WO1999043821A1 - Genes activant le systeme de defense de plantes contre les elements pathogenes - Google Patents

Genes activant le systeme de defense de plantes contre les elements pathogenes Download PDF

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Publication number
WO1999043821A1
WO1999043821A1 PCT/US1999/003337 US9903337W WO9943821A1 WO 1999043821 A1 WO1999043821 A1 WO 1999043821A1 US 9903337 W US9903337 W US 9903337W WO 9943821 A1 WO9943821 A1 WO 9943821A1
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promoter
plant
sequence
nucleotide sequence
maize
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PCT/US1999/003337
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English (en)
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Steven P. Briggs
Jonathan P. Duvick
Carl R. Simmons
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Pioneer Hi-Bred International, Inc.
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Priority to AU26833/99A priority Critical patent/AU2683399A/en
Publication of WO1999043821A1 publication Critical patent/WO1999043821A1/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
    • 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

Definitions

  • the invention relates to the genetic manipulation of plants, particularly to transforming plants with genes that enhance disease resistance.
  • Biotic causes include fungi, viruses, bacteria, and nematodes. Of these, fungi are the most frequent causative agent of disease on plants.
  • Abiotic causes of disease in plants include extremes of temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution.
  • a host of cellular processes enables plants to defend themselves from disease caused by pathogenic agents. These processes apparently form an integrated set of resistance mechanisms that is activated by initial infection and then limits further spread of the invading pathogenic microorganism.
  • plants can activate an array of biochemical responses. Generally, the plant responds by inducing several local responses in the cells immediately surrounding the infection site. The most common resistance response observed in both nonhost and race-specific interactions is termed the "hypersensitive response" (HR). In the hypersensitive response, cells contacted by the pathogen, and often neighboring cells, rapidly collapse and dry in a necrotic fleck. Other responses include the deposition of callose, the physical thickening of cell walls by lignification, and the synthesis of various antibiotic small molecules and proteins. Genetic factors in both the host and the pathogen determine the specificity of these local responses, which can be very effective in limiting the spread of infection.
  • HR hypersensitive response
  • the hypersensitive response in many plant-pathogen interactions results from the expression of a resistance (R) gene in the plant and a corresponding avirulence
  • R genes that respond to specific bacterial, fungal, or viral pathogens have been isolated from a variety of plant species and several appear to encode cytoplasmic proteins.
  • the resistance gene in the plant and the avirulence gene in the pathogen often conform to a gene-for-gene relationship. That is, resistance to a pathogen is only observed when the pathogen carries a specific avirulence gene and the plant carries a corresponding or complementing resistance gene.
  • R gene The function of a given R gene is dependent on the genotype of the pathogen. Plant pathogens produce a diversity of potential signals, and in a fashion analogous to the production of antigens by mammalian pathogens, some of these signals are detectable by some plants.
  • the avirulence gene causes the pathogen to produce a signal that triggers a strong defense response in a plant with the appropriate R gene.
  • expressing an avirulence gene does not stop the pathogen from being virulent on hosts that lack the corresponding R gene.
  • a single plant can have many R genes, and a pathogen can have many avr genes.
  • phytopathogenic fungi play the dominant role. Phytopathogenic fungi cause devastating epidemics, as well as causing significant annual crop yield losses. All of the approximately 300,000 species of flowering plants are attacked by pathogenic fungi. However, a single plant species can be host to only a few fungal species, and similarly, most fungi usually have a limited host range.
  • Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change.
  • the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose.
  • the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack.
  • compositions and methods of the invention can be used for enhancing resistance to plant pests.
  • the method involves stably transforming a plant with a gene capable of inducing the plant pathogen defense system operably linked with a promoter capable of driving expression of a gene in a plant cell. It is recognized that a variety of promoters will be useful in the invention, the choice of which will depend in part upon the desired level of expression of the disclosed genes.
  • the levels of gene expression can be controlled to induce the disease resistance pathway resulting in levels of immunity in the plant that impart resistance to the pathogen or induce cell death.
  • the methods of the invention find use in controlling plant pests, including fungal pathogens, viruses, nematodes, insects, and the like.
  • Figure 1 schematically illustrates the plasmid construct comprising the ubiquitin promoter and CRC fusion protein gene.
  • Figure 2 schematically illustrates the plasmid construct comprising the ubiquitin promoter and an activator sequence.
  • the invention is drawn to compositions and methods for inducing resistance in a plant to plant pests. Accordingly, the compositions and methods are also useful in protecting plants against fungal pathogens, viruses, nematodes, insects and the like.
  • Disease resistance is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions.
  • pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen is minimized or lessened.
  • the methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by plant pathogens.
  • Pathogens of the invention include, but are not limited to, viruses or viroids, bacteria, insects, nematodes, fungi, and the like. Viruses include tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specific fungal and viral pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani,
  • Xanthomonas campestris p.v. phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus.
  • Tobacco Streak virus Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibater michiganese subsp.
  • Puccinia graminis f.sp. tritici Puccinia recondita f.sp. tritici, Puccinia striiformis, Pyrenophora tritici- repentis, Septoria nodorum, Septoria tritici, Septoria avenae.
  • Pseudocercosporella herpotrichoides Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var.
  • Ustilago tritici Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, High Plains Virus, European wheat striate virus; Sunflower: Plasmophora halstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv.
  • Curvularia inaequalis Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia corotovora, Cornstunt spiroplasma,
  • Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternate, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa.
  • Pseudomonas avenae Pulseudomonas alboprecipitans
  • Ramulispora sorghi Ramulispora sorghicola
  • Phyllachara sacchari Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola, etc.
  • Nematodes include parasitic nematodes such as root knot, cyst and lesion nematodes. etc.
  • Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga. Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.
  • Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp.
  • Rhopalosiphum maidis corn leaf aphid
  • Anuraphis maidiradicis corn root aphid
  • Blissus leucopterus leucopterus chinch bug
  • Melanoplus femur rubrum redlegged grasshopper
  • Melanoplus sanguinipes migratory grasshopper
  • Hylemya platura seedcorn maggot
  • Agromyza parvicornis corn blot leafminer
  • Anaphothrips obscrurus
  • cereal leaf beetle Hyper a punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum. greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly: Sitodiplosis mosellana, wheat midge; Meromyza americana.
  • wheat stem maggot Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower: Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm: Pectinophora gossypiella, pink bollworm; Anthonomus grandis.
  • compositions of the invention include genes that are involved in activating plant systems for defense against pathogens.
  • the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, and 12, or the nucleotide sequences encoding the DNA sequence deposited in a bacterial host as ATCC Accession No. 207013.
  • Plasmids containing the nucleotide sequences of the invention were deposited with American Type Culture Collection (ATCC), Manassas, Virginia, and assigned Accession No. 207013. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the invention encompasses isolated or substantially purified nucleic acid or protein compositions.
  • An "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0J kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein.
  • culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • the gene products function to activate the defense system.
  • the activation of the defense system may involve inducing resistance of the plant to pathogen invasion or alternatively, the gene products may turn on the hypersensitive response in the plant.
  • the hypersensitive response is a localized lesion of necrotic tissue that forms around the site of pathogen infection. This plant-induced necrosis thwarts progression of the disease by depriving the pathogen of plant material for its consumption.
  • activation of the plant defense system may involve the induced production of gene products, such as PR proteins and various secondary metabolites, many of which are antipathogenic.
  • the induction may involve inducing the accumulation of cytotoxic phytoalexins, the deposition of callose and lignin in cell
  • the induction may involve the activation of transcription factors, reactive oxygen species, ion fluxes, G proteins, salicylic acids, and other HR and plant defense regulators. It is recognized that the present invention is not dependent upon a particular mechanism of defense. Rather, the genes and methods of the invention work to increase resistance of the plant to pathogens independent of how that resistance is brought about.
  • fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention.
  • fragment is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby.
  • Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence activate the plant defense system.
  • fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity.
  • fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the entire nucleotide sequence encoding the proteins of the invention.
  • a fragment of an activator nucleotide sequence that encodes a biologically active portion of an activator protein of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, 250 contiguous amino acids, or up to the total number of amino acids present in a full-length activator protein of the invention (for example, 287, 284, 284, 176, 158, or 157 amino acids for SEQ ID NO: 2, 4, 6, 8, 10, or 12, respectively).
  • Fragments of an activator nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of an activator protein.
  • a fragment of an activator nucleotide sequence may encode a biologically active portion of an activator protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
  • a biologically active portion of an activator protein can be prepared by isolating a portion of one of the activator nucleotide sequences of the invention, expressing the encoded portion of the activator protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the activator protein.
  • Nucleic acid molecules that are fragments of an activator nucleotide sequence comprise at least 16, 20, 50, 75, 100, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100,
  • nucleotide sequences 10 1J00, 1J00, or 1,400 nucleotides. or up to the number of nucleotides present in a full-length activator nucleotide sequence disclosed herein (for example, 1187, 1020, 1450, 974, 1117, or 965 nucleotides for SEQ ID NOs: 1, 3, 5, 7, 9, or 11, respectively).
  • variants are intended substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the defense activator polypeptides of the invention.
  • nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, 70%, generally, 80%, preferably 85%, 90% up to 95%, 98% sequence identity to its respective native nucleotide sequence.
  • variant protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N- terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • the proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the activator proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, T. (1985) Proc. Natl. Acad. Sci. USA 52:488-492: Kunkel et al. (1987) Methods in Enzymol. 154:367-382; US Patent No. 4,873,192; Walker and Gaastra (eds.) Techniques in Molecular Biology.
  • genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired activator activity.
  • mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
  • the present invention encompasses the activator proteins as well as components and fragments thereof. That is, it is recognized that component polypeptides or fragments of the proteins may be produced which retain activator activity. These fragments include truncated sequences, as well as N-terminal, C-terminal, internal and internally deleted amino acid sequences of the proteins.
  • the deletions, insertions, and substitutions of the protein sequence encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by the induction of the plant defense system. See, for example U.S. Patent No. 5,614,395, herein incorporated by reference.
  • nucleotide sequences of the invention can be used to isolate other homologous sequences in other plant species. Methods are readily available in the art for the hybridization of nucleic acid sequences. Coding sequences from other plants may be isolated according to well known techniques based on their sequence homology to the coding sequences set forth herein. In these techniques all or part of the known coding sequence is used as a probe which selectively hybridizes to other activator coding sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e. genomic or cDNA libraries) from a chosen organism.
  • the entire defense activator sequence or portions thereof may be used as probes capable of specifically hybridizing to corresponding coding sequences and messenger RNAs.
  • probes include sequences that are unique and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length.
  • Such probes may be used to amplify the activator coding sequences of interest from a chosen organism by the well-known process of polymerase chain reaction (PCR). This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism.
  • PCR polymerase chain reaction
  • Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New
  • hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 37°C; conditions represented by a wash stringency of 40-45% Formamide with 5x Denhardt's solution, 0.5% SDS, and lx SSPE at 42°C; and conditions represented by a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42°C, respectively), to DNA encoding the activator genes disclosed herein in a standard hybridization assay. See Sambrook et al.
  • sequences which code for the defense activators and other activator proteins of the invention and hybridize to the sequences disclosed herein will be at least 40% to 50% homologous, 60% to 70% homologous, and even 85%, 90% to 98% homologous or more with the disclosed sequence. That is, the sequence similarity of sequences may range, sharing at least about 40% to 50%, about 60% to 70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • additions or deletions i.e., gaps
  • reference sequence which does not comprise additions or deletions
  • comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; by the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 55:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View. California; GAP, BESTFIT, BLAST, FASTA. and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Drive, Madison. Wisconsin, USA; the CLUSTAL program is well described by Higgins et al. (1988) Gene
  • sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences, which differ by such conservative substitutions, are said to have “sequence similarity” or “similarity”. Means for making this adjustment are
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i. e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity preferably at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%.
  • Polypeptides which are "substantially similar” share sequences as, noted above except that residue positions, which are not identical, may differ by conservative amino acid changes. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5°C to about 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. 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.
  • stringent wash conditions are those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 50, 55, or 60°C.
  • nucleic acids, which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is that the polypeptide, which the first nucleic acid encodes, is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443.
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • the activator molecules described herein may be used alone or in combination with other proteins or agents to protect against plant diseases and pathogens.
  • Other plant defense proteins include those described in the copending applications entitled “Methods for Enhancing Disease Resistance in Plants", U.S. Application Serial No. 60/076,151, filed February 26, 1998, and U.S. Application Serial No. 60/092,464, filed July 11, 1998, both of which are herein incorporated by reference.
  • the expression of the activator molecules in the plant cell induces the disease resistance pathway or induces immunity, i.e., disease resistance, in the plant. That is, the expression of the genes can induce a defense response in the cell or can turn on the disease resistance pathway to obtain cell death.
  • the end result can be controlled by the level of expression of the activator sequences in the plant. Where the expression is sufficient to cause cell death, such response is a receptor-
  • a number of promoters can be used in the practice of the invention.
  • the promoters can be selected based on the desired outcome.
  • an inducible promoter can be used to drive the expression of the genes of the invention.
  • the inducible promoter must be tightly regulated to prevent unnecessary cell death yet be expressed in the presence of a pathogen to prevent infection and disease symptoms.
  • Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-lJ-glucanase, chitinase, etc.
  • PR proteins pathogenesis-related proteins
  • promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335- 342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331 ; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 55:2427-2430; Somsisch et al. (1988) Molecular and General Genetics 2:93-98; and Yang, Y (1996) Proc. Natl. Acad. Sci. USA 95:14972-14977. See also, Chen et al. (1996) Plant J. 70:955-966; Zhang et al.
  • Such wound inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan, C, Ann. Rev. Phytopath. 25:425-449; Duan et al. Nature Biotechnology 14:494-498); wunl and wun2, US Patent No. 5,428,148; winl and
  • Such weak promoters cause activation of the plant defense system short of hypersensitive cell death.
  • there is at least a partial activation of the plant defense system wherein the plant produces increased levels of antipathogenic factors such as PR proteins, i.e., PR1, chitinases, ⁇ -glucanases, etc.; secondary metabolites; phytoalexins; reactive oxygen species; and the like.
  • weak promoter a promoter that drives expression of a coding sequence at a low level.
  • low level is intended at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts.
  • weak promoters also encompasses promoters that are expressed in only a few cells and not in others to give a total low level of expression. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.
  • Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 (copending application serial number 08/661,601), the core 35S CaMV promoter, and the like.
  • Other constitutive promoters include, for example, U.S. Patent Nos. 5,608.149; 5,608,144; 5,604.121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142. See also, copending application entitled “Constitutive Maize Promoters", U.S.. Application Serial No. 60/076,075, filed February 26, 1998, and herein incorporated by reference.
  • Tissue-specific promoters include Yamamoto et al. (1997) Plant J. 12(2)255- 265; Kawamata et al. (1997) Plant Cell Physiol. 38 (7) :792-803; Hansen et al. (1997) Mol Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 60:157-168; Rinehart et al. (1996) Plant Physiol. 7720:1331-1341; Van Camp et al. (1996) Plant Physiol. 7720:525-535; Canevascini et al. (1996) Plant Physiol. 7720:513-524; Yamamoto et al. (1994) Plant Cell Physiol. 550:773-778; Lam et al. (1994) Results
  • the activator genes of the invention can be introduced into any plant.
  • the genes to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest.
  • Such expression cassettes will comprise a transcriptional initiation region linked to the activator sequence of interest.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the transcriptional initiation region may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • the transcriptional cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671- 674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 97:151-158; Ballas et al. (1989) Nuc. Acids Res. 77:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 75:9627-9639.
  • the genes of the invention are provided in expression cassettes for expression in the plant of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest.
  • the cassette may additionally contain at least
  • the additional gene(s) can be provided on another expression cassette.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant preferred codons for improved expression. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nuc. Acids Res. 17:477-498, herein incorporated by reference.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences, which may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the expression cassettes may additionally contain 5' leader sequences in the expression cassette construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) RNJS USA 5(5:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al.
  • EMCV leader Engelphalomyocarditis 5' noncoding region
  • potyvirus leaders for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20
  • the various D ⁇ A fragments may be manipulated, so as to provide for the D ⁇ A sequences in the proper orientation and, as appropriate, in the proper reading frame. Towards this end, adapters or linkers may be employed to join the D ⁇ A fragments or other manipulations may be involved to
  • the genes of the present invention can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols may vary depending on the type of plant or plant cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl.
  • the cells which have been transformed, may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
  • the methods of the invention can be used with other methods available in the art for enhancing disease resistance in plants.
  • the present invention also provides isolated nucleic acids comprising polynucleotides of sufficient length and complementarity to a gene of the invention to use as probes or amplification primers in the detection, quantitation, or isolation of gene transcripts.
  • isolated nucleic acids of the present invention can be used as probes in detecting deficiencies in the level of mRNA in screenings for desired transgenic plants, for detecting mutations in the gene (e.g., substitutions, deletions, or additions), for monitoring upregulation of expression or changes in enzyme activity in screening assays of compounds, for detection of any number of allelic variants (polymorphisms) of the gene, or for use as molecular markers in plant breeding programs.
  • the isolated nucleic acids of the present invention can also be used for recombinant expression of polypeptides, or for use as immunogens in the preparation and/or screening of antibodies.
  • the isolated nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more genes of the invention in a host cell, tissue, or plant. Attachment of chemical agents, which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation.
  • a primer specific to an insertion sequence e.g. , transposon
  • a primer which specifically hybridizes to an isolated nucleic acid of the present invention one can use nucleic acid amplification to identify insertion sequence inactivated genes of the invention from a cDNA library prepared from insertion
  • Progeny seed from the plants comprising the desired inactivated gene can be grown to a plant to study the phenotypic changes characteristic of that inactivation. See, Tools to Determine the Function of Genes, 1995 Proceedings of the Fiftieth Annual Corn and Sorghum Industry Research Conference, American Seed Trade Association, Washington, D.C., 1995.
  • nontranslated 5' or 3' regions of the polynucleotides of the present invention can be used to modulate turnover of heterologous mRNAs and/or protein synthesis.
  • the codon preference characteristic of the polynucleotides of the present invention can be employed in heterologous sequences, or altered in homologous or heterologous sequences, to modulate translational level and/or rates.
  • the present invention provides a method of genotyping a plant comprising a polynucleotide of the present invention.
  • the plant is a monocot, such as maize or sorghum.
  • Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
  • the particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques such as, but not limited to, restriction fragment length polymorphisms (RFLPs).
  • RFLPs restriction fragment length polymorphisms
  • the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis.
  • Linked chromosomal sequences are within 50 centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of a gene of the invention.
  • nucleic acid probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective
  • the probes are selected from polynucleotides of the present invention.
  • these probes are cDNA probes or Pst I genomic clones.
  • the length of the probes is discussed in greater detail, supra, but are typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length.
  • the probes are single copy probes that hybridize to a unique locus in a haploid chromosome complement.
  • the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe.
  • the sample is a plant sample; preferably, a sample suspected of comprising a maize polynucleotide of the present invention (e.g., gene, mRNA).
  • the nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymo ⁇ hic marker. Selective hybridization of the nucleic acid probe to the polymo ⁇ hic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymo ⁇ hic marker in the sample.
  • the nucleic acid probe comprises a polynucleotide of the present invention.
  • EXPERIMENTAL Example 1 Disease Resistant Transient Gene Expression Assay Using Biolistics Particle Bombardment.
  • a transient gene expression assay as modified from Nelson et al. (1997) Transgenic Res. 6:233-244, is used to evaluate the ability of an introduced activator gene, whose expression product would induce the pathogen defense system in a host plant cell, to confer a hypersensitive response within the host cell.
  • a particle bombardment system is used to simultaneously introduce a construct comprising a reporter gene driven by a constitutive promoter and a construct
  • the first construct comprises a ubiquitin promoter driving the expression of the reporter CRC fusion protein gene, which when expressed causes cells to turn red due to anthocyanin production.
  • Other reporter genes such as GUS, luciferase, or green fluorescent protein, can be used in this assay.
  • the second construct comprises one of the activator genes of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, driven by the constitutive ubiquitin promoter.
  • Immature maize embryos are isolated from ears 9-11 days after pollination using a scalpel. Prior to embryo isolation, pollinated ears are surface-sterilized with a microdetergent and 25% commercial bleach mixture, then washed with 3 exchanges of sterile H 2 O. Isolated immature embryos, approximately 1.5-1.8 mm long, are placed on a high sucrose culture medium and aligned in a target grid about 1.4 cm wide in preparation for bombardment.
  • Plasmid pi 1416 ( Figure 1), comprising the ubiquitin promoter (ubi) and the CRC fusion protein gene (ubi::CRC fusion), the expression of which yields the anthocyanin- producing, or red cell, phenotype
  • plasmid pi 0249 Figure 2, comprising the ubiquitin promoter and an activator sequence of the invention (ubi::activator), the expression of which yields the activator product.
  • Plasmid p7770 (not shown), comprising an empty ubiquitin promoter construct (ubi::pinll terminator), is used as a control to balance promoter site molarity; and plasmid p7731 (not shown), an inert
  • 25 DNA filler is used to balance the amount of DNA shot with each bombardment episode.
  • Embryos are transformed by the tungsten particle biolistic method (Tomes et al. (1995) supra; Koziel et al. (1993) Bio/Technology 77:194-200) using a high pressure particle delivery system (Biolistic Particle Delivery System Model PDS-100 by DuPont). Forty-five embryos, arranged in 5 plates, each with 9 embryos, are subjected to bombardment with the ubi::CRC fusion construct alone (Treatment A) or to cobombardment with the ubi::CRC fusion construct and the ubi:: activator construct (Treatment B). Following bombardment, embryos are stored in the dark for 36 hours at 23°C.
  • CRC fusion gene Expression of the CRC fusion gene is quantified by visual means 16 to 48 hours, more usually 36 hours, following bombardment. Cells expressing the CRC fusion protein gene are red in color.
  • Activation of the defense system using the maize PRl protein as a marker, is verified with an antibody Western blot for the PRl class of pathogenesis-related proteins. Forty-eight hours after bombardment, 18 embryos for each treatment are pooled and their protein extracted and run on SDS-PAGE, electroblotted onto 0J micron PVDV membrane, and probed with antibodies raised against tobacco PRl protein.
  • An activator sequence is cloned into a plant expression vector as shown in Figure 2.
  • the nucleotide sequence is under transcriptional control of the maize ubiquitin promoter.
  • the selectable marker gene PAT is used.
  • Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing the activator sequence operably linked to the ubiquitin promoter plus a plasmid containing the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to the herbicide Bialaphos. Transformation is performed as follows. All media recipes are in the Appendix.
  • the ears are surface sterilized in 30% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water.
  • the immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 -cm target zone in preparation for bombardment.
  • a plasmid vector comprising the activator sequence operably linked to the ubiquitin promoter is made.
  • This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 ⁇ m (average diameter) tungsten pellets using a CaCl 2 precipitation procedure as follows:
  • Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer.
  • the final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes.
  • the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 ⁇ l 100% ethanol is added to the final tungsten particle pellet.
  • the tungsten DNA particles are briefly sonicated and 10 ⁇ l spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
  • sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.
  • the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection- resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone- free medium in tubes for 7-10 days until plantlets are well established.
  • Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1 -2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for enhanced disease resistance.
  • Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for one month, unless contamination or precipitation occurs, then make fresh stock.
  • Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for one month, unless contamination or precipitation occurs, then make fresh stock.

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Abstract

L'invention porte sur des compositions et des procédés renforçant la résistance des plantes aux maladies. Lesdits procédés consistent à transformer la plante à l'aide d'une séquence d'activateur propre à l'invention. Ladite séquence induit dans les cellules de la plante des défenses. On utilise à cet effet un promoteur inducteur d'éléments pathogènes ou bien un promoteur constitutif de préférence faible pour réguler le niveau désiré de résistance aux maladies de la plante. L'invention porte également sur des plantes transformées, des cellules de plantes, des tissus et des semences présentant une résistance renforcée aux maladies.
PCT/US1999/003337 1998-02-26 1999-02-17 Genes activant le systeme de defense de plantes contre les elements pathogenes WO1999043821A1 (fr)

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Cited By (2)

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WO2003000863A2 (fr) 2001-06-22 2003-01-03 Pioneer Hi-Bred International, Inc. Polynucleotides de defensine et methodes d'utilisation
WO2012030759A1 (fr) 2010-09-01 2012-03-08 Pioneer Hi-Bred International, Inc. Peptides de ciblage de vacuole et leurs procédés d'utilisation

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000863A2 (fr) 2001-06-22 2003-01-03 Pioneer Hi-Bred International, Inc. Polynucleotides de defensine et methodes d'utilisation
EP2270186A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270185A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270184A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270165A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270187A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270188A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2333070A2 (fr) 2001-06-22 2011-06-15 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
WO2012030759A1 (fr) 2010-09-01 2012-03-08 Pioneer Hi-Bred International, Inc. Peptides de ciblage de vacuole et leurs procédés d'utilisation

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