oxford, IRI Press, 1985)), (2) the particle delivery system (see, e.g., Gordon-Kamm et al.
  • Agrobacterium- mediated transformation A. tumefaciens or A. rhizogenes
  • Lichtenstein and Fuller In: Genetic Engineering, vol 6, PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C.P, and Dra
  • the general process for manipulating genes to be transferred into the genome of plant cells is carried out in two phases.
  • the plasmid contains an origin of replication that allows it to replicate in Agrobacterium and a high copy number origin of replication functional in E. coli. This permits facile production and testing of transgenes in E. coli prior to transfer to Agrobacterium for subsequent introduction into plants.
  • Resistance genes can be carried on the vector, one for selection in bacteria, for example, streptomycin, and another that will function in plants, for example, a gene encoding kanamycin resistance or herbicide resistance. Also present on the vector are restriction endonuclease sites for the addition of one or more transgenes and directional T-DNA border sequences which, when recognized by the transfer functions of Agrobacterium, delimit the DNA region that will be transferred to the plant.
  • plant cells may be transformed by shooting into the cell tungsten microprojectiles on which cloned DNA is precipitated.
  • a gunpowder charge 22 caliber Power Piston Tool Charge
  • an air-driven blast drives a plastic macroprojectile through a gun barrel.
  • An aliquot of a suspension of tungsten particles on which DNA has been precipitated is placed on the front of the plastic macroprojectile.
  • the latter is fired at an acrylic stopping plate that has a hole through it that is too small for the macroprojectile to pass through.
  • the plastic macroprojectile smashes against the stopping plate, and the tungsten microprojectiles continue toward their target through the hole in the plate.
  • the target can be any plant cell, tissue, seed, or embryo.
  • the DNA introduced into the cell on the microprojectiles becomes integrated into either the nucleus or the chloroplast.
  • Plant cells transformed with a plant expression vector can be regenerated, for example, from single cells, callus tissue, or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant; such techniques are described, e.g., in
  • Vasil supra Green et al, supra; Weissbach and Weissbach, supra; and Gelvin et al, supra.
  • a cloned CDPK polypeptide (or PK domain or CaM-K) construct under the control of the nos promoter and the nopaline synthase terminator and carrying a selectable marker (for example, kanamycin resistance) is transformed into Agrobacterium. Transformation of leaf discs (for example, of tobacco or potato leaf discs), with vector- containing Agrobacterium is carried out as described by Horsch et al. (Science 227: 1229, 1985). Putative transformants are selected after a few weeks (for example, 3 to 5 weeks) on plant tissue culture media containing kanamycin (e.g. 100 ⁇ g/mL). Kanamycin-resistant shoots are then placed on plant tissue culture media without hormones for root initiation.
  • kanamycin e.g. 100 ⁇ g/mL
  • Kanamycin-resistant plants are then selected for greenhouse growth. If desired, seeds from self- fertilized transgenic plants can then be sowed in a soil-less medium and grown in a greenhouse. Kanamycin-resistant progeny are selected by sowing surfaced sterilized seeds on hormone-free kanamycin-containing media. Analysis for the integration of the transgene is accomplished by standard techniques (see, for example, Ausubel et al. supra; Gelvin et al. supra).
  • Transgenic plants expressing the selectable marker are then screened for transmission of the transgene DNA by standard immunoblot and DNA detection techniques. Each positive transgenic plant and its transgenic progeny are unique in comparison to other transgenic plants established with the same transgene. Integration of the transgene DNA into the plant genomic DNA is in most cases random, and the site of integration can profoundly affect the levels and the tissue and developmental patterns of transgene expression.
  • transgenic lines are usually screened for each transgene to identify and select plants with the most appropriate expression profiles.
  • Transgenic lines are evaluated for levels of transgene expression. Expression at the RNA level is determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis are employed and include PCR amplification assays using oligonucleotide primers designed to amplify only transgene RNA templates and solution hybridization assays using transgene-specific probes (see, e.g., Ausubel et al, supra). The RNA-positive plants are then analyzed for protein expression by Western immunoblot analysis using specific antibodies (see, e.g., Ausubel et al, supra). In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using transgene-specific nucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue.
  • the recombinant regulator protein e.g., CDPK, PK domain, or CaM-K
  • a transgenic plant for example, as described above
  • an anti-regulator polypeptide antibody e.g., produced as described in Ausubel et al, supra, or by any standard technique
  • Lysis and fractionation of regulator-producing cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al, supra).
  • the recombinant protein can, if desired, be further purified, for example, by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, eds. Work and Burdon, Elsevier, 1980).
  • plasmid constructs designed for the expression of regulator gene products are useful for generating transgenic plants having an increased level of tolerance to environmental stress. To achieve such tolerance, it is important to express a regulator gene product at an effective level. Evaluation of the level of stress protection conferred to a plant by expression of regulator gene is determined according to conventional methods and assays.
  • constitutive expression of the PK domain gene in tomato is used to enhance salt stress tolerance.
  • a plant expression vector is constructed that contains a PK cDNA sequence expressed under the control of the nos promoter, a low constitutive promoter. This expression vector is then used to transform tomato according to standard methods.
  • transformed tomato plants and appropriate controls are evaluated according to methods described in Lilus et al. (BioTechnology 14: 177, 1996) and Tarczynski et al. (Science 259:508, 1993).
  • Transformed tomato plants that express a PK domain gene having an increased level of salt tolerance relative to control plants are taken as being useful in the invention.
  • the invention described herein is useful for a variety of agricultural and commercial purposes including, but not limited to, improving tolerance to a variety of environmental stresses, including but not limited to, drought, salinity, cold, and heat, increasing crop yields, improving crop and ornamental quality, and reducing agricultural production costs.
  • a regulator gene e.g., a CDPK, a PK domain, or a CaM-K gene
  • the invention therefore affords a means for producing plants that can live in environments where growth would otherwise be impaired by adverse environmental factors.

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Abstract

L'invention a trait à des procédés permettant de renforcer la résistance de plantes aux agressions du milieu.
PCT/US1997/023019 1996-12-13 1997-12-12 Plantes transgeniques a l'epreuve des agressions du milieu Ceased WO1998026045A1 (fr)

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US6794561B2 (en) 1998-07-10 2004-09-21 E. I. Du Pont De Nemours And Company Plant protein kinases
WO2001007592A3 (fr) * 1999-07-27 2001-10-11 Syngenta Ltd Plantes resistant aux herbicides et procedes de production desdites plantes
WO2001045492A3 (fr) * 1999-12-22 2002-08-01 Basf Plant Science Gmbh Proteines de proteine kinase liees au stress et procedes d'utilisation dans les plantes
US7223903B2 (en) 1999-12-22 2007-05-29 Da Costa E Silva Oswaldo Protein kinase stress-related proteins and methods of use in plants
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US6720477B2 (en) 2000-04-07 2004-04-13 Basf Plant Science Gmbh Signal transduction stress-related proteins and methods of use in plants
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EP1015556A4 (fr) 2004-05-26
EP1015556A1 (fr) 2000-07-05
AU5898298A (en) 1998-07-03

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