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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
• • 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
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  • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8217—Gene switch

Definitions

• the present invention relates to compositions and methods useful for regulation of gene expression in plants through a transactivation system using a chimeric transactivator.
• transgenic plants characteristically have recombinant DNA constructs in their genome that have a protein-coding region operably linked to at least one regulatory region, e.g., a promoter.
• Novel strategies need to be developed for transgene expression that provide for precise spatiotemporal control.
• Conventional approaches to the regulation of plant transgene expression by the fusing of a highly expressed promoter element directly with the protein-coding sequence have proved insufficient to meet the stringent safety and technical demands of plant biotechnology today.
• Commercially the exploitation of plants by transgenic modification is hampered by the inability to introduce and coordinately regulate multiple transgenes in transgenic crops.
• a two component transcription factor/target promoter system could be used to address the above limitations of transgene expression with existing promoters.
• Many positive transcriptional regulatory factors are modular, consisting of a DNA-binding domain and transactivation domain that interacts with components of the transcriptional machinery assembling at the promoter (Ptashne, M. (1988) Nature. Oct 20; 335(6192):683-9.). Fusing combinations of these elements, derived from different kingdoms, results in production of diverse hybrid factors having defined DNA-binding specificity and transcriptional activation function (Moore et al. (2006) Plant J.45:651 -683; Weinmann et al (1994) Plant J 5(4):559- 569; US Patent No. 7,285,416, US Patent No.6,252,136, US Patent No.6,576,422)
• One embodiment of this invention is a recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein, wherein the polynucleotide comprises: (a) a first nucleotide sequence encoding a first amino acid sequence having a DNA-binding domain wherein the first amino acid sequence is SEQ ID NO:15, 32, 34, 36 or 38 or a functional fragment of SEQ ID NO:15, 32, 34, 36 or 38; and (b) a second nucleotide sequence encoding a second amino acid sequence having a transactivation domain wherein the second amino acid sequence is SEQ ID NO:1 1 ,13, 90-96 or 97 or a functional fragment of SEQ ID NO:1 1 ,13, 90-96 or 97 wherein the fusion protein capable of activating transcription of a heterologous polynucleotide that is operably linked to a LexA operator.
• the fusion protein encoded by this recombinant DNA construct is capable of activating transcription of a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence; and further wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell.
• the at least one LexA operator sequence comprises at least one sequence selected from the group consisting of: a recA-type operator sequence and a caa-type operator sequence.
• the recA-type operator sequence comprises the sequence given in SEQ ID NO:22 and the caa-type operator sequence comprises the sequence given in SEQ ID NO:23.
• the transactivator domain is operably linked to a nuclear localization sequence (NLS).
• NLS nuclear localization sequence
• the NLS comprises the sequence of SEQ ID NO:39.
• this invention concerns a vector, cell, plant, or seed comprising at least one of the recombinant DNA constructs described in the present invention.
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) obtaining a plant comprising a first recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein, and a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; and (b) growing the plant of step (a) under conditions in which the first recombinant DNA construct is expressed.
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) obtaining a plant comprising a first recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein; (b) introducing into the plant of step (a) the second recombinant DNA construct comprising a
• heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; and (c) growing the plant under conditions in which the first recombinant DNA construct is expressed.
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) introducing into a regenerable plant cell the first recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein, and a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; and (b) regenerating a transgenic plant from the regenerable plant cell of step (a) wherein the transgenic plant comprises in its genome the first recombinant DNA construct and the second recombinant DNA construct.
• the plant may be grown under conditions in which the first recombinant
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) introducing into a regenerable plant cell the first recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein, and a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; (b) regenerating a transgenic plant from the regenerable plant cell of step (a) wherein the transgenic plant comprises in its genome the first recombinant DNA construct and the second recombinant DNA construct; and (c) obtaining a progeny plant derived from the transgenic plant
• recombinant DNA constructs of step (a) may be introduced into the regenerable plant cell sequentially or simultaneously.
• the invention encompasses regenerated, mature and fertile transgenic plants comprising the recombinant DNA constructs described above, transgenic seeds produced therefrom, T1 and subsequent generations.
• the transgenic plant cells, tissues, plants, and seeds may comprise at least one recombinant DNA construct of interest.
• the plant comprising the recombinant constructs described in the present invention is selected from the group consisting of:
• Arabidopsis maize, soybean, sunflower, sorghum, canola, mustard, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
• FIG.1 shows a map of the MiniOmega_GUS-pBC construct.
• FIG.2 shows a map of the pZGG vector.
• FIG.3 shows a schematic depicting the mechanism of the transactivation system disclosed in the present invention.
• FIG.4 shows the MUG analysis for Driver 4 with different operator combinations (Driver4 and 6XrecA operator, Driver4 and 2Xcaa operator, Driver4 and hybrid operator) compared to the negative control (Driver4-miniQ; no operator).
• FIG.5 shows the MUG analysis for Driver 6 with different operator
• SEQ ID NOS:1 and 2 are the forward and reverse primers, respectively, for amplifying the full-length LexA gene.
• SEQ ID NOS:3 and 4 are the forward and reverse primers, respectively, for amplifying the LexA ORF.
• SEQ ID NOS:5 and 6 are the forward and reverse primers, respectively, for amplifying the AtCBFI ORF.
• SEQ ID NOS:7 and 8 are the forward and reverse primers, respectively, for amplifying the AtCBF3 ORF.
• SEQ ID NO:9 is the sequence of the Arabidopsis thaliana CBF1 cDNA.
• SEQ ID NO:10 is the nucleotide sequence of the Arabidopsis thaliana CBF1
• SEQ ID NO:1 1 is the amino acid sequence of the Arabidopsis thaliana CBF1 , encoded by SEQ ID NO:10.
• SEQ ID NO:12 is the nucleotide sequence of the Arabidopsis thaliana CBF3
• SEQ ID NO:13 is the amino acid sequence of the Arabidopsis thaliana CBF3, encoded by SEQ ID NO:12.
• SEQ ID NO:14 is the nucleotide sequence of the LexA ORF.
• SEQ ID NO:15 is the amino acid sequence of the LexA protein, encoded by SEQ ID NO:14.
• SEQ ID NO:16 is the sequence of the CaMV 35S promoter minimal sequence.
• SEQ ID NO:17 s the TMV omega sequence.
• SEQ ID NO:18 s the sequence of the Minicorepromoter upper oligo.
• SEQ ID NO:19 s the sequence of the Minicorepromoter lower oligo.
• SEQ ID NO:20 s the LexA full-length sequence.
• SEQ ID NO:21 s the CaMV 35S promoter-TMV omega sequence.
• SEQ ID NO:22 s the sequence of a recA operator.
• SEQ ID NO:23 s the sequence of a caa operator.
• SEQ ID NO:24 is the sequence of a hybrid operator comprising 2 copies of a recA operator (SEQ ID NO:22) operably linked to 2 copies of a caa operator (SEQ ID NO:23.
• SEQ ID NO:25 and SEQ ID NO:26 are the sequences of the oligonucleotides used for recA operator multimerization.
• SEQ ID NO:27 and SEQ ID NO:28 are the sequences of the oligonucleotides used for caa operator multimerization.
• SEQ ID NO:29 and SEQ ID NO:30 are the sequences of the oligonucleotides used for hybrid operator synthesis.
• SEQ ID NO:31 is the nucleotide sequence of a LexA wt DBD.
• SEQ ID NO:32 is the amino acid sequence of the LexA wt DBD, encoded by the sequence given in SEQ ID NO:31 .
• SEQ ID NO:33 is the nucleotide sequence of the LexAMNF mutant of LexA.
• SEQ ID NO:34 is the amino acid sequence of LexAMNF mutant of LexA, encoded by the sequence given in SEQ ID NO:33.
• SEQ ID NO:35 is the nucleotide sequence of the LexAFL mutant of LexA.
• SEQ ID NO:36 is the amino acid sequence of the LexAFL mutant of LexA, encoded by the sequence given in SEQ ID NO:35.
• SEQ ID NO:37 is the nucleotide sequence of the LexAFLNLS mutant of LexA.
• SEQ ID NO:38 is the amino acid sequence of the LexAFLNLS mutant of LexA, encoded by the sequence given in SEQ ID NO:37.
• SEQ ID NO:39 is the consensus NLS sequence.
• SEQ ID NOS:40-46 are the sequences of Arabidopsis thaliana CBF1 fragments A-G respectively, as given in Table 3.
• SEQ ID NO:47 is the sequence of Arabidopsis thaliana CBF3 fragment I, as given in Table 3.
• SEQ ID NOS:48-74 and SEQ ID NOS: 81 -89 are the sequences of the primers used for amplifying the various LexA and CBF fragments and creating their fusions, as given in the tables 4-7.
• SEQ ID NOS:75-80 are the sequences of the different LexAFL and LexAFLNLS fragments used for amplifying LexAFL and LexAFLNLS.
• SEQ ID NOS:90-96 are the amino acid sequences of Arabidopsis thaliana CBF1 fragments A-G respectively, encoded by the nucleotide sequences given in SEQ ID NOS:40-46.
• SEQ ID NO:97 is the amino acid sequence of the Arabidopsis thaliana CBF3 fragment I, encoded by the nucleotide sequence given in SEQ ID NO:47.
• SEQ ID NO:98 is the LexA binding consensus sequence in Gram negative bacteria.
• SEQ ID NO:99 is the LexA binding consensus sequence in Gram positive bacteria.
• a monocot of the current invention includes the Gramineae.
• a dicot of the current invention includes the following families:
• nucleotide sequence refers to a complement of a given nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
• Transgenic refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event.
• a heterologous nucleic acid such as a recombinant DNA construct
• the term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross- fertilization, non-recombinant viral infection, non-recombinant bacterial
• Gene as it applies to plant cells encompasses not only chromosomal
• components e.g., mitochondrial, plastid
• Plant includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same.
• Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
• Progeny comprises any subsequent generation of a plant.
• Transgenic plant includes reference to a plant which comprises within its genome a heterologous polynucleotide.
• heterologous polynucleotide For example, the heterologous
• polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
• the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.
• Heterologous with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human
• nucleic acid sequence refers to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
• Nucleotides are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), "K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
• Polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• the terms “polypeptide”, “peptide”, “amino acid sequence”, and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
• mRNA essential RNA
• mRNA RNA that is without introns and that can be translated into protein by the cell.
• cDNA refers to a DNA that is complementary to and synthesized from an mRNA template using the enzyme reverse transcriptase.
• the cDNA can be single- stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
• EST is a DNA sequence derived from a cDNA library and therefore is a sequence which has been transcribed.
• An EST is typically obtained by a single sequencing pass of a cDNA insert.
• the sequence of an entire cDNA insert is termed the "Full-Insert Sequence” (“FIS").
• FIS Frull-Insert Sequence
• a "Contig” sequence is a sequence assembled from two or more sequences that can be selected from, but not limited to, the group consisting of an EST, FIS and PCR sequence.
• a sequence encoding an entire or functional protein is termed a
• CCS Complete Gene Sequence
• “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product has been removed.
• Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.
• isolated refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment.
• Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
• Recombinant refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
• Recombinant also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural
• transformation/transduction/transposition such as those occurring without deliberate human intervention.
• Recombinant DNA construct refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a
• recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
• “Operably linked” refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other.
• a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.
• “Expression” refers to the production of a functional product.
• expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.
• “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in a null segregating (or non- transgenic) organism from the same experiment.
• Phenotype means the detectable characteristics of a cell or organism.
• a “transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.
• Transformation refers to both stable transformation and transient transformation.
• “Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.
• Transient transformation refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.
• crossing means the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants).
• progeny e.g., cells, seeds or plants.
• the term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant).
• crossing refers to the act of fusing gametes via pollination to produce progeny.
• a "favorable allele” is the allele at a particular locus that confers, or contributes to, a desirable phenotype, e.g., increased cell wall digestibility, or alternatively, is an allele that allows the identification of plants with decreased cell wall digestibility that can be removed from a breeding program or planting
• a favorable allele of a marker is a marker allele that
• introductiond means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into a cell, means “transfection" or
• transformation or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
• the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
• PENALTY 10).
• KTUPLE 2
• GAP PENALTY 5
• DIAGONALS SAVED 5.
• KTUPLE 2
• GAP PENALTY 5
• WINDOW 4 and
• DIAGONALS SAVED 4.
• Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook”).
• a recombinant DNA construct (including a suppression DNA construct) of the present invention may comprise at least one regulatory sequence.
• regulatory sequences or “regulatory elements” are used interchangeably and refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.
• Promoter refers to a nucleic acid fragment capable of controlling
• Promoter functional in a plant is a promoter capable of controlling
• tissue-specific promoter and “tissue-preferred promoter” are used interchangeably to refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell.
• “Developmentally regulated promoter” refers to a promoter whose activity is determined by developmental events.
• Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
• 35S or UBI promoter may have pleiotropic effects, although candidate gene efficacy may be estimated when driven by a constitutive promoter.
• Use of tissue-specific and/or stress-specific promoters may eliminate undesirable effects but retain the ability to produce a desired phenotype, e.g., to enhance drought tolerance. This effect has been observed in Arabidopsis (Kasuga et al. (1999) Nature Biotechnol. 17:287-91 ).
• Suitable constitutive promoters for use in a plant host cell include, but are not limited to, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.
• viral promoters may be utilized in plant expression vectors. These include the 35S RNA and 19S RNA promoters of CaMV (Brisson, et al., Nature, 310:51 1 , 1984, Odell, et al., Nature, 313:810, 1985); the promoter from Figwort Mosaic Virus (FMV) (Gowda, et al., J. Cell Biochem.,
• tissue-specific or developmentally regulated promoter it may be desirable to use a tissue-specific or developmentally regulated promoter.
• a tissue-specific or developmentally regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in the cells/tissues of a plant critical to tassel development, seed set, or both, and limits the expression of such a DNA sequence to the period of tassel development or seed maturation in the plant. Any identifiable promoter may be used in the methods of the present invention which causes the desired temporal and spatial expression.
• Promoters which are seed or embryo-specific and may be useful in the invention include, but are not limited to, soybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1 :1079-1093 (1989)), patatin (potato tubers) (Rocha-Sosa, M., et al. (1989) EMBO J. 8:23-29), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W.G., et al. (1991 ) Mo/. Gen. Genet. 259:149-157;
• Such examples include, but are not limited to, Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds (Vanderkerckhove et ai, Bio/Technology 7 ⁇ 929-932 (1989)), bean lectin and bean beta-phaseolin promoters to express luciferase (Riggs et al., Plant Sci. 63:47-57 (1989)), and wheat glutenin promoters to express chloramphenicol acetyl
• Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals.
• Inducible or regulated promoters include, but are not limited to, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
• inducible promoters useful in plants include those induced by chemical means, such as the yeast metallothionein promoter which is activated by copper ions (Mett, et al., (1993) Proc. Natl. Acad. Sci., U.S.A., 90:4567); In2-1 and ln2-2 regulator sequences which are activated by substituted benzenesulfonamides, e.g., herbicide safeners (Hershey, et al., (1991 ) Plant Mol. Biol., 17:679): and the GRE regulatory sequences which are induced by glucocorticoids (Schena, et al. (1991 ), Proc. Natl. Acad. Sci., U.S.A., 88:10421 ). Plant promoters also include the light-inducible promoter from the small subunit of ribulose bis-phosphate
• hormones such as abscisic acid (Marcotte et al., (1989) Plant Cell 1 : 969); wounding (e.g., wunl, Siebertz et al., (1989) Plant Cell 1 : 961 ; and chemicals such as methyl jasminate or salicylic acid (Gatz et al., (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48: 89- 108).
• tissue specific promoters may also be utilized for expression of genes in plants.
• tissue specific promoters useful in transgenic plants include, but are not limited to the cdc2a promoter and cyc07 promoter (Ito, et al.,(1994) Plant Mol. Biol., 24:863; Martinez, et al. (1992) Proc. Natl. Acad. Sci. USA, 89:7360;
• tissue specific promoters that are utilized in plants include, but are not limited to, the histone promoter (Atanassova, et al ., (1992) Plant Journal, 2:291 ); the cinnamyl alcohol dehydrogenase (CAD) promoter (Feuillet, et al., (1995) Plant Mol. Biol., 27:651 ); the mustard CHS1 promoter (Kaiser, et al., (1995) Plant Mol.
• CAD cinnamyl alcohol dehydrogenase
• expression can be controlled by using promoters such as those acting at
• promoters include, but are not limited to, root-specific promoters such as root-specific promoters disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186 or the prxEa promoter in Wanapu and Shinmyo (1996) Ann. N. Y. Acad. Sci. 782:107-1 13 or Miao et al. (1991 ) P/anf Cell 3:1 1 -22 or Hirel et al. (1992) Plant Mol. Biol. 20:207-218), seed-specific promoters such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697, the oleate 12- hydroxylase: desaturase promoter from Lesquerella (Broun et al (1998) Plant J.
• root-specific promoters such as root-specific promoters disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186 or the prxEa promoter
• inducible or tissue-specific promoters include, but are not limited to, Rd 29a (Yamaguchi-Shinozaki and Shinozaki (1993) Plant Physiol. Mar; 101 :1 1 19-20), LTP1 (Thoma et al. (1994) Plant Physiol, 105(1 ):35-45), STM (Long et al. (1996) Nature. 1996 379:66-9), rbcS (Krebbers (1 988) Plant Mol Biol. 1 1 : 745-759), sucrose synthase (Martin et al. (1993) Plant J. 4:367-77), EIR1
• Plant RNA polymerase II promoters like those of other higher eukaryotes, are comprised of several distinct “cis-acting transcriptional regulatory elements,” or simply “cis-elements,” each of which appears to confer a different aspect of the overall control of gene expression. Examples of such cis-acting elements include, but are not limited to, such as TATA box and CCAAT or AGGA box.
• the promoter can roughly be divided in two parts: a proximal part, referred to as the core, and a distal part.
• the proximal part is believed to be responsible for correctly assembling the RNA polymerase II complex at the right position and for directing a basal level of transcription, and is also referred to as "minimal promoter" or "basal promoter”.
• the distal part of the promoter is believed to contain those elements that regulate the spatio-temporal expression.
• other regulatory regions have also been described, that contain enhancer and/or repressors elements
• the latter elements can be found from a few kilobase pairs upstream from the transcription start site, in the introns, or even at the 3' side of the genes they regulate (Rombauts, S. et al.
• Enhancer sequences refer to the sequences that can increase gene expression. These sequences can be located upstream, within introns or
• the transcribed region is comprised of the exons and the intervening introns, from the promoter to the transcription termination region.
• the enhancement of gene expression can be through various mechanisms which include, but are not limited to, increasing transcriptional efficiency,
• Recombinant DNA constructs of the present invention may also include other regulatory sequences, including but not limited to, translation leader sequences, introns, and polyadenylation recognition sequences.
• an "intron” is an intervening sequence in a gene that is transcribed into RNA and then excised in the process of generating the mature mRNA. The term is also used for the excised RNA sequences.
• An "exon” is a portion of the sequence of a gene that is transcribed and is found in the mature messenger RNA derived from the gene, and is not necessarily a part of the sequence that encodes the final gene product.
• An "enhancing intron” is an intronic sequence present within the transcribed region of a gene which is capable of enhancing expression of the gene when compared to an intronless version of an otherwise identical gene.
• An enhancing intronic sequence might also be able to act as an enhancer when located outside the transcribed region of a gene, and can act as a regulator of gene expression independent of position or orientation (Chan et. al. (1999) Proc. Natl. Acad. Sci. 96: 4627-4632; Flodby et al. (2007) Biochem. Biophys. Res. Commun. 356: 26-31J.
• Eukaryotic transcriptional activators are modular proteins that are typically composed of a sequence-specific DNA binding domain and an activating region (or domain). Activators interact through their activating regions with components of the RNA polymerase II transcriptional apparatus that bind promoters. It has been shown that some activators can also interact with factors that modify or remodel nucleosomes (chromatin). According to their amino acid constitution, activation domains have been classified as acidic (e.g. the viral VP16 and the yeast Gal4), glutamine-rich (e.g. SP1 ) or proline-rich (e.g. CTF).
• acidic e.g. the viral VP16 and the yeast Gal4
• glutamine-rich e.g. SP1
• proline-rich e.g. CTF
• Binding of the activator to DNA serves merely to locate it to a position from which the activation domain can activate transcription. DNA binding per se is clearly not sufficient for activating transcription.
• Many genes, especially in higher eukaryotes, are activated not by just one but by several activators that act in concert. The use of several, and often different, activators to activate one gene establishes a combinatorial control that allows differential regulation of many genes with a relatively small number of transcriptional activators.
• Transactivation refers herein to the activation of the expression of a heterologous polynucleotide (effector locus) by the introduction of a “transcriptional activator” (activating locus or driver locus).
• the "transcriptional activator” referred to herein may be a chimeric transcriptional activator, comprising DNA-binding domain and transactivation domains from different proteins.
• the "DNA-binding domain” referred to herein may be the DNA-binding domain from a LexA protein.
• the transactivator domain may be a CBF1 or CBF3 "transactivator domain”.
• the "transactivation system” described herein comprises generation of two different loci.
• the first locus comprises the “activator locus” or the “driver locus” and the second locus comprises the "effector locus”.
• the "activator locus” comprises a promoter functional in a plant cell, operably linked to a chimeric transcriptional activator comprising a LexA DNA-binding domain and a transactivator domain, e.g., the activation domain of a CBF protein, wherein the LexA DNA-binding domain and the activation domain are operably linked to each other.
• the chimeric transactivator may comprise the LexA N-terminal DNA binding domain.
• the chimeric transactivator may comprise residues 1 -87 of the LexA polypeptide.
• the chimeric transactivator may comprise the full length LexA polypeptide.
• chimeric transcriptional activator fusion protein or “chimeric protein” are used interchangeably herein and refer to a fusion protein comprising the DNA-binding domain of a LexA repressor protein and a transactivation domain, e.g., the activator domain of the C-repeat binding factor (CBF) protein.
• the chimeric transactivator protein may comprise the DNA-binding domain (DBD) or the full length LexA protein.
• the fusion protein comprises a first nucleotide sequence encoding a first amino acid sequence having a DNA binding domain wherein the first amino acid sequence is SEQ ID NO:15, 32, 34, 36 or 38 or a functional fragment of SEQ ID NO:15, 32, 34, 36 or 38; and a second nucleotide sequence encoding a second amino acid sequence encoding a second amino acid sequence having a transactivation domain wherein the second amino acid sequence is SEQ ID NO:1 1 , 13, 90-96 or 97 or a functional fragment of SEQ ID NO:1 1 , 13, 90-96 or 97.
• effector locus comprises a second recombinant construct comprised of at least one LexA binding site (operator sequence) operably linked to a minimal promoter and a heterologous polynucleotide.
• heterologous polynucleotide is regulated by the expression of the fusion protein.
• the heterologous polynucleotide is operably linked to a minimal promoter.
• a minimal promoter contains only the proximal promoter elements for binding of the basal transcription apparatus but lacks binding sites for endogenous transcription factors. It is therefore transcriptionally inactive allowing the construct to be introduced into plants and maintained without inducing expression of the
• heterologous polynucleotide The minimal promoter is used to minimize the background expression in the absence of the transactivator.
• minimal promoters are well known in the art and several are described in U.S. patent application publication numbers 2007/0009487 and 2006/0242717.
• Examples of minimal promoters include, but are not limited to, CaMV 35S minimal promoter, SV40 minimal promoter, and Hsp70 minimal promoter.
• a minimal or basal promoter is a polynucleotide molecule that is capable of recruiting and binding the basal transcription machinery.
• the "transactivation system” described herein may comprise generation of two different kinds of plant lines. One is referred to herein as the “activator line”, “activator plant line” or the “driver line” and the other line is referred to herein as the "effector line” or “effector plant line”.
• the “activator line” comprises the activator locus and the "effector line” comprises the effector locus comprising the
• heterologous polynucleotide the expression of which is regulated by the expression of the chimeric transcriptional activator.
• Each of these "driver” (or “activator”) and “effector” genes can be separately transformed into the genome of a plant species such that transformants harboring one component are distinct from transformants harboring the second component.
• the two components can then be combined into the genome of one plant by genetic crossing of the two transformants harboring the two separate components.
• the expression of the heterologous polynucleotide in the progeny plant which comprises the driver locus as well as the activator locus is regulated by the expression of the chimeric transcriptional activator.
• the expression of the chimeric transcriptional activator may be regulated by constitutive, tissue-specific or inducible promoter or regulatory elements.
• Crossing a driver plant line with an effector plant line generates double transgenic offspring capable of expressing the heterologous polynucleotide regulated by the expression of the chimeric transactivator.
• combinatorial experiments can be done for bringing the driver locus and the effector locus together in the same plant: one driver line can be crossed to different effector lines to express different heterologous polynucleotides with the same expression pattern and regulatory control. Conversely, the same effector line can be crossed to various drivers to express one heterologous polynucleotide in different spatiotemporal manner.
• the plant may be a crop plant such as maize, soybean, wheat, corn, potato, cotton, rice, oilseed rape (including canola), sunflower, alfalfa, sugarcane and turf; or a fruit or vegetable plant, such as apple, banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grape, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits (such as peach, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, brussel sprouts and kohlrabi).
• a crop plant such as maize, soybean, wheat, corn, potato, cotton, rice, oilseed rape (including canola), sunflower, alfalfa, sugarcane and turf
• a fruit or vegetable plant such as apple, banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot,
• Other crops, fruits and vegetables whose trait may be improved include barley, sorghum, currant, avocado, citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, currant, cherries, nuts such as the walnut and peanut, pear, endive, leek, roots, such as arrowroot, beet, cassava, turnip, radish, yam, sweet potato and beans.
• heterologous polynucleotide can be combined into the genome of a plant cell by transforming a first plant cell with the first construct, transforming a second plant cell with the second construct, growing fertile mature plants from the first and second transformed plant cells and genetically crossing the transformed plants to produce progeny whose genome contains the first and second constructs.
• the LexA (locus for X-ray sensitivity A) protein is a regulator of the SOS response to DNA damage in Escherichia coli.
• RecA recombinase A
• the LexA repressor stimulates cleavage of the LexA repressor, inducing more than 40 genes that comprise the SOS global regulatory network.
• LexA gene encodes a 202 amino acid protein that folds into two structurally defined domains, which are linked by a flexible hinge region.
• LexA protein is a repressor protein, which, during normal bacterial growth downregulates its own expression and, in E. coli, the expression of at least 43 unlinked genes.
• CCD carboxy-terminal domain
• NTD amino-terminal domain
• LexA binds as a dimer with varying affinities to single or multiple copies of gene-specific LexA DNA-binding motifs (LexAop) found upstream of its target genes.
• the key catalytic residues in LexA are Ser1 19 and Lys156.
• the Ser-Lys dyad catalyzes the cleavage of the bond between residues Ala84 and Gly85.
• the consensus DNA target for E. coli LexA is CTGT-N 8 -ACAG (SEQ ID NO:98), which consists of a palindrome. This consensus is conserved in many Gram-negative bacteria, whilst in Gram-positive bacteria; the consensus is GAAC- N 4 -GTTC (SEQ ID NO:99). In E. coli, approximately 30 LexA binding sites have been identified, but LexA also appears to interact with at least 19 targets that lack a conventional site. Promoters of different genes of the SOS response pathway in E.Coli have different LexA operator sequences, the LexA operator sequence upstream of the recA gene is called the "rec operator” or “recA operator”, and that found in the colicin A promoter is called the "caa operator”. The binding affinity of LexA towards these sequences is different.
• LexA operator or "LexA DNA-binding motif as used herein refers to the polynucleotide sequence to which the LexA binds through its DNA-binding domain (DBD).
• DBD DNA-binding domain
• rec operator and "recA operator”, used interchangeably herein refer to the LexA operator sequence found within the recA promoter and
• One or multiple copies of the recA operator can be operably linked to each other to comprise the target sequence that LexA can bind to.
• caa operator refers to herein as the LexA operator sequence found within the colicin A promoter and corresponds to SEQ ID NO:23. Single or multiple copies of the recA operator can be operably linked to each other to comprise the target sequence that LexA can bind to.
• hybrid operator refers to a target sequence that LexA can bind to, that is comprised of at least one copy each of at least two non- identical LexA operator sequences.
• the "hybrid operator” can comprise one copy each of a recA operator (SEQ ID NO:22) and a caa operator (SEQ ID NO: 23).
• the “hybrid operator” can comprise two copies each of a recA operator (SEQ ID NO:22) and a caa operator (SEQ ID NO:23).
• the “hybrid operator” can comprise more than two copies each of a recA operator (SEQ ID NO:22) and a caa operator (SEQ ID NO:23).
• the chimeric transactivator protein may comprise the DNA-binding domain (DBD) or the full-length LexA protein.
• the LexA protein may comprise one or more amino acid changes to increase its activity and stability.
• the LexA polypeptide may have a mutation at residue 85 to change the amino acid Glycine (Gly) to Alanine (G85 to A85).
• the LexA polypeptide may have a mutation at residue 71 to change the amino acid Glutamine (Glu) to Lysine (E71 to K71 ).
• the LexA polypeptide may have a mutation at residue 1 19 to change the amino acid Serine (Ser) to Alanine (S1 19 to A1 19).
• NLS consensus sequence may be operably linked to the chimeric transactivator.
• sequence of the NLS is SEQ ID NO:39
• CBF C-repeat/dehydration responsive element binding factor protein
• DREB dehydration responsive element binding factor
• the polynucleotide sequence of the transactivation domain of the CBF protein comprises the nucleotide sequence of SEQ ID NOS:1 1 , 13, 90-96 or 97.
• DREB proteins namely, DREB1 and DREB2, involved in two separate signal transduction pathways under low temperature and dehydration, respectively, are important APETALA2 (AP2)/ethylene responsive factor (ERF) plant
• DREB genes play an important role in the ABA-independent stress-tolerance pathways that induce the expression of various stress-responsive genes in plants.
• the first isolated cDNAs encoding DRE (C-repeat/dehydration responsive element) binding proteins, CBF1 (CRT binding factorl ), DREB1A and DREB2A were first isolated by using yeast one-hybrid screening (Stockinger, E.J et al. (1997) Proc Natl Acad Sci. 94:1035-1040; Liu et al., 1998) from Arabidopsis. Since then, numerous DREB genes have been isolated from a number of plants. These proteins specifically bind to the DRE sequence and activate the expression of genes driven by it.
• DREB1 B/CBF1/ At4 g25490, DREB1 A/CBF3/ At4 g25480, and DREB1 C/CBF2/ At4 g25470 genes lie in tandem on chromosome 4 of Arabidopsis.
• DREB1 F/DDF1 are induced by osmotic stress, suggesting the existence of cross- talk between the DREB1 and the DREB2 pathways.
• the Arabidopsis CBF proteins activate expression of a set of genes whose upstream regulatory sequences typically harbor one or more copies of the
• CRT/DRE C-repeat/dehydration responsive element
• COR the cis-acting DNA regulatory element controlling low temperature induced expression of the Arabidopsis cold- regulated (COR) genes.
• the NH3-terminal 1 15 amino acids direct CBF1 to target genes and the COOH-terminal 98 amino acids function in trans-activation.
• the CBFs are members of the AP2 domain-containing protein superfamily that has about 145 members in Arabidopsis (Riechmann and Meyerowitz, 1998 Biol Chem. 379(6):633-46.) and are distinguished by the presence of the CBF signature sequences (Jaglo, K.R et al., 2001 Plant Physiol. 127(3): 910-917). These two motifs flank the AP2 domain and are thought to impart specificity and targeting of CBFs to their cognate cis-acting DNA binding site, the CRT/DRE.
• the CBFs are not unique to Arabidopsis; cDNAs encoding CBF-like proteins have been identified and characterized in numerous species across the plant kingdom (Jaglo et al., 2001 ; Choi et al., 2002; Gao et al., 2002; Dubouzet et al., 2003;Zhang et al., 2004). The CBFs have been shown to function across species barriers.
• nucleic acid of interest can be expressed using the methods of the present invention.
• nucleic acids include, but are not limited to, disease and insect resistance genes, genes conferring nutritional value, genes to confer male and/or female sterility, antifungal, antibacterial or antiviral genes, and the like.
• the method can be used to express any nucleic acid that controls gene expression.
• nucleic acids that could be used to control gene expression include, but are not limited to, antisense oligonucleotides, suppression DNA constructs, or nucleic acids encoding transcription factors.
• Genes of interest can be genes conferring modified agronomic traits and characteristics including, but not limited to, yield, heterosis, oil content and nutritional value.
• the modified agronomic trait may be an increase in the trait.
• genes of interest include, for example, those genes involved in information, such as Zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat-shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics and commercial products.
• the gene of interest may also be a screenable or selectable marker.
• Agronomically important traits such as oil, starch and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing or decreasing the content of oleic acid, saturated and
• the recombinant constructs can also contain a selectable or a screenable marker, or both.
• the nucleic acid sequence serving as the selectable or a screenable marker functions to produce a phenotype in cells that facilitates their identification relative to cells not containing the marker.
• Useful selectable and screenable markers include, but are not limited to,
• GUS green fluorescent protein
• LUX luciferase
• Selectable marker genes may be utilized for the selection of transformed cells or tissues.
• selectable marker genes include, but are not limited to, genes encoding antibiotic resistance, such as nptll which encodes neomycin phosphotransferase II (NEO), hpt which encodes hygromycin phosphotransferase
• HPT monocot-optimized cyanamide hydratase gene
• moCAH monocot-optimized cyanamide hydratase gene
• selectable marker genes are not meant to be limiting. Any selectable marker gene can be used in the present invention.
• the gene sequence(s) may be modified to optimize for increased expression in the transformed plant. Examples of such modifications include, but are not limited to, synthesizing the genes using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1 -1 1 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831 , and 5,436,391 and Murray et al. (1989) Nucleic Acids Res. 17:477-498.
• One embodiment of this invention is a recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein, wherein the polynucleotide comprises: (a) a first nucleotide sequence encoding a first amino acid sequence having a DNA-binding domain wherein the first amino acid sequence is SEQ ID NO:15, 32, 34, 36 or 38 or a functional fragment of SEQ ID NO:15, 32, 34, 36 or 38; and (b) a second nucleotide sequence encoding a second amino acid sequence having a transactivation domain wherein the second amino acid sequence is SEQ ID NO:1 1 ,13, 90-96 or 97 or a functional fragment of SEQ ID NO:1 1 ,13, 90-96 or 97 wherein the fusion protein capable of activating transcription of a heterologous polynucleotide that is operably linked to a LexA operator.
• the fusion protein encoded by this recombinant DNA construct is capable of activating transcription of a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence; and further wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell.
• the at least one LexA operator sequence comprises at least one sequence selected from the group consisting of: a recA-type operator sequence and a caa-type operator sequence.
• the recA-type operator sequence comprises the sequence given in SEQ ID NO:22 and the caa-type operator sequence comprises the sequence given in SEQ ID NO:23.
• the transactivator domain is operably linked to a nuclear localization sequence (NLS).
• the transactivator domain may be from a CBF protein.
• the NLS comprises the sequence of SEQ ID NO:39.
• this invention concerns a vector, cell, plant, or seed comprising at least one of the recombinant DNA constructs described in the present invention.
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) obtaining a plant comprising a first recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein, and a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; and (b) growing the plant of step (a) under conditions in which the first recombinant DNA construct is expressed.
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) obtaining a plant comprising a first recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein; (b) introducing into the plant of step (a) a second recombinant DNA construct comprising a
• heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; and (c) growing the plant under conditions in which the first recombinant DNA construct is expressed.
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) obtaining a plant comprising a first recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; (b) introducing into the plant of step (a) a second recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein; and (c) growing the plant of step (b) under conditions in which the second
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) introducing into a regenerable plant cell a first recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein, and a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; (b) regenerating a transgenic plant from the regenerable plant cell of step (a) wherein the transgenic plant comprises in its genome the first recombinant DNA construct and the second recombinant DNA construct; and (c) growing the transgenic plant of step (b) under conditions in which
• One embodiment of the present invention is a method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of: (a) introducing into a regenerable plant cell a first recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein as described herein, and a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; (b) regenerating a transgenic plant from the regenerable plant cell of step (a) wherein the transgenic plant comprises in its genome the first recombinant DNA construct and the second recombinant DNA construct; and (c) obtaining a progeny plant derived from the transgenic
• recombinant DNA constructs of step (a) may be introduced into the regenerable plant cell sequentially or simultaneously.
• the invention encompasses regenerated, mature and fertile transgenic plants comprising the recombinant DNA constructs described above, transgenic seeds produced therefrom, T1 and subsequent generations.
• the transgenic plant cells, tissues, plants, and seeds may comprise at least one recombinant DNA construct of interest.
• the plant comprising the recombinant constructs described in the other embodiments is selected from the group consisting of:
• Arabidopsis maize, soybean, sunflower, sorghum, canola, mustard, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
• the present invention concerns a cell, a
• the cell may be eukaryotic, e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell.
• the microorganism may be
• Agrobacterium e.g. Agrobacterium tumefaciens or Agrobacterium rhizogenes.
• One embodiment is a method for regulating the expression of a heterologous polynucleotide in a cell (or microorganism) comprising introducing into the cell (or microorganism) with the recombinant DNA constructs of the present invention.
• the cell (or microorganism) comprising the recombinant DNA construct is also included.
• the cell is eukaryotic cell, e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell.
• the microorganism may be Agrobacterium, e.g. Agrobacterium tumefaciens or Agrobacterium rhizogenes.
• the promoter operably linked to the fusion protein is a constitutive promoter.
• the promoter is a tissue specific promoter.
• the promoter is a developmental ⁇ regulated promoter.
• the promoter is an inducible promoter.
• any promoter can be used for regulating the expression of the fusion protein. The choice of promoter depends on the
• the transactivation construct containing the fusion protein is operatively linked a splice acceptor sequence.
• Splice acceptor sequences are well known to skilled artisans.
• other regulatory elements that can modulate expression of the chimeric transcriptional activator can be operably linked to it.
• a recombinant DNA construct of the present invention comprises other regulatory sequences.
• a recombinant construct of the present invention comprises an enhancer or silencer.
• the present invention provides recombinant constructs for use in expressing heterologous polynucleotides of interest in transgenic plants.
• the construct comprises at least one LexA operator sequence, a promoter and a heterologous polynucleotide of interest wherein the operator sequence, the heterologous polynucleotide and the promoter are operably linked to each other.
• the LexA operator is a caa operator.
• the LexA operator is a recA operator.
• the LexA operator comprises multiple adjacent copies (in tandem, separated by a few nucleotides, or overlapping) of the caa operator sequence.
• the LexA operator comprises multiple adjacent copies (in tandem, separated by a few nucleotides, or overlapping) of the recA operator sequence.
• the LexA operator is a recA operator that is modified or changed to a different sequence containing LexA binding sites (e.g., LexA, recA operator sequences or consensus).
• the LexA operator is a caa operator that is modified or changed to a different sequence containing LexA binding sites (e.g., LexA, caa operator sequences or consensus).
• copies of different or non-identical LexA operator sequences may be located adjacently for LexA binding.
• the number of the operator sequences or LexA binding sites can be changed to optimize the performance.
• the operator is a synthetic LexA operator operatively linked to a minimal promoter.
• the fusion protein encoded by this recombinant construct is capable of activating transcription of a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence; and further wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell.
• the fusion protein is capable of increasing transcription of a heterologous polynucleotide operably linked to: (a) a promoter functional in a plant cell; and (b) at least one LexA operator sequence; and further wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell.
• the at least one LexA operator sequence comprises at least one sequence selected from the group consisting of: a recA-type operator sequence and a caa-type operator sequence.
• the at least one LexA operator sequence may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 20, 30,
• LexA operator sequences may comprise a combination of non-identical LexA operator sequences, such as at least one recA-type operator sequence and at least one caa-type operator sequence.
• this invention concerns a vector, cell, plant, or seed comprising the recombinant DNA constructs described in the present invention.
• the fusion protein in the activator locus recognizes an
• the expression of the fusion protein does not adversely affect plant function when expressed at levels required for transactivation.
• the "transactivation system” described herein comprises generation of two different kinds of plant lines. One is referred to herein as the "activator line” or the “driver line” and the other line is referred to herein as the "effector” line.
• the "activator line” comprises the activator locus and the “effector line” comprises the effector locus comprising the heterologous polynucleotide, the expression of which is regulated by the expression of the chimeric transcriptional activator.
• a plant line comprising the driver locus is transformed to introduce the effector locus to generate a plant line comprising the driver locus as well as the effector locus.
• a plant line comprising the effector locus is transformed to introduce the driver locus to generate a plant line comprising the driver locus as well as the effector locus.
• Crossing a driver plant line with an effector plant line generates double transgenic offspring capable of expressing the heterologous polynucleotide regulated by the expression of the chimeric transactivator.
• combinatorial experiments can be done for bringing the driver locus and the effector locus together in the same plant: one driver line can be crossed to different effector lines to express different heterologous polynucleotides with the same expression pattern and regulatory control. Conversely, the same effector line can be crossed to various drivers to express one heterologous polynucleotide in different spatiotemporal manner.
• a large number of driver lines with various expression patterns can be easily generated using different enhancers or additional regulatory elements.
• the chimeric transactivator may comprise the LexA N- terminal DNA binding domain. In one embodiment the chimeric transactivator may comprise residues 1 -87 of the LexA polypeptide. In one embodiment the chimeric transactivator may comprise the full length LexA polypeptide.
• the use of the transactivation system disclosed herein allows regulated expression of transgenes. In one embodiment, it allows gene stacking and allows expression of these genes without using multiple promoters. In one embodiment, the transactivation system disclosed herein can be used with gene-trap and enhancer trapping systems. In one embodiment, the system of gene expression disclosed herein allows expression of developmental ⁇ regulated genes which if expressed otherwise may lead to lethality. In one embodiment, this transactivation system may be used for activation tagging screens for identifying key genes for drought tolerance, yield, nitrogen use efficiency, abiotic stress tolerance, root architecture, etc. This system can also be useful for lead optimization (using a bank of promoters). In one embodiment, this system can be used to regulate the trait-conferring transgene expression differently in successive generations.
• transactivation compositions and methods disclosed herein there are a number of circumstances in which it is advantageous to use the transactivation compositions and methods disclosed herein. Examples where such transactivation would be advantageous include, but are not limited to, situations where the transgene is likely to compromise plant viability or fertility, when a gene is to be expressed or a mutant phenotype is to be complemented in diverse patterns or at distinct developmental stages. A single transgene can be activated in the desired pattern simply by crossing to pre-existing activator lines or retransforming with existing plasmids.
• the present invention encompasses functional fragments and variants of the first amino acid sequence having the DNA-binding domain wherein the first amino acid sequence is SEQ ID NO:15, 32, 34, 36 or 38.
• the present invention also encompasses functional fragments and variants of the second amino acid sequence having the transactivation domain wherein the second amino acid sequence is SEQ ID NO:1 1 , 13, 90-96 or 97.
• a “functional fragment” as used herein is defined as any subset of
• “functional fragment” with substantially similar function to a sequence disclosed herein refers to a functional fragment that retains largely the same level of activity as the sequence and exhibits the same pattern of expression as the sequence disclosed.
• a “functional fragment” of the DNA-binding domain sequence disclosed herein exhibits DNA-binding activity to a LexA operator sequence.
• a “functional fragment” of the transactivation domain sequence disclosed herein exhibits transactivator activity.
• a "variant”, as used herein, is the sequence of the DNA-binding domain or the transactivator domain or the sequence of a functional fragment of the DNA- binding domain or the transactivator domain containing changes in which one or more amino acids of the original sequence is deleted, added, and/or substituted, while substantially maintaining the function of the sequence disclosed herein.
• One or more amino acids can be inserted, deleted, or substituted internally to the sequence disclosed herein.
• Variant domains can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant DNA-binding domain or a portion thereof. Substitutions, deletions, insertions or any combination thereof can be combined to produce a final construct.
• a variant polypeptide may have an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to the sequence of the DNA-binding domain or the transactivator domain or the sequence of a functional fragment of the DNA-binding domain or the transactivator domain.
• a variant of the DNA-binding domain substantially maintains DNA-binding activity to the LexA operator.
• a variant of the transactivator domain substantially maintains transactivator activity.
• the present invention provides a method to manipulate and improve a plant trait.
• the method entails transforming a first plant with a member of a first pool of driver constructs.
• Each driver construct includes a chimeric transactivator that is placed under the control of a different promoter so that the expression of the chimeric transactivator can be controlled under different conditions.
• the method entails transforming a second plant with a member of a second pool of effector constructs.
• the invention encompasses regenerated, mature and fertile transgenic plants comprising the recombinant DNA constructs described above, transgenic seeds produced therefrom, T1 and subsequent generations.
• the transgenic plant cells, tissues, plants, and seeds may comprise at least one recombinant DNA construct of interest.
• the plant comprising the recombinant constructs, described in any of the above embodiments is selected from the group consisting of: Arabidopsis, maize, soybean, sunflower, sorghum, canola, mustard, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
• the plant comprising the recombinant DNA constructs is a monocotyledenous plant. In another embodiment, the plant comprising the recombinant DNA constructs is a corn or rice plant.
• genomic DNA was prepared from a derivative of E. coli K12 strain (Strain deleted for araC). Preparation of genomic DNA was accomplished using the PROMEGA® Genomic DNA extraction Kit. Briefly, single colonies of E. coli were inoculated from freshly streaked LB agar plates and grown in 3ml LB broth overnight at 37°C shaker, and bacterial suspension harvested by centrifugation. E.coli genomic DNA was prepared as described in the PROMEGA® WIZARD® Genomic DNA extraction Kit.
• Arabidopsis cDNA library used for amplification of target cDNA was a single stranded cDNA library pool, prepared from total RNA derived from Columbia ecotype. The first strand cDNA synthesis was performed with oligo dT primer as described by in the SUPERSCRIPT®!!! cDNA synthesis Kit (INVITROGENTM). To amplify the E. coli LexA sequences the following primers sets at the PCR conditions described below. Primer pairs for amplification of E. coli LexA and Arabidopsis are given in Table 1 .
• the reaction was incubated at 25°C for 15 min and transformed into chemically competent E.coli cells via heat shock transformation, followed by revival and plating on LB plates supplemented with carbenicillin (50mg/L) and X-Gal- IPTG for blue-white selection of transformants.
• Transformants were analyzed by colony PCR with gene-specific primers, and positives were sequenced with combinations of vector specific and gene specific primers. Analysis of sequence was performed using software application in
• AtCBFI ORF SEQ ID NO:10; AtCBF3 ORF (SEQ ID NO:12) and E.Coli LexAORF (SEQ ID NO:14). Amplification of AtCBF2 was not accomplished in the conditions marked above.
• LexAORF SEQ ID NO:14
• AtCBFI SEQ ID NO:10
• AtCBF3 SEQ ID NO:12
• AtCBF3 SEQ ID NO:12
• a Mini- ⁇ sequence consisting of a 82bp CaMV 35S promoter minimal sequence (SEQ ID NO:16), proximal to the transcription start site and 67bp Tobacco mosaic virus (TMV) derived omega sequences (SEQ ID NO:17) were generated. Due to the smaller molecular size of this sequence, the fragment was derived through oligonucleotide synthesis that was subsequently cloned into the cloning vector PBLU ESCRIPT®KSI I .
• MiniCorePromU SEQ ID NO:18
• MiniCorePromL SEQ ID NO:19
• amplification primer fwd Am pi i Prom F; SEQ ID NO:87
• AmpliPromR AmpliPromR
• the amplicon (SEQ ID NO:21 ) was digested with BamH ⁇ and Xbal, after PCR purification (QIAGEN® PCR purification kit); following this the fragment was ligated to PBLUESCRIPT®KSI I as a
• Transformants were screened with colony PCR, and verified with restriction digestion.
• mini promoter-containing vector To create the pBC-Mini-omega::GUS-Pin construct, the mini promoter-TMV omega sequences (SEQ ID NO:21 ) were subcloned from Mini-Omega- PBLUESCRIPT®KSI I to pBC sans Hind ⁇ . Directional cloning was by performed using the BamHI -Xbal sited compatible with the mini-omega fragment released from the cloning vector. Presence of insert was verified using digestion with Ncol.
• the reporter GUS and PIN and terminator sequences were excised from the vector INDIA3R/ PHP31993 as a 2442bp Hind ⁇ fragment. This was ligated to the pBC Mini-omega backbone (intermediate clone), that was restricted with Hindlll.
• the mini-omega::GUS clone was sequenced using both vector specific primers as well as primers designed within the GUS and PIN sequences to verify the construct.
• RecA Operator dimer (recA operator single unit is given in SEQ ID NO:22).
• Caa Operator dimer (Caa operator single unit is given in SEQ ID NO:23).
• Hybrid Operator (2X Caa type operator + 2X RecA type operator) is given in SEQ ID NO:24.
• Multimerization of the RecA type operator was accomplished through a PCR driven multimerization reaction.
• a dimer of RecA was synthesized, and multimer formation was though partial annealing, followed by fill-in reaction at each iteration of PCR.
• the PCR mediated multimerization reaction is described below.
• the oligonucleotides used were RecAopU (SEQ ID NO:25) and RecAopL (SEQ ID NO:26).
• Oligonucleotides mix 10 ⁇ each 10 ⁇
• Hot start master mix (QIAGEN®) 25 ul
• RecA type operator multimers To clone and screen RecA type operator multimers, the PCR amplicons were cloned in PCR®2.1 -TOPO®, and transformed in to E.coli cells. Putative multimers were identified and sorted by colony PCR using vector specific M13F and M13R primers. The formation of RecA multimers was verified using sequence analysis Caa type operator was formed by annealing two partially complimentary oligo-nucleotide sequences.
• the sequences of the oligonucleotides are given in SEQ ID NO:27 (CaaOpU) and SEQ ID NO:28 (CaaOpL).
• Oligonucleotides mix 10 ⁇ each 10 ⁇
• Hot start master mix (QIAGEN®) 25 ul
• the Caa type operator dimer was cloned into PCR®2.1 -TOPO® vector and transformed in to E.coli cells. Putative clones were identified by colony PCR using vector specific M13F and M13R primers. The formation of caa type operator dimer was verified by sequencing.
• oligonucleotide consisting of both RecA type and Caa type operators.
• two partially complementary oligonucleotides were designed.
• the annealed oligonucleotide was filled-in using a polymerase as described below, the sequences of the oligonucleotides are given in SEQ ID NO:29 (HybOpUhalf) and SEQ ID NO:30 (HybOpLHalf).
• Oligonucleotides mix 10 ⁇ each 10 ⁇
• Hot start master mix (QIAGEN®) 25 ul
• Step 2 Denaturation 94°C for 45 sec
• Step 3 Annealing 50°C for 30 sec
• the formation of the desired 100 bp fragment was confirmed by loading 2.5 ⁇ of the reaction in a 2% agarose gel.
• Hybrid type operator was PCR purified, restriction digested with EcoRI and BamHI, and ligated into Mini-Omega- PBLUESCRIPT®KSI I, to create Hybop- MiniOmega fragment.
• E.coli transformants were identified with colony PCR using vector specific M13F and M13R primers.
• the operator multimers for RecA type operator or Caa type operator were excised from their cloning vector backbones as EcoRI fragments and were cloned into the EcoRI site of the basal operator-less vector MiniQ::GUS.
• Ligation reaction for these reactions was performed using 1 :50 to 1 :100 molar ratios of vector to insert:
• Hybop-MinQ :GUS
• Hybrid-type operator sequences were excised from PBLUESCRIPT®KSI I, or the Caa type operator or RecA type operators were mixed in equimolar ratios and ligated at the EcoRI site upstream to mini-promoter-Omega: :GUS sequences.
• Ligation reaction were transformed into E.co// XL1 -BLUE® cells and plated on LB plates supplemented with Kanamycin.
• LexAwtl -87 aa DBD SEQ ID NO:31 ; amino acid sequence SEQ ID NO:32
• LexA 1 -87MNF engineered NLS and Gly-Ala cleavage blocked mutant
• LexA 1 -202 full length LexAFL; SEQ ID NO:35
• mutant (LexAmut FL; and amino acid sequence SEQ ID NO:36).
• LexA 1 -202 full length LexA 1 -202 full length (LexAFLNLS), cleavage blocked, containing NLS
• N terminal 1 -87 amino-acid region has been shown to be involved in DNA binding. This region has been used for create LexA-VP16 based induction systems.
• the LexA DNA binding region displays auto-cleavage, at the Ala-Gly bond. G 8 5 to D mutation stabilized this bond from cleavage in full length LexA protein.
• an NLS has been introduced at the domain linker region, to enhance nuclear localization of the fusions.
• LexA full length protein has been shown to bind at higher affinity, as a protein dimer to its operator sequences with high binding affinity.
• native LexA protein displays autocatalytic cleavage.
• auto-cleavage mutations were generated. Besides these mutations silent mutation to remove an EcoRV site for facilitating molecular cloning has been engineered. The various mutations generated are described in detail below.
• LexA full-length protein stabilized for auto-cleavage has been engineered to include mutation at E71 -K, that has been shown to enhance DNA binding, as well as insert an eukaryotic NLS consensus KKRK, in a loop region of the LexA protein.
• Trans-activator fusion comprised of various deletions of the plant DREB/CBF sequences derived from the hydrophobic cluster region.
• the sequences taken for creation of trans-activators are given in Table 3.
• Fusion fragments marked A-G are derived from AtCBFI/ AtDREBI b and fragment I (SEQ ID NO:47) is derived from the AtCBF3/AtDREB1 a sequences.
• the nucleotide sequences for these selected sequences and their protein translations are given in Table 3.
• fusion proteins were accomplished by a modified overlap fusion PCR based approach that involved two PCR amplification reactions.
• the linker sequences comprised of the GC rich sequences.
• LexA 1 -87 domain (SEQ ID NO:31 ) was amplified and the various deletions of CFB1/CBF3 were individually amplified with the primer pair listed below Table 4.
• PCR amplification components for the primary reaction were:
• Step3 63-58°C 1 min (Touch down 1 °C /cycle)
• Step2 94°C 45 sec-60 sec
• reaction 2.5 ⁇ of reaction were checked on a 1 .5% agarose gel, to determine the formation of fused sequences based on their molecular sizes.
• Step3 63-58°C 1 min (Touch down 1 °C /cycle)
• first step required the creation of site-directed mutations in the LexA gene, prior to generation of sequence fusion.
• Site-directed mutation was through overlap PCR approach.
• wild-type LexA gene template was amplified as three overlapping fragments (fragments 1 , 2 and 3, given below), encompassing the mutations. The map of the fragment is described above with the description of the mutation. The PCR conditions for creating these fragments and the primer pairs are marked below
• Step3 63-58°C 1 min (Touch down 1 °C /cycle)
• Fwd primer SEQ ID NO: 48:
• reaction was analyzed by to determine the formation of appropriate sized amplicons.
• LexA full-length containing the engineered mutations was amplified with 3' reverse primer, containing sequences predicted to display flexibility, represented by the type II linker sequences (described in Patent Application No. PCT/US201 1 /050191 ).
• the reconstituted LexA full-length (LexAFL) amplicon was fused to the CFB1 /CBF3 fragments (A-G and I; SEQ ID NO:40-46 and 47 respectively), containing the Type II linker tag, that were individually amplified with the same primer pairs as above in Table 5. 2.5 ⁇ of reaction were checked on a 2% agarose gel, and appropriately sized amplicons representing each deletion were recovered via gel elution (QIAGEN®).
• LexA full-length protein with the mutations described before (LexAFLNLS; SEQ ID NO:37) were used as template to create additional site-directed mutation and insertion mutation of the NLS in a loop region of LexA.
• the mutations were accomplished by using overlap extension PCR approaches.
• LexA sequences were amplified as three overlapping fragments with the following primer sets, to create the fragments 4, 5, and 6. Primer sets used to amplify the fragments were
• the desired site-directed LexA full-length NLS mutant sequences were reconstituted by mixing equimolar amounts of the three amplicons (4+5+6; SEQ ID NOS:78-80 respectively), that was amplified with the primer set marked below:
• Fwd primer SEQ ID NO:81
• LexA full-length with NLS LexAFLNLS
• the reconstituted LexA full-length (LexAFLNLS) amplicon was fused to the CFB1/CBF3 fragments (A- G; I (SEQ ID NOS:40-46 and 47 respectively)), containing the Type II linker tag, that were individually amplified with the same primer pairs as above in Table 5.
• 2.5 ⁇ of reaction were checked on a 2% agarose gel, and appropriately sized amplicons representing each deletion were recovered via gel elution (QIAGEN®).
• the transformed cells were centrifuged at 4000 rpm for 4-5 minutes. The supernatant was discarded (retaining 200 ⁇ of the media). The cells were resuspended and then plated on LB plates with X-gal and IPTG added in the medium for blue white selection. Carbenicillin was the selection marker added.
• colony PCR analysis was performed in 96 well microtitre plates or PCR strips (Axygen, USA). Single, XL1 -BLUE® transformants, growing on antibiotic carbenicillin supplemented LB-agar plates were individually picked and patched onto LB-Agar carbenicillin plates and then dropped into 5 ⁇ of sterile water. A total of 12 clones were screened for each ligation reaction done, and in each instance M13 forward and reverse primers were used for screening the clones.
• Step 1 95°C 5:00 minutes (Initial enzyme activation)
• Step 3 55°C 00:45 seconds (Annealing)
• Step 5 72°C 15:00 minutes (Final elongation)
• the fused fragments with the vector were then resolved on 1 % agarose gels containing DNA visualization dye ethidium bromide. Gel was documented with transmitted UV using Alphalmager system. Image contrast and exposure were corrected using the software. After analysis of the gel image four positive colonies for each CBF fragment (A, B, C, D, E, F, G and I fusion constructs) were selected from the patched LB-Agar carbenicillin plates and inoculated into culture tubes. The culture tubes contained 6 ml of 2XYT media with 6 ⁇ of carbenicillin antibiotic. They were then incubated in the orbital shaker at 250 rpm at 37 ° C overnight. Plasmids were isolated from all the colonies using the Wizard Plus SV Minipreps Plasmid Purification Kit.
• the plasmids were eluted in 60 ⁇ of sterile distilled water. The concentrations were checked using the Nanodrop. All the concentrations were found to be of an average of 200 ng/ ⁇ . The plasmids were then sent for sequencing to check for any mutations in the length of the fragment.
• Sequence analysis was performed using analysis software, SEQUENCHER® version 4.7, developed by Gene Codes, USA.
• LexA-CBF fusions from pBSKSII were cloned in to pZGG binary vector (FIG.2) and subsequently transformed in to Agrobacterium tumefaciens.
• the harvested operator seeds were sown in the organic soil mixture. Once the seedlings reach two leaf stage three consecutive BASTA® sprays were administered with an interval of 3 days each. The concentration of BASTA® used was 3mg/liter. The plants were allowed to grow till the flowering stage and used for the transformation with driver constructs. After transformation with the driver constructs these lines were allowed to complete their life cycle and seeds were harvested.
• the seeds were dried at room temperature for two weeks and then selected for kanamycin resistance on MS media.
• the details of the protocol used are given below:
• MS media was prepared with the following composition and the pH was set to 5.8.
• Vitamin B5 1 ml/L
• the media was autoclaved and kanamycin was added in (50mg/L) concentrations and poured into the plates.
• the wild-type and the transgenic seeds were surface sterilized with hypochlorite (with Tween 20) and 70% ethanol with the following protocol
• Transgenic seeds containing driver operator constructs were taken in a 1 .7 ml microfuge tube. 1 ml of ethanol was added to each tube and they were inverted a few times to wash the seeds. The ethanol treatment was given for 1 :30 minutes, followed by a brief spin in the microfuge such that the seeds settled down and the ethanol could be poured out into the beaker. Wash the seeds with 1 ml sterile water, followed by a brief spin and removal of the water.1 ml of hypochlorite containing 2% TWEEN®-20 was added to the tubes containing ethanol treated seeds for a 2:30 minute treatment. Hypochlorite was removed and the seeds were washed multiple (4-5) times in sterile water to remove any remnants of detergent. The seeds were suspended in 500 ⁇ of sterile water after the final wash and stratified overnight for two days at 4°C.
• the seeds were then inoculated into MS plates supplemented with (50mg/L) kanamycin. An approximate of 100 ⁇ of Arabidopsis seeds are plated in a single plate. The plates were then transferred to the growth chamber.
• the seedlings that had grown on the kanamycin selection were selected after 7 days. Positive seedlings were selected based on the green color and survival on the selection media. These were then taken for the assay for both qualitative with GUS histochemical staining and quantitative by fluorescent MUG assay.
• GUS assay was done by collecting the leaf tissue by cutting with a pair of sharp scissors and incubated with 200 ⁇ of GUS Histochemical staining solution at 37°C for overnight in incubator and then imaged, the stained tissue sample after decolorizing the chlorophyll pigment of the sample with 95% ethanol.
• GUS staining protocol used was as per materials and methods in Jefferson et ai, (1987) EMBO Journal, vol.6 no.13 pp. 3901 -3907.
• MUG Assay was done by extraction of protein from collected tissue sample and assay was carried out in triplicates for every sample as per the materials and methods in (Richard A. Jefferson, Tony A. Kavanagh and Michael W. Bevan (1987) EMBO Journal, vol.6 no.13 pp. 3901 -3907)
• MUG fluorescent assay data was analyzed for the positive samples from GUS assay which had shown GUS expression for the Driver4 (Table 1 1 and FIG.4) and Driver6 (Table 12 and FIG.5), with three operators combinations
• Protein cone mg/ml protein concentration in mg/ml of sample
• transactivation system described above can be used for regulating expression of other genes that can be used in place of GUS and may be used in other plant species such as rice, soybean and maize.

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