EP1159424A2 - Nouveaux genes de vegetaux et leurs utilisations - Google Patents

Nouveaux genes de vegetaux et leurs utilisations

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Publication number
EP1159424A2
EP1159424A2 EP00909334A EP00909334A EP1159424A2 EP 1159424 A2 EP1159424 A2 EP 1159424A2 EP 00909334 A EP00909334 A EP 00909334A EP 00909334 A EP00909334 A EP 00909334A EP 1159424 A2 EP1159424 A2 EP 1159424A2
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EP
European Patent Office
Prior art keywords
seq
sequence
plant
nucleotide sequence
nucleic acid
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EP00909334A
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German (de)
English (en)
Inventor
John Manuel Salmeron
Laura Jean Weislo
Michael G. Willits
Tesfaye Mengiste
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Syngenta Participations AG
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Syngenta Participations AG
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Publication of EP1159424A2 publication Critical patent/EP1159424A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • the present invention relates to broad-spectrum disease resistance in plants, including the phenomenon of systemic acquired resistance (SAR). More particularly, the present invention relates to the identification, isolation and characterization of homologues of the Arabidopsis NIM1 gene involved in the signal transduction cascade leading to systemic acquired resistance in plants.
  • SAR systemic acquired resistance
  • Plants are constantly challenged by a wide variety of pathogenic organisms including viruses, bacteria, fungi, and nematodes. Crop plants are particularly vulnerable because they are usually grown as genetically-uniform monocultures; when disease strikes, losses can be severe. However, most plants have their own innate mechanisms of defense against pathogenic organisms. Natural variation for resistance to plant pathogens has been identified by plant breeders and pathologists and bred into many crop plants. These natural disease resistance genes often provide high levels of resistance to or immunity against pathogens.
  • SAR Systemic acquired resistance
  • the SAR response can be divided into two phases.
  • initiation phase a pathogen infection is recognized, and a signal is released that travels through the phloem to distant tissues. This systemic signal is perceived by target cells, which react by expression of both SAR genes and disease resistance.
  • the maintenance phase of SAR refers to the period of time, from weeks up to the entire life of the plant, during which the plant is in a quasi steady state, and disease resistance is maintained (Ryals etal., 1996). Salicylic acid (SA) accumulation appears to be required for SAR signal transduction.
  • SA Salicylic acid
  • SAR can be activated in Arabidopsis by both pathogens and chemicals, such as SA, 2,6-dichloroisonicotinic acid (INA) and benzo(1 ,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) (Uknes et al., 1992; Vernooij etal., 1995; Lawton etal., 1996).
  • SA 2,6-dichloroisonicotinic acid
  • BTH benzo(1 ,2,3)thiadiazole-7-carbothioic acid S-methyl ester
  • pathogenesis-related (PR) protein genes namely, PR-1, PR-2, and PR-5 are coordinately induced concomitant with the onset of resistance (Uknes et al., 1992, 1993).
  • PR-1, PR-2, and PR-5 are coordinately induced concomitant with the onset of resistance.
  • pathogen or an immunization compound induces the expression of at least nine sets of genes (Ward et al., 1991).
  • Transgenic disease-resistant plants have been created by transforming plants with various SAR genes (U.S. Patent No. 5,614,395).
  • a number of Arabidopsis mutants have been isolated that have modified SAR signal transduction (Delaney, 1997) The first of these mutants are the so-called Isd (lesions simulating disease) mutants and acd2 (accelerated cell death) (Dietrich et al., 1994; Greenberg etal., 1994). These mutants all have some degree of spontaneous necrotic lesion formation on their leaves, elevated levels of SA, mRNA accumulation for the SAR genes, and significantly enhanced disease resistance. At least seven different Isd mutants have been isolated and characterized (Dietrich etal., 1994; Weymann et al., 1995).
  • mutants are elm (constitutive Immunity) mutants (Lawton et al., 1993). See also, U.S. Patent No. 5,792,904 and International PCT Application WO 94/16077. Like Isd mutants and acd2, cim mutants have elevated SA and SAR gene expression and resistance, but in contrast to Isd or acd2, do not display detectable lesions on their leaves.
  • cpr7 (constitutive expresser of PR genes) may be a type of cim mutant; however, because the presence of microscopic lesions on the leaves of cprl has not been ruled out, cprl might be a type of ted mutant (Bowling et al., 1994).
  • ndrl non-race- specific disease resistance
  • nprl nonexpresser of PR genes
  • eds (enhanced disease susceptibility) mutants have been isolated based on their ability to support bacterial infection following inoculation of a low bacterial concentration (Glazebrook et al., 1996; Parker et al., 1996). Certain eds mutants are phenotypically very similar to nprl, and, recently, eds ⁇ and eds53 have been shown to be allelic to nprl (Glazebrook etal., 1996).
  • nim1 noninducible immunity
  • P. parasitica i.e., causal agent of downy mildew disease
  • nim1 can accumulate SA following pathogen infection, it cannot induce SAR gene expression or disease resistance, suggesting that the mutation blocks the pathway downstream of SA. nim1 is also impaired in its ability to respond to INA or BTH, suggesting that the block exists downstream of the action of these chemicals (Delaney et al., 1995; Lawton et al., 1996).
  • Allelic Arabidopsis genes have been isolated and characterized, mutants of which are responsible for the nim1 and nprl phenotypes, respectively (Ryals et al., 1997; Cao et al., 1997).
  • the wild-type NIM1 gene product is involved in the signal transduction cascade leading to both SAR and gene-for-gene disease resistance in Arabidopsis (Ryals et al., 1997).
  • Ryals etal., 1997 also report the isolation of five additional alleles of n tr?7 that show a range of phenotypes from weakly impaired in chemically induced PR-1 gene expression and fungal resistance to very strongly blocked.
  • Associated With / Operatively Linked refers to two DNA sequences that are related physically or functionally.
  • a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
  • Chimeric Gene A recombinant DNA sequence in which a promoter or regulatory DNA sequence is operatively linked to, or associated with, a DNA sequence that codes for an mRNA or which is expressed as a protein, such that the regulator DNA sequence is able to regulate transcription or expression of the associated DNA sequence.
  • the regulator DNA sequence of the chimeric gene is not normally operatively linked to the associated DNA sequence as found in nature.
  • Coding Sequence a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
  • RNA is then translated in an organism to produce a protein.
  • Complementary refers to two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences.
  • Expression refers to the transcription and/or translation of an endogenous gene or a transgene in plants.
  • expression may refer to the transcription of the antisense DNA only.
  • Expression Cassette A nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue, or organ, or stage of development.
  • Gene A defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion.
  • a gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
  • heterologous DNA sequence refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • Homologous DNA Sequence A DNA sequence naturally associated with a host cell into which it is introduced.
  • Isocoding A nucleic acid sequence is isocoding with a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
  • Isolated In the context of the present invention, an isolated nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
  • Minimal Promoter promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.
  • Native refers to a gene that is present in the genome of an untransformed cell.
  • Naturally occurring is used to describe an object that can be found in nature as distinct from being artificially produced by man.
  • a protein or nucleotide sequence present in an organism which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
  • NIM1 Gene described in Ryals et al., 1997, which is involved in the SAR signal transduction cascade.
  • NIM1 Protein encoded by the NIM1 gene
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)).
  • the terms "nucleic acid” or “nucleic acid sequence” may also be used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • the nucleic acid molecule is preferably a segment of DNA. Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). ORF: Open Reading Frame.
  • Plant Any whole plant.
  • Plant Cell Structural and physiological unit of a plant, comprising a protoplast and a cell wall.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.
  • Plant Cell Culture Cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
  • Plant Material Refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
  • Plant Organ A distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
  • Plant tissue A group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
  • Promoter An untranslated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase II and initiates transcription of the DNA.
  • the promoter region may also include other elements that act as regulators of gene expression.
  • Protoplast An isolated plant cell without a cell wall or with only parts of the cell wall.
  • purified when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • nucleic acid or protein is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.
  • Recombinant DNA molecule a combination of DNA molecules that are joined together using recombinant DNA technology
  • Regulatory Elements Sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
  • Selectable marker gene a gene whose expression in a plant cell gives the cell a selective advantage.
  • the selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non- transformed cells.
  • the selective advantage possessed by the transformed cells, compared to non-transformed cells may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source.
  • Selectable marker gene also refers to a gene or a combination of genes whose expression in a plant cell gives the cell both, a negative and a positive selective advantage.
  • an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.
  • nucleic acid or protein sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • substantially identical in the context of two nucleic acid or protein sequences, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90-95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • substantially identical nucleic acid or protein sequences perform substantially the same function.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by visual inspection (see generally, Ausubel et al., infra).
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff , Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Bod(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5 9 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but to no other sequences.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 9 C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.1 5M NaCI at 72 9 C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65 9 C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1x SSC at 45 9 C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6x SSC at 40 9 C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 9 C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodec
  • nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid.
  • a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised to the protein with the amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins except for polymorphic variants.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York "Harlow and Lane"), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • Consatively modified variations of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein.
  • nucleic acid variations are "silent variations" which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein which encodes a protein also describes every possible silent variation, except where otherwise noted.
  • each codon in a nucleic acid except ATG, which is ordinarily the only codon for methionine
  • each "silent variation" of a nucleic acid which encodes a protein is implicit in each described sequence.
  • a “subsequence” refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., protein) respectively.
  • Nucleic acids are "elongated" when additional nucleotides (or other analogous molecules) are incorporated into the nucleic acid. Most commonly, this is performed with a polymerase (e.g., a DNA polymerase), e.g., a polymerase which adds sequences at the 3' terminus of the nucleic acid.
  • a polymerase e.g., a DNA polymerase
  • a polymerase e.g., a polymerase which adds sequences at the 3' terminus of the nucleic acid.
  • Two nucleic acids are “recombined” when sequences from each of the two nucleic acids are combined in a progeny nucleic acid.
  • Two sequences are “directly” recombined when both of the nucleic acids are substrates for recombination.
  • Two sequences are "indirectly recombined” when the sequences are recombined using an intermediate such as a cross-over oligonucieotide.
  • no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.
  • a "specific binding affinity" between two molecules means a preferential binding of one molecule for another in a mixture of molecules.
  • the binding of the molecules can be considered specific if the binding affinity is about 1 x 10 4 M "1 to about 1 x 10 6 M "1 or greater.
  • Transformation a process for introducing heterologous DNA into a host cell or organism.
  • Transformed refers to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • non-transformed refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
  • the present invention addresses the aforementioned needs by providing several homologues of the Arabidopsis NIM1 gene from additional species of plants.
  • the present invention concerns the isolation of Nicotiana tabacum (tobacco), Lycopersicon esculentum (tomato), Brassica napus (oilseed rape), Arabidopsis thallana, Beta vulgaris (sugarbeet), Helianthus annuus (sunflower), and Solanum tuberosum (potato) homologues of the NIM1 gene, which encode proteins believed to be involved in the signal transduction cascade responsive to biological and chemical inducers that lead to systemic acquired resistance in plants.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes SEQ ID NO:2, 4, 6, 8, 16, 18, 20, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 62, 64, 66, 68, 70, 72, or 74.
  • the present invention is directed to an isolated nucleic acid molecule comprising SEQ ID NO:1 , 3, 5, 7, 15, 17, 19, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 61 , 63, 65, 67, 69, 71 , or 73.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that comprises an at least 20, 25, 30, 35, 40, 45, or 50 (preferably 20) consecutive base pair portion identical in sequence to an at least 20, 25, 30, 35, 40, 45, or 50 (preferably 20) consecutive base pair portion of SEQ ID NO:1 , 3, 5, 7, 15, 17, 19, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 61 , 63, 65, 67, 69, 71 , or 73.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from a Lycopersicon esculentum DNA library using the polymerase chain reaction with the pair of primers set forth as SEQ ID NO:9 and 10, SEQ ID NO:21 and 24, SEQ ID NO:22 and 24, SEQ ID NO:25 and 28, SEQ ID NO:26 and 28, or SEQ ID NO:59 and 60.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from a Beta vulgaris DNA library using the polymerase chain reaction with the pair of primers set forth as SEQ ID NO:22 and 24 or SEQ ID NO:26 and 28.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from a Helianthus annuus DNA library using the polymerase chain reaction with the pair of primers set forth as SEQ ID NO:26 and 28.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from a Solanum tuberosum DNA library using the polymerase chain reaction with the pair of primers set forth as SEQ ID NO:21 and 24, SEQ ID NO:21 and 23, SEQ ID NO:22 and 24, SEQ ID NO:25 and 28, or SEQ ID NO:26 and 28.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from a Brassica napus DNA library using the polymerase chain reaction with the pair of primers set forth as SEQ ID NO:9 and 10 or SEQ ID NO:26 and 28.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from an Arabidopsis thallana DNA library using the polymerase chain reaction with the pair of primers set forth as SEQ ID NO:13 and 14, SEQ ID NO:21 and 24, or SEQ ID NO:22 and 24.
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from an Nicotiana tabacum DNA library using the polymerase chain reaction with the pair of primers set forth as SEQ ID NO:9 and 10, SEQ ID NO:11 and 12, SEQ ID NO:21 and 24, SEQ ID NO:22 and 24, SEQ ID NO:25 and 28, or SEQ ID NO:26 and 28; or
  • the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from an plant DNA library using the polymerase chain reaction with a pair of primers comprising the first 20 nucleotides and the reverse complement of the last 20 nucleotides of the coding sequence (CDS) of SEQ ID NO:1 , 3, 5, 7, 15, 17, 19, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 61 , 63,
  • CDS coding sequence
  • the present invention also encompasses a chimeric gene comprising a promoter active in plants operatively linked to a NIM1 homologue coding sequence of the present invention, a recombinant vector comprising such a chimeric gene, wherein the vector is capable of being stably transformed into a host, as well as a host stably transformed with such a vector.
  • the host is a plant such as one of the following agronomically important crops: rice, wheat, barley, rye, canola, sugarcane, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum, and sugarcane.
  • the present invention also encompasses seed from a plant of the invention.
  • the present invention is directed to a method of increasing SAR gene expression in a plant by expressing in the plant a chimeric gene that itself comprises a promoter active in plants operatively linked to a NIM1 homologue coding sequence of the present invention, wherein the encoded protein is expressed in the transformed plant at higher levels than in a wild type plant.
  • the present invention is directed to a method of enhancing disease resistance in a plant by expressing in the plant a chimeric gene that itself comprises a promoter active in plants operatively linked to a NIM1 homologue coding sequence of the present invention, wherein the encoded protein is expressed in the transformed plant at higher levels than in a wild type plant.
  • the present invention is directed to a PCR primer selected from the group consisting of SEQ ID NO:9-14, 21-28, 59, and 60.
  • the present invention also encompasses a method for isolating a NIM1 homologue involved in the signal transduction cascade leading to systemic acquired resistance in plants comprising amplifying a DNA molecule from a plant DNA library using the polymerase chain reaction with a pair of primers corresponding to the first 20 nucleotides and the reverse complement of the last 20 nucleotides of the coding sequence (CDS) of SEQ ID NO:1 , 3, 5, 7, 15, 17, 19, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 61 , 63, 65, 67, 69, 71 , or 73 or with the pair of primers set forth as SEQ ID NO:9 and 10, SEQ ID NO:11 and 12, SEQ ID NO:13 and 14, SEQ ID NO:21 and 24, SEQ ID NO:22 and 24, SEQ ID NO:21 and 23, SEQ ID NO:25 and 28, SEQ ID NO:26 and 28, or SEQ ID NO:59 and 60
  • the plant DNA library is a Nicotiana tabacum (tobacco), Lycopersicon esculentum (tomato), Brassica napus (oilseed rape), Arabidopsis thallana, Beta vulgaris (sugarbeet), Helianthus annuus (sunflower), or Solanum tuberosum (potato) DNA library.
  • Northern data on several of the NIM1 homologues described herein indicates constitutive expression or BTH-inducibility.
  • the homologues of the NIM1 gene described herein are predicted to encode proteins involved in the signal transduction cascade responsive to biological and chemical inducers, which leads to systemic acquired resistance in plants.
  • the present invention also concerns the transgenic expression of such NIM1 homologues in plants to increase SAR gene expression and enhance disease resistance.
  • the DNA sequences of the invention can be isolated using the techniques described in the examples below, or by PCR using the sequences set forth in the sequence listing as the basis for constructing PCR primers.
  • oligonucleotides having the sequence of approximately the first and last 20-25 consecutive nucleotides of SEQ ID NO:7 e.g., nucleotides 1 -20 and 1742-1761 of SEQ ID NO:7 can be used as PCR primers to amplify the cDNA sequence (SEQ ID NO:7) directly from a cDNA library from the source plant (Arabidopsis thaliana).
  • the other DNA sequences of the invention can likewise be amplified by PCR from cDNA or genomic DNA libraries of the respective plants using the ends of the DNA sequences set forth in the sequence listing as the basis for PCR primers.
  • the transgenic expression of the NIM1 homologues of the invention in plants is predicted to result in immunity to a wide array of plant pathogens, which include, but are not limited to viruses or viroids, e.g. tobacco or cucumber mosaic virus, ringspot virus or necrosis virus, pelargonium leaf curl virus, red clover mottle virus, tomato bushy stunt virus, and like viruses; fungi, e.g. oomycetes such as Phythophthora parasitica and Peronospora tabacina; bacteria, e.g. Pseudomonas syringae and Pseudomonas tabaci; insects such as aphids, e.g.
  • viruses or viroids e.g. tobacco or cucumber mosaic virus, ringspot virus or necrosis virus, pelargonium leaf curl virus, red clover mottle virus, tomato bushy stunt virus, and like viruses
  • fungi e.g. oomycetes such as Phythophthora parasitica and Per
  • the vectors and methods of the invention are useful against a number of disease organisms of maize including but not limited to downy mildews such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari and Peronosclerospora maydis; rusts such as Puccinia sorphi, Puccinia polysora and Physopella zeae; other fungi such as Cercospora zeae-maydis, Colletotrichum graminicola, Fusarium monoliforme, Gibberella zeae, Exsero
  • downy mildews such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Per
  • the methods of the present invention can be utilized to confer disease resistance to a wide variety of plants, including gymnosperms, monocots, and dicots. Although disease resistance can be conferred upon any plants falling within these broad classes, it is particularly useful in agronomically important crop plants, such as rice, wheat, barley, rye, rape, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
  • agronomically important crop plants such as rice, wheat, barley, rye, rape, corn, potato, carrot, sweet potato, sugar beet
  • a NIM1 homologue coding sequence of the present invention may be inserted into an expression cassette designed for plants to construct a chimeric gene according to the invention using standard genetic engineering techniques.
  • the choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the achieving the desired pattern and level of expression in the chosen plant host is within the level of skill of the routineer in the art.
  • the resultant molecule, containing the individual elements linked in proper reading frame, may be inserted into a vector capable of being transformed into a host plant cell.
  • promoters capable of functioning in plants or plant cells include the Arabidopsis and maize ubiquitin promoters; cauliflower mosaic virus (CaMV) 19S or 35S promoters and CaMV double promoters; rice actin promoters; PR-1 promoters from tobacco, Arabidopsis, or maize; nopaline synthase promoters; small subunit of ribulose bisphosphate carboxylase (ssuRUBISCO) promoters, and the like.
  • Arabidopsis ubiquitin promoter Especially preferred is the Arabidopsis ubiquitin promoter.
  • the promoters themselves may be modified to manipulate promoter strength to increase expression of the associated coding sequence in accordance with art-recognized procedures.
  • Preferred promoters for use with the present invention are those that confer high level constitutive expression.
  • Signal or transit peptides may be fused to the NIM1 homologue coding sequence in the chimeric DNA constructs of the invention to direct transport of the expressed protein to the desired site of action.
  • signal peptides include those natively linked to the plant pathogenesis-related proteins, e.g. PR-1 , PR-2, and the like. See, e.g., Payne et al., 1988.
  • Examples of transit peptides include the chloroplast transit peptides such as those described in Von Heijne etal.
  • the chimeric DNA construct(s) of the invention may contain multiple copies of a promoter or multiple copies of a NIM1 homologue coding sequence of the present invention.
  • the construct(s) may include coding sequences for markers and coding sequences for other peptides such as signal or transit peptides, each in proper reading frame with the other functional elements in the DNA molecule. The preparation of such constructs are within the ordinary level of skill in the art.
  • Useful markers include peptides providing herbicide, antibiotic or drug resistance, such as, for example, resistance to protoporphyrinogen oxidase inhibitors, hygromycin, kanamycin, G418, gentamycin, lincomycin, methotrexate, glyphosate, phosphinothricin, or the like. These markers can be used to select cells transformed with the chimeric DNA constructs of the invention from untransformed cells.
  • Other useful markers are peptidic enzymes which can be easily detected by a visible reaction, for example a color reaction, for example luciferase, ⁇ -glucuronidase, or ⁇ -galactosidase.
  • Chimeric genes designed for plant expression such as those described herein can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant (i.e. monocot or dicot) and/or organelle (i.e. nucleus, chloroplast, mitochondria) targeted for transformation.
  • organelle i.e. nucleus, chloroplast, mitochondria
  • Suitable methods of transforming plant cells include microinjection (Crossway etal., 1986), electroporation (Riggs et al., 1986), Agrobacterium mediated transformation (Hinchee et al., 1988; Ishida etal., 1996), direct gene transfer (Paszkowski et al., 1984; Hayashimoto et al., 1990), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for example, U.S. Patent 4,945,050; and McCabe etal., 1988). See also, Weissinger et al. (1988); Sanford etal.
  • a particularly preferred set of embodiments for the introduction of recombinant DNA molecules into maize by microprojectile bombardment can be found in Koziel et al. (1993); Hill etal. (1995) and Koziel etal. (1996).
  • An additional preferred embodiment is the protoplast transformation method for maize as disclosed in EP 0292435.
  • a chimeric gene comprising a NIM1 homologue coding sequence may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.
  • Particularly preferred plants of the invention include the agronomically important crops listed above. The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction and can thus be maintained and propagated in progeny plants.
  • Example 1 Isolation of a NIM1 Homologue from Nicotiana tabacum
  • Plasmid DNA from a mass excision of phage from a tobacco cDNA library is used as a template for PCR using the following primer pairs: 5'-AGATTATTGTCAAGTCTAATG-3' (SEQ ID NO:9) + 5'-TTCCATGTACCTTTGCTTC-3' (SEQ ID NO: 10), and 5'-GCGGATCCATGGATAATAGTAGG-3' (SEQ ID NO:11) + 5'-GCGGATCCTATTTCCTAAAAGGG-3' (SEQ ID NO:12). Cycling conditions are preferably 94 degrees for one minute, 40 degrees for one minute, and 72 degrees for 1.5 minutes, and the reaction is preferably carried out for 40 cycles. PCR products are run out on agarose gels, excised, and cloned into pCRII-TOPO (Invitrogen).
  • the full-length cDNA sequence of this tobacco NIM1 homologue is shown in SEQ ID NO:1 , and the protein encoded by this cDNA sequence is shown in SEQ ID NO:2.
  • a tobacco NIM1 homologue comprising SEQ ID NO:1 has been deposited as pNOV1206 with the NRRL (Agricultural Research Service, Patent Culture Collection, Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A) on August 17, 1998, and assigned accession no. NRRL B-30051.
  • Example 2 Isolation of a NIM1 Homologue from Lycopersicon esculentum
  • Phagemids are excised from ⁇ ZAPII cDNA libraries of tomato using a protocol from Stratagene. Phagemids (plasmids) are mass-transformed into E. co//XL1 -Blue in 10 pools of about 80,000 clones each and DNA is extracted from these pools. The pools are screened by PCR for the presence of NIM1 homologues by PCR using the following primers: 5'-AGATTATTGTCAAGTCTAATG-3' (SEQ ID NO:9) and 5'- TTCCATGTACCTTTGCTTC-3' (SEQ ID NO:10).
  • Sequences amplified from the pools are confirmed to contain NIM1 homologues by cloning the PCR-amplified DNA fragment and sequencing. Pools are made successively smaller and screened by PCR using the same primers mentioned above for the presence of the NIM1 homologues until a single clone containing the homologue is obtained. In the event that the cDNA clone contains a partial gene missing the 5' end, 5' RACE (Rapid Amplification of cDNA Ends) is used to obtain the full-length sequence of the gene.
  • 5' RACE Rapid Amplification of cDNA Ends
  • the full-length cDNA sequence of this tomato NIM1 homologue is shown in SEQ ID NO:3, and the protein encoded by this cDNA sequence is shown in SEQ ID NO:4.
  • a tomato NIM1 homologue comprising SEQ ID NO:3 has been deposited as pNOV1204 with the NRRL (Agricultural Research Service, Patent Culture Collection, Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A) on August 17, 1998, and assigned accession no. NRRL B-30050.
  • Phagemids are excised from ⁇ ZAPII cDNA libraries of Brassica napus using a protocol from Stratagene. Phagemids (plasmids) are mass-transformed into E. co// XL1 - Blue in 10 pools of about 80,000 clones each and DNA is extracted from these pools. The pools are screened by PCR for the presence of NIM1 homologues by PCR using the following primers: 5'-AGATTATTGTCAAGTCTAATG-3' (SEQ ID NO:9) and 5'- TTCCATGTACCTTTGCTTC-3' (SEQ ID NO:10).
  • Sequences amplified from the pools are confirmed to contain NIM1 homologues by cloning the PCR-amplified DNA fragment and sequencing. Pools are made successively smaller and screened by PCR using the same primers mentioned above for the presence of the NIM1 homologues until a single clone containing the homologue is obtained. In the event that the cDNA clone contains a partial gene, missing the 5' end, 5' RACE (Rapid Amplification of cDNA Ends) is used to obtain the full-length sequence of the gene.
  • a partial cDNA sequence of this Brassica napus NIM1 homologue is shown in SEQ ID NO:5, and the protein encoded by this cDNA sequence is shown in SEQ ID NO:6.
  • a Brassica napus NIM1 homologue comprising SEQ ID NO:5 has been deposited as pNOV1203 with the NRRL (Agricultural Research Service, Patent Culture Collection, Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A) on August 17, 1998, and assigned accession no. NRRL B-30049.
  • Example 4 Isolation of a NIM1 Homologue from Arabidopsis thallana
  • GenBank entry B26306 contains Arabidopsis genomic sequence from the Bacterial Artificial Chromosome (BAC) F18D8.
  • BAC Bacterial Artificial Chromosome
  • Part of the BAC sequence is predicted to encode a protein with significant similarity (47% amino acid identity) to NIM1.
  • the following primers are designed to regions of the F18D8 sequence: 5'- TCAAGGCCTTGGATTCAGATG-3' (SEQ ID NO:13) and 5'- ATTAACTGCGCTACGTCCGTC-3' (SEQ ID NO:14).
  • the primers are used in a PCR reaction with DNA from a pFL61 -based Arabidopsis cDNA library as a template.
  • Preferable cycling conditions are 94 degrees for 30 seconds, 53 degrees for 30 seconds, 72 degrees for 30 seconds.
  • the reaction is preferably run for 40 cycles.
  • a PCR product of the predicted size (290 base pairs) is detected, and the cDNA clone corresponding to the F18D8 primers is purified from the cDNA library by sequential purification by passage of increasingly smaller amounts of the library through E. coli and re- diagnosis of the presence of the clone by PCR.
  • a single positive clone is obtained and sequenced.
  • the sequence of the clone confirms the presence of an open reading frame with significant homology to NIM1.
  • a full-length cDNA sequence of this Arabidopsis thaliana NIM1 homologue is shown in SEQ ID NO:7, and the protein encoded by this cDNA sequence is shown in SEQ ID NO:8.
  • An Arabidopsis thaliana NIM1 homologue comprising SEQ ID NO:7 has been deposited as A tNMLc ⁇ in E. coli with the NRRL (Agricultural Research Service, Patent Culture Collection, Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A) on May 25, 1999, and assigned accession no. NRRL B- 30139.
  • Arabidopsis thaliana contains three other M-like (NML) genomic sequences: AtNMLc2 (SEQ ID NO:15), AtNMLc4-1 (SEQ ID NO:17), and AtNMLc4-2 (SEQ ID NO:19), where c[#] stands for the chromosome number on which the particular NML gene is located.
  • NIM1 sequences from Arabidopsis thaliana (Ryals et al., 1997), Nicotiana tabacum (Example 1 - SEQ ID NO:1), and Lycopersicon esculentum (Example 2 - SEQ ID NO:3), as well as the NML sequences from Arabidopsis thaliana (SEQ ID NO:7, 15, 17, and 19) are aligned. Based on this alignment, three regions emerge with sufficient conservation to design degenerate PCR primers for PCR amplification of NIM1 homologues from other crop species, including sugarbeet, sunflower, potato, and canola.
  • the primers designed from these conserved regions are listed below in Table 1.
  • the NIM 1(A-D) primers are designed using a lineup with only the NIM1 genes from Arabidopsis thaliana (Ryals et al., 1997), Nicotiana tabacum (Example 1 - SEQ ID NO:1), and Lycopersicon esculentum (Example 2 - SEQ ID NO:3).
  • the NIM 2(A-D) primers are designed using a lineup with these three sequences in addition to the four NML sequences from Arabidopsis thaliana (SEQ ID NO:7, 15, 17, and 19).
  • Primers are preferably synthesized by Genosys Biotechnologies, Inc. (The Woodlands, Texas).
  • Positions of degeneracy are indicated in Table 1 by the notation of more than one base at a single site in the oligonucieotide. Orientation" designates whether the primer is directed towards the 3' end (Downstream) or the 5' end (Upstream) of the cDNA.
  • -like DNA fragments are amplified from Arabidopsis, tomato, tobacco, sugarbeet, sunflower, potato, and canola, using either genomic DNA or cDNA as templates.
  • the primer combinations used, along with the expected fragment sizes, are listed below in Table 2.
  • Degenerate primer PCR is preferably performed with Ready-To-Go PCR Beads (Amersham, Piscataway, NJ) in a GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA). 20 to 40 ng of genomic DNA or 5 to 10 ng of cDNA is used in each reaction, with each primer at a final concentration of 0.8 ⁇ M.
  • Preferable cycling parameters are as follows: 94°C for 1 minute; 3 cycles of [94°C for 30 seconds; 37°C for 30 seconds; 72°C for 2 minutes]; 35 cycles of [94°C for 30 seconds; 60°C for 30 seconds; 72°C for 2 minutes]; 72°C for 7 minutes; 4°C hold.
  • DNA fragments are excised.
  • DNA fragments are isolated from agarose bands using, for example, the Geneclean III Kit (BIO 101 , Inc., Carlsbad, CA) and cloned using, for example, the TOPO TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA).
  • Plasmids are isolated using, for example, the CONCERT Rapid Plasmid Miniprep System (Life Technologies, Inc., Rockville, MD) and sequenced by standard protocols. /W-like DNA fragments are obtained from all plant species attempted, and in many cases multiple, unique NIM-Wke sequences are isolated. Table 3 and Figure 2 detail the MM-like fragments that are isolated. Table 3: M ⁇ like PCR fragments
  • the degenerate primer PCR described above can amplify ⁇ WM-like fragments from a wide variety of plant species.
  • the primer combination of NIM 2B/NIM 2D is successful with cDNA as a template from all species attempted.
  • the use of Ready-To-Go PCR Beads is especially preferably for obtaining products.
  • using cDNA as a template is preferable for all samples except Arabidopsis, tomato and sugarbeet, where genomic DNA is sufficient.
  • a new pair of degenerate primers is designed based on a sequence alignment of the four tobacco fragments (SEQ ID NO: 29, 31 , 33, and 35) and the tomato sequence (SEQ ID NO: 37) for use in determining whether tomato also contains similar NIM-Wke sequences that are not amplified with the degenerate primers listed in Table 1.
  • the primers designed from these fragments are listed below in Table 3 and are preferably synthesized by Genosys Biotechnologies, Inc. (The Woodlands, Texas). Positions of degeneracy are indicated in Table 3 by the notation of more than one base at a single site in the oligonucieotide. Orientation" designates whether the primer is directed towards the 3' end (Downstream) or the 5' end (Upstream) of the cDNA.
  • Degenerate primer PCR is performed as described above using tomato cDNA, and potential products are cloned and sequenced.
  • the sequence analysis reveals two classes of MM-like fragments: the first is identical to the tomato sequence shown in SEQ ID NO: 37, and the second is unique in tomato and 88% identical to the tobacco sequences shown in SEQ ID NO:31 and 33.
  • the sequence of this new tomato sequence is presented in SEQ ID NO:61.
  • Corresponding cDNA sequences upstream and downstream from MM-like PCR fragments are preferably obtained by RACE PCR using the SMART RACE cDNA Amplification Kit (Clontech, Palo Alto, CA). Preferably, at least three independent RACE products are sequenced for each 5'- or 3'-end in order to eliminate PCR errors. Resulting full-length cDNA sequences for Sugarbeet, Sunflower B, and Tobacco B NIM1 homologues, which correspond to the MM-like PCR fragments shown in SEQ ID NO:39, 43, and 31 are presented as SEQ ID NO:63, 65, and 73 respectively.
  • MM-like Arabidopsis thaliana cDNA's corresponding to the MM-like genomic sequences AtNMLc2 (SEQ ID NO:15), AtNMLc4-1 (SEQ ID NO:17), and AtNMLc4-2 (SEQ ID NO:19), are preferably cloned by RT-PCR.
  • Total RNA from Arabidopsis thaliana is reverse transcribed using oligo dT primer.
  • the resulting first strand cDNA is amplified by PCR using specific sense and antisense oligonucieotide primers designed based on the 5' and 3' ends of the coding region of each genomic sequence (SEQ ID NO:15, 17, and 19).
  • PCR fragments of the predicted sizes are cloned into a vector and sequenced to confirm that these cDNA clones correspond to the MM-like genomic sequences.
  • a cDNA sequence corresponding to the MM-like genomic sequence AtNMLc2 (SEQ ID NO:15) is presented as SEQ ID NO:67; a full-length cDNA sequence corresponding to the MM-like genomic sequence AtNMLc4-1 (SEQ ID NO:17) is presented as SEQ ID NO:69; and a full-length cDNA sequence corresponding to the MM-like genomic sequence AtNMLc4-2 (SEQ ID NO:19) is presented as SEQ ID NO:71.
  • Northern data shows that expression of the sugarbeet MM-like clone (SEQ ID NO:39 and 63) increases three to seven fold after 100 ⁇ M or 300 ⁇ M BTH (benzo(1 ,2,3)thiadiazole- 7-carbothioic acid S-methyl ester) treatment. Also, Northern data shows that expression of the Sunflower A MM-like clone (SEQ ID NO:41) is constitutive. Furthermore, Northern data shows that expression of the Sunflower B MM-like clone (SEQ ID NO:43 and 65) increases two fold after 100 ⁇ M or 300 ⁇ M BTH treatment.
  • a MM7 homologue of the present invention can be incorporated into plant cells using conventional recombinant DNA technology. Generally, this involves inserting a coding sequence of the invention into an expression system to which the coding sequence is heterologous (i.e., not normally present) using standard cloning procedures known in the art.
  • the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • a large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses.
  • Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems ⁇ gtH , ⁇ gtIO and Charon 4; plasmid vectors such as pBI121 , pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101 , pCDNAII; and other similar systems.
  • the components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed.
  • Coding sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants.
  • the expression cassettes may also comprise any further sequences required or selected for the expression of the transgene.
  • Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
  • the selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non- limiting examples of promoters that may be used in expression cassettes.
  • Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - Binet et a/., 1991 ; maize - Christensen et al., 1989; and Arabidopsis - Norris et al., 1993).
  • the maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol). Taylor et al.
  • a vector that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment.
  • the Arabidopsis ubiquitin promoter is especially preferred for use with the MM7 homologues of the present invention.
  • the ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons.
  • Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.
  • pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone.
  • a derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761 ENX.
  • pCGN1761 ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants.
  • the entire 35S promoter-coding sequence-ttn/ terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites 5' to the promoter and Xbal, BamHI and Bgll sites 3' to the terminator for transfer to transformation vectors such as those described below.
  • the double 35S promoter fragment can be removed by 5' excision with Hindlll, Sphl, Sail, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter.
  • modifications around the cloning sites can be made by the introduction of sequences that may enhance translation. This is particularly useful when overexpression is desired.
  • pCGN1761 ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Patent No. 5,639,949.
  • actin promoter is a good choice for a constitutive promoter.
  • the promoter from the rice >4ctV gene has been cloned and characterized (McElroy et al., 1990).
  • a 1.3kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts.
  • numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy etal., 1991).
  • promoter-containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences.
  • the fusion genes thus constructed can then be transferred to appropriate transformation vectors.
  • the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al., 1993).
  • the double 35S promoter in pCGN1761 ENX may be replaced with any other promoter of choice that will result in suitably high expression levels.
  • one of the chemically regulatable promoters described in U.S. Patent No. 5,614,395 may replace the double 35S promoter.
  • the promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector.
  • the chemically/pathogen regulatable tobacco PR- la promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104) and transferred to plasmid pCGN1761 ENX (Uknes et al., 1992).
  • pCIB1004 is cleaved with Ncol and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase.
  • the fragment is then cleaved with Hindlll and the resultant PR-1 a promoter-containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed.
  • Various chemical regulators may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395.
  • a promoter inducible by certain alcohols or ketones, such as ethanol, may also be used to confer inducible expression of a coding sequence of the present invention.
  • a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et al., 1998).
  • the a/c>4 gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer.
  • the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter are replaced by a coding sequence of the present invention to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods well known in the art.
  • glucocorticoid-mediated induction system is used (Aoyama and Chua, 1997) and gene expression is induced by application of a glucocorticoid, for example a synthetic glucocorticoid, preferably dexamethasone, preferably at a concentration ranging from 0.1 mM to 1mM, more preferably from 10mM to 100mM.
  • the luciferase gene sequences are replaced by a gene sequence encoding a NIM1 homologue to form an expression cassette having the gene sequence encoding a NIM1 homologue under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter.
  • the trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al., 1986) fused to the transactivating domain of the herpes viral protein VP16 (Triezenberg etal., 1988) fused to the hormone- binding domain of the rat glucocorticoid receptor (Picard et al., 1988).
  • the expression of the fusion protein is controlled by any promoter suitable for expression in plants known in the art or described here.
  • This expression cassette is also comprised in the plant comprising the gene sequence encoding a NIM1 homologue fused to the 6xGAL4/minimal promoter.
  • tissue- or organ-specificity of the fusion protein is achieved leading to inducible tissue- or organ-specificity of the NIM1 homologue.
  • a suitable root promoter is described by de Framond (1991) and also in the published patent application EP 0 452 269. This promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.
  • Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described (e.g. Xu et al., 1993); Logemann et al., 1989; Rohrmeier & Lehle, 1993; Firek et al., 1993; Warner et al., 1993) and all are suitable for use with the instant invention. Logemann etal. describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice.
  • Rohrmeier & Lehle describe the cloning of the maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similar, Firek et al. and Warner et al. have described a wound-induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to this invention, and used to express these genes at the sites of plant wounding.
  • Patent Application WO 93/07278 describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells.
  • the gene sequence and promoter extending up to -1726 bp from the start of transcription are presented.
  • this promoter, or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner.
  • fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
  • a maize gene encoding phosphoenol carboxylase has been described by Hudspeth & Grula (1989). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.
  • WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene which is expressed in pollen cells.
  • CDPK calcium-dependent protein kinase
  • the gene sequence and promoter extend up to 1400 bp from the start of transcription.
  • this promoter or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a NIM1 homologue of the present invention in a pollen-specific manner.
  • transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation.
  • Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
  • a gene's native transcription terminator may be used.
  • intron sequences have been shown to enhance expression, particularly in monocotyledonous cells.
  • the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells.
  • Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., 1987).
  • the intron from the maize bronzel gene had a similar effect in enhancing expression.
  • Intron sequences have been routinely inco ⁇ orated into plant transformation vectors, typically within the non-translated leader.
  • leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
  • TMV Tobacco Mosaic Virus
  • MCMV Maize Chlorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized. See also, the section entitled “Expression With Chloroplast Targeting" in Example 37 of U.S. Patent No. 5,639,949.
  • cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular protein bodies has been described by Rogers et al. (1985).
  • amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, 1990). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., 1990).
  • the transgene product By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment.
  • chloroplast targeting for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene.
  • the signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence.
  • Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. (1982) and Wasmann et al. (1986). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.
  • the above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.
  • transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors.
  • the selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred.
  • Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra, 1982; Bevan et al., 1983), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., 1990; Spencer et al., 1990), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., 1983), and the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642).
  • vectors Suitable for Agrobacterium Transformation Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.
  • the binary vectors pclB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner.
  • pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, 1985) allowing excision of the tetracydine-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Messing & Vierra, 1982; Bevan et al., 1983; McBride et al., 1990).
  • Xhol linkers are ligated to the EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., 1987), and the Nbo/-digested fragment are cloned into Sa//-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19).
  • pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail.
  • pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites.
  • Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bell, Avrll, Apal, Hpal, and Stul.
  • pCIB2001 in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for /4c7robacter/ ' ut7T-mediated transformation, the RK2-derived trf A function for mobilization between E. coli and other hosts, and the Or/Tand Or/Vfunctions also from RK2.
  • the pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
  • the binary vector pCIBIO contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (1987). Various derivatives of pCIBIO are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al, 1983). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717). 2.
  • Vectors Suitable for non-Agrobacterium Transformation Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for non-Agrobacterium transformation is described.
  • pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin).
  • the plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278.
  • the 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull.
  • the new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site.
  • the resultant derivative of pCIB246 is designated pCIB3025.
  • the GUS gene is then excised from pCIB3025 by digestion with Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060.
  • the plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al., 1987).
  • This generated pCIB3064 which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, Hindlll, and BamHI.
  • This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
  • pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate.
  • DFR E. coli gene dihydrofolate reductase
  • PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adhl gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250-bp fragment encoding the E.
  • coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which comprises the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generates pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Pstl and EcoRI sites available for the cloning of foreign substances.
  • MCMV Maize Chlorotic Mottle Virus
  • the gene sequence of interest Once the gene sequence of interest has been cloned into an expression system, it is transformed into a plant cell.
  • Methods for transformation and regeneration of plants are well known in the art.
  • Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles.
  • bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants.
  • Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium.
  • Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., 1984; Potrykus et al., 1985; Reich et al., 1986; and Klein et al., 1987. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
  • Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species.
  • Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001 ) to an appropriate Agrobacterium strain which may depend of the complement of wr genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes etal., 1993).
  • the transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E.
  • helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain.
  • the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (H ⁇ fgen & Willmitzer, 1988).
  • Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T- DNA borders.
  • Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells.
  • This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792.
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford inco ⁇ oration within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the desired gene.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced
  • Transformation of most monocotyledon species has now also become routine.
  • Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co- transformation) and both these techniques are suitable for use with this invention.
  • Co- transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable.
  • a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al., 1986).
  • Patent Applications EP 0 292 435, EP 0392225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts.
  • Gordon-Kamm etal. (1990) and Fromm et al. (1990) have published techniques for transformation of A188-derived maize line using particle bombardment.
  • WO 93/07278 and Koziel et al. (1993) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1 OOOHe Biolistics device for bombardment.
  • Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment.
  • Protoplast-mediated transformation has been described for Japon/ca-types and Indica-types (Zhang etal., 1988; Shimamoto et al., 1989; Datta etal., 1990). Both types are also routinely transformable using particle bombardment (Christou et al., 1991 ).
  • WO 93/21335 describes techniques for the transformation of rice via electroporation.
  • Patent Application EP 0332581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (1992) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil etal. (1993) and Weeks etal. (1993) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery.
  • any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, 1962) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark.
  • MS medium with 3% sucrose (Murashiga & Skoog, 1962) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark.
  • embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical.
  • An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures.
  • Each plate of embryos is shot with the DuPont Biolistics® helium device using a burst pressure of -1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration.
  • the plants obtained via tranformation with a gene of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention are preferably selected from the list of agronomically important target crops set forth supra.
  • the expression of a gene of the present invention in combination with other characteristics important for production and quality can be inco ⁇ orated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R. (1981); Wood D. R. (Ed.) (1983); Mayo O. (1987); Singh, D.P. (1986); and Wricke and Weber (1986).
  • the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants.
  • said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting.
  • Specialized processes such as hydroponics or greenhouse technologies can also be applied.
  • measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield.
  • Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering.
  • the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants.
  • different breeding measures are taken.
  • the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
  • Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means.
  • Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.
  • the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow one to dispense with said methods due to their modified genetic properties.
  • new crops with improved stress tolerance can be obtained, which, due to their optimized genetic "equipment” , yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.
  • Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD ® ), methalaxyl (Apron ® ), and pirimiphos-methyl (Actellic ® ). If desired, these compounds are formulated together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests.
  • the protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.
  • the seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds.
  • the bag, container or vessel may be designed for either short term or long term storage, or both, of the seed.
  • a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal.
  • the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type.
  • the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed.
  • the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering.
  • water absorbent materials are placed between or adjacent to packaging material layers.
  • the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed.
  • An example of such treatment is sterilization, for example by chemical means or by exposure to radiation.
  • a commercial bag comprising seed of a transgenic plant comprising a gene of the present invention that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with label instructions for the use thereof for conferring broad spectrum disease resistance to plants.
  • Assays for resistance to Phytophthora parasitica the causative organism of black shank, are performed on six-week-old plants grown as described in Alexander et al. (1993). Plants are watered, allowed to drain well, and then inoculated by applying 10 ml of a sporangium suspension (300 sporangia/ml) to the soil. Inoculated plants are kept in a greenhouse maintained at 23-25°C day temperature, and 20-22°C night temperature.
  • Example 17 Cercospora nicotianae Resistance Assay
  • a spore suspension of Cercospora nicotianae (ATCC #18366) (100,000-150,000 spores per ml) is sprayed to imminent run-off onto the surface of the leaves.
  • the plants are maintained in 100% humidity for five days. Thereafter the plants are misted with water 5-10 times per day.
  • Six individual plants are evaluated at each time point. Cercospora nicotianae is rated on a % leaf area showing disease symptoms basis.
  • a T-test (LSD) is conducted on the evaluations for each day and the groupings are indicated after the Mean disease rating value. Values followed by the same letter on that day of evaluation are not statistically significantly different.
  • Assays for resistance to Peronospora parasitica are performed on plants as described in Uknes et al, (1993). Plants are inoculated with a compatible isolate of P. parasitica by spraying with a conidial suspension (approximately 5 x 10 4 spores per milliliter). Inoculated plants are incubated under humid conditions at 17° C in a growth chamber with a 14-hr day/10-hr night cycle. Plants are examined at 3-14 days, preferably 7-12 days, after inoculation for the presence of conidiophores. In addition, several plants from each treatment are randomly selected and stained with lactophenol-trypan blue (Keogh et al., 1980) for microscopic examination.
  • SEQ ID NO:1 Full length cDNA sequence of a MM7 homologue from Nicotiana tabacum.
  • SEQ ID NO:2 Protein sequence of the Nicotiana tabacum NIM1 homologue encoded by
  • SEQ ID NO:1 SEQ ID NO:3 - Full length cDNA sequence of a NIM1 homologue from Lycopersicon esculentum.
  • SEQ ID NO:4 Protein sequence of the Lycopersicon esculentum NIM1 homologue encoded by SEQ ID NO:3.
  • SEQ ID NO:5 Partial cDNA sequence of a MM7 homologue from Brassica napus.
  • SEQ ID NO:6 Partial protein sequence of the Brassica napus NIM1 homologue encoded by SEQ ID NO:5.
  • SEQ ID NO:7 Full length cDNA sequence of a NIM1 homologue (AtNMLcS) from
  • Arabidopsis thaliana SEQ ID NO:8 - Full length protein sequence of the Arabidopsis thaliana NIM1 homologue
  • SEQ ID NO:15 Genomic DNA sequence of a MM7 homologue (AtNMLc2) from
  • Arabidopsis thaliana SEQ ID NO:16 - Protein sequence of the Arabidopsis thaliana NIM1 homologue AtNMLc2 encoded by SEQ ID NO:15.
  • SEQ ID NO:17 Genomic DNA sequence of a MM7 homologue (AtNMLc4-1) from
  • Arabidopsis thaliana SEQ ID NO:18 - Protein sequence of the Arabidopsis thaliana NIM1 homologue AtNMLc4-1 encoded by SEQ ID NO:17.
  • SEQ ID NO:19 Genomic DNA sequence of a MM7 homologue (AtNMLc4-2) from
  • Arabidopsis thaliana SEQ ID NO:20 - Protein sequence of the Arabidopsis thaliana NIM1 homologue AtNMLc4-2 encoded by SEQ ID NO:19.
  • SEQ ID NO:24 - PCR primer NIM 1D SEQ ID NO:25 - PCR primer NIM 2A.
  • SEQ ID NO:28 - PCR primer NIM 2D SEQ ID NO:29 - 659 bp MM-like DNA fragment amplified from Nicotiana tabacum (Tobacco).
  • SEQ ID NO:30 Protein sequence encoded by SEQ ID NO:29.
  • SEQ ID NO:32 Protein sequence encoded by SEQ ID NO:31.
  • SEQ ID NO:36 Protein sequence encoded by SEQ ID NO:35.
  • Tomato A which is a consensus of 8 sequences and has 67% sequence identity to the Arabidopsis thaliana NIM1 gene sequence.
  • SEQ ID NO:38 Protein sequence encoded by SEQ ID NO:37.
  • SEQ ID NO:39 - 498 bp MM-like DNA fragment amplified from Beta vulgaris (Sugarbeet), which is a consensus of 24 sequences and has 66% sequence identity to the Arabidopsis thaliana NIM1 gene sequence.
  • SEQ ID NO:40 Protein sequence encoded by SEQ ID NO:39.
  • SEQ ID NO:50 Protein sequence encoded by SEQ ID NO:49.
  • Arabidopsis thaliana NIM1 gene sequence SEQ ID NO:52 - Protein sequence encoded by SEQ ID NO:51.
  • SEQ ID NO:54 Protein sequence encoded by SEQ ID NO:53.
  • SEQ ID NO:55 - 498 bp MM-like DNA fragment amplified from Brassica napus (Canola C), which has 56% sequence identity to the Arabidopsis thaliana NIM1 gene sequence.
  • SEQ ID NO:56 Protein sequence encoded by SEQ ID NO:55.
  • SEQ ID NO:57 - 498 bp MM-like DNA fragment amplified from Brassica napus (Canola D), which has 73% sequence identity to the Arabidopsis thaliana NIM1 gene sequence.
  • SEQ ID NO:58 Protein sequence encoded by SEQ ID NO:57.
  • SEQ ID NO:59 - PCR primer NIM 3A SEQ ID NO:60 - PCR primer NIM 3B.
  • Tomato B which is a consensus of 3 sequences and has 72% sequence identity to the Arabidopsis thaliana NIM1 gene sequence.
  • SEQ ID NO:62 Protein sequence encoded by SEQ ID NO:61.
  • SEQ ID NO:63 Full length cDNA sequence of a MM7 homologue from Beta vulgaris
  • SEQ ID NO:65 Full length cDNA sequence of a MM7 homologue from Helianthus annuus (Sunflower B), which corresponds to the PCR fragment of SEQ ID NO:43.
  • SEQ ID NO:66 Protein sequence of the Helianthus annuus NIM1 homologue encoded by SEQ ID NO:65.
  • SEQ ID NO:68 Protein sequence encoded by SEQ ID NO:67.
  • SEQ ID NO:69 cDNA sequence corresponding to the Arabidopsis thaliana MM-like genomic sequence AtNMLc4-1 (SEQ ID NO: 17).
  • SEQ ID NO:70 Protein sequence encoded by SEQ ID NO:69.
  • SEQ ID NO:71 cDNA sequence corresponding to the Arabidopsis thaliana MM-like genomic sequence AtNMLc4-2 (SEQ ID NO:19).
  • SEQ ID NO:72 Protein sequence encoded by SEQ ID NO:71.
  • SEQ ID NO:73 Full length cDNA sequence of a MM7 homologue from Nicotiana tabacum (Tobacco B), which corresponds to the PCR fragment of SEQ ID NO:31.
  • SEQ ID NO:74 Protein sequence of the Nicotiana tabacum NIM1 homologue encoded by SEQ ID NO:73.

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Abstract

L'invention concerne des homologues du gène Arabidopsis NIM1, impliqué dans la cascade de transduction des signaux menant à la résistance systémique acquise (RSA), qui sont isolés à partir de Nicotiana tabacum (tabac), de Lycopersicon esculentum (tomate), de Brassica napus (colza oléagineux), d'Arabidopsis thaliana, de Beta vulgaris (betterave à sucre), d'Helianthus annuus (tournesol) et de Solanum tuberosum (pomme de terre). L'invention concerne également des vecteurs de transformation et des processus permettant d'exprimer les homologues de NIM1 dans des végétaux transgéniques afin d'accroître l'expression du gène RSA et d'élargir le large spectre de résistance aux maladies.
EP00909334A 1999-03-09 2000-03-07 Nouveaux genes de vegetaux et leurs utilisations Withdrawn EP1159424A2 (fr)

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US26514999A 1999-03-09 1999-03-09
PCT/EP2000/001978 WO2000053762A2 (fr) 1999-03-09 2000-03-07 Nouveaux genes de vegetaux et leurs utilisations

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KR100423262B1 (ko) * 2000-12-18 2004-03-19 세미니스코리아주식회사 담배 유래의 Ｔｓｉ1 유전자를 도입하여 제조된 형질전환균주
KR100447813B1 (ko) * 2000-12-18 2004-09-08 세미니스코리아주식회사 담배 유래의 Ｔｓｉｐ1 유전자 도입 재조합 벡터 및 그 형질전환균주
JP4739672B2 (ja) 2001-12-21 2011-08-03 ネクター セラピューティクス 湿気のバリアを有するカプセルパッケージ
US20090137390A1 (en) * 2004-06-30 2009-05-28 Eric Wendell Triplett Materials and methods for enhancing nitrogen fixation in plants
KR102583332B1 (ko) * 2020-11-13 2023-09-25 서울대학교산학협력단 셀룰로오스 합성 유전자 Solyc07g043390이 과발현된 토마토 황화잎말림 바이러스병에 대한 내성이 증진된 형질전환 식물체
CN117088948B (zh) * 2023-08-23 2024-05-14 中国农业大学 辣椒疫霉调控游动孢子发育相关蛋白及其编码基因与应用

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US20020138872A1 (en) * 1996-08-09 2002-09-26 Xinnian Dong Acquired resistance genes and uses thereof
FR2757875A1 (fr) * 1996-12-13 1998-07-03 Ciba Geigy Ag Procedes d'utilisation du gene nim1 pour conferer aux vegetaux une resistance aux maladies
CA2275854A1 (fr) * 1996-12-27 1998-07-09 Novartis Ag Procede de protection de plantes

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WO2000053762A3 (fr) 2001-05-31
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