EP0944728A1 - PROCEDE D'UTILISATION DU GENE $i(NIM1) POUR CONFERER A DES PLANTES UNE RESISTANCE AUX MALADIES - Google Patents

PROCEDE D'UTILISATION DU GENE $i(NIM1) POUR CONFERER A DES PLANTES UNE RESISTANCE AUX MALADIES

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Publication number
EP0944728A1
EP0944728A1 EP97952940A EP97952940A EP0944728A1 EP 0944728 A1 EP0944728 A1 EP 0944728A1 EP 97952940 A EP97952940 A EP 97952940A EP 97952940 A EP97952940 A EP 97952940A EP 0944728 A1 EP0944728 A1 EP 0944728A1
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EP
European Patent Office
Prior art keywords
nim1
plant
plants
gene
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97952940A
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German (de)
English (en)
Inventor
John Andrew Ryals
Kay Ann Lawton
Scott Joseph Uknes
Henry-York Steiner
Michelle Denise Hunt
Leslie Bethards Friedrich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngenta Participations AG
Original Assignee
Novartis AG
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Publication date
Priority claimed from US08/880,179 external-priority patent/US6091004A/en
Application filed by Novartis AG filed Critical Novartis AG
Publication of EP0944728A1 publication Critical patent/EP0944728A1/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/38—Solanaceae [Potato family], e.g. nightshade, tomato, tobacco or chilli pepper
    • 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
    • C12N15/8281—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
    • 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 generally relates to broad-spectrum disease resistance in plants, including the phenomenon of systemic acquired resistance (SAR). More particularly, the present invention relates to the recombinant expression of wild-type and altered forms of the NIM1 gene, which is involved in the signal transduction cascade leading to SAR to create transgenic plants having broad-spectrum disease resistance. The present invention relates further to high-level expression of the cloned NIM1 gene in transgenic plants that have broad-spectrum disease resistance.
  • 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 et al., 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 et al., Mol. Plant-Microbe Interact.
  • SA 2,6-dichloroisonicotinic acid
  • BTH benzo(1 ,2,3)thiadiazole-7-carbothioic acid S-methyl ester
  • cprl constitutive expresser of P_R genes
  • cprl might be a type of Isd mutant (Bowling et al., Plant Cell 6, 1845-1857 (1994), incorporated by reference herein in its entirety).
  • ndrl non-race- specific disease resistance
  • ndrl non-race- specific disease resistance
  • nprl nonexpresser of PR genes
  • INA treatment Cao et al., Plant Cell 6, 1583-1592 (1994), incorporated by reference herein in its entirety
  • 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., Genetics 143, 973-982 (1996), incorporated by reference herein in its entirety; Parker et al., Plant Cell 8, 2033- 2046 (1996), incorporated by reference herein in its entirety).
  • nim1 noninducible immunity
  • P. parasitica i.e., causal agent of downy mildew disease
  • INA treatment i.e., INA treatment
  • 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., Plant Cell 9, 425-439 (1997), incorporated by reference herein in its entirety; Cao et al., Cell 88, 57-63 (1997), incorporated by reference herein in its entirety).
  • 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).
  • NF- ⁇ B/l ⁇ B signaling pathways have been implicated in disease resistance responses in a range of organisms from Drosophila to mammals.
  • NF- ⁇ B/l ⁇ B signal transduction can be induced by a number of different stimuli including exposure of cells to lipopolysaccharide, tumor necrosis factor, interleukin 1 (IL-1), or virus infection (Baeuerle and Baltimore, Ce//87, 13-20 (1996); Baldwin, Annu. Rev. Immunol. 14, 649-681 (1996)).
  • the activated pathway leads to the synthesis of a number of factors involved in inflammation and immune responses, such as IL-2, IL-6, IL-8 and granulocyte/macrophage- colony stimulating factor (deMartin et al., Gene 152, 253-255 (1995)).
  • IL-2 IL-2
  • IL-6 IL-6
  • IL-8 granulocyte/macrophage- colony stimulating factor
  • granulocyte/macrophage- colony stimulating factor granulocyte/macrophage- colony stimulating factor
  • SAR is functionally analogous to inflammation in that normal resistance processes are potentiated following SAR activation leading to enhanced disease resistance (Bi et al., 1995; Cao et al., 1994; Delaney et al., 1995; Delaney et al., 1994; Gaffney et al., 1993; Mauch-Mani and Slusarenko 1996; Delaney, 1997).
  • inactivation of the pathway leads to enhanced susceptibility to bacterial, viral and fungal pathogens.
  • SA has been reported to block NF- ⁇ B activation in mammalian cells (Kopp and Ghosh, Science 265, 956-959 (1994)), while SA activates signal transduction in
  • Bacterial infection of Drosophila activates a signal transduction cascade leading to the synthesis of a number of antifungal proteins such as cercropin B, defensin, diptericin and drosomycin (Ip et al., Ce//75, 753-763 (1993); Lemaitre et al., Ce//86, 973- 983 (1996)).
  • This induction is dependent on the gene product of dorsal and dif, two NF- ⁇ B homologs, and is repressed by cactus, an l ⁇ B homolog, in the fly. Mutants that have decreased synthesis of the antifungal and antibacterial proteins have dramatically lowered resistance to infection.
  • Plant cell the structural and physiological unit of plants, consisting of a protoplast and the cell wall.
  • the term "plant cell” refers to any cell which is either part of or derived from a plant.
  • Some examples of cells include differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; the cells of undifferentiated tissue such as callus or tumors; differentiated cells of seeds, embryos, propagules and pollen.
  • 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.
  • Protoplast a plant cell without a cell wall.
  • Descendant plant a sexually or asexually derived future generation plant which includes, but is not limited to, progeny plants.
  • Transgenic plant a plant having stably incorporated recombinant DNA in its genome.
  • Recombinant DNA Any DNA molecule formed by joining DNA segments from different sources and produced using recombinant DNA technology.
  • Recombinant DNA technology Technology which produces recombinant DNA in vitro and transfers the recombinant DNA into cells where it can be expressed or propagated (See, Concise Dictionary of Biomedicine and Molecular Biology, Ed. Juo, CRC Press, Boca Raton (1996)), for example, transfer of DNA into a protoplast(s) or cell(s) in various forms, including, for example, (1) naked DNA in circular, linear or supercoiled forms, (2) DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, (3) DNA complexed or associated with other molecules, (4) DNA enclosed in liposomes, spheroplasts, cells or protoplasts or (5) DNA transferred from organisms other than the host organism (ex.
  • Agrobacterium tumefiaciens Agrobacterium tumefiaciens. These and other various methods of introducing the recombinant DNA into cells are known in the art and can be used to produce the transgenic cells or transgenic plants of the present invention.
  • Recombinant DNA technology also includes the homologous recombination methods described in Treco et al., WO 94/12650 and Treco et al., WO 95/31560 which can be applied to increasing peroxidase activity in a monocot.
  • regulatory regions can be introduced into the plant genome to increase the expression of the endogenous peroxidase.
  • Chimeric gene A DNA molecule containing at least two heterologous parts, e.g., parts derived from pre-existing DNA sequences which are not associated in their pre-existing states, these sequences having been preferably generated using recombinant DNA technology.
  • Expression cassette a DNA molecule comprising a promoter and a terminator between which a coding sequence can be inserted.
  • Coding seguence a DNA molecule which, when transcribed and translated, results in the formation of a polypeptide or protein.
  • Gene a discrete chromosomal region comprising a regulatory DNA sequence responsible for the control of expression, i.e. transcription and translation, and of a coding sequence which is transcribed and translated to give a distinct polypeptide or protein.
  • the present invention describes the identification, isolation, and characterization of the NIM1 gene, which encodes a protein involved in the signal transduction cascade responsive to biological and chemical inducers that leads to systemic acquired resistance in plants.
  • the present invention discloses an isolated DNA molecule (NIM1 gene) that encodes a NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants.
  • a DNA molecule that encodes the NIM1 protein hybridizing under the following conditions to clone BAC-04, ATCC Deposit No. 97543: hybridization in 1%BSA; 520mM NaPO 4 , pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • the NIM1 gene is comprised within clone BAC-04, ATCC Deposit No. 97543.
  • DNA molecule that encodes the NIM1 protein hybridizes under the following conditions to cosmid D7, ATCC Deposit No. 97736: hybridization in 1%BSA; 520mM NaPO 4 , pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • the NIM1 gene is comprised within cosmid D7, ATCC Deposit No. 97736.
  • the NIM1 gene described herein may be isolated from a dicotyledonous plant such as Arabidopsis, tobacco, cucumber, or tomato. Alternately, the NIM1 gene may be isolated from a monocotyledonous plant such as maize, wheat, or barley.
  • NIM1 protein comprising the amino acid sequence set forth in SEQ ID NO:3. Further described is the NIM1 gene coding sequence hybridizing under the following conditions to the coding sequence set forth in SEQ ID NO:2: hybridization in 1%BSA; 520mM NaPO 4 , pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • the NIM1 gene coding sequence comprises the coding sequence set forth in SEQ ID NO:2.
  • the present invention also describes a chimeric gene comprising a promoter active in plants operatively linked to a NIM1 gene coding sequence, 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, 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 crops rice, wheat, barley, rye, 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,
  • the NIM1 protein is expressed in a transformed plant at higher levels than in a wild type plant.
  • the present invention is also directed to a method of conferring a CIM phenotype to a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to a NIM1 gene coding sequence, wherein the encoded NIM1 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 activating systemic acquired resistance in a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to a NIM1 gene coding sequence, wherein the encoded NIM1 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 conferring broad spectrum disease resistance to a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to a NIM1 gene coding sequence, wherein the encoded NIM1 protein is expressed in the transformed plant at higher levels than in a wild type plant.
  • Another aspect of the present invention exploits both the recognition that the SAR pathway in plants shows functional parallels to the NF- ⁇ B/l ⁇ B regulation scheme in mammals and flies, as well as the discovery that the NIM1 gene product is a structural homologue of the mammalian signal transduction factor l ⁇ B subclass ⁇ . Mutations of l ⁇ B have been described that act as super-repressors or dominant-negatives of the NF- ⁇ B/l ⁇ B regulation scheme.
  • the present invention encompasses altered forms of wild-type NIM1 gene (SEQ NO: 2) that act as dominant-negative regulators of the SAR signal transduction pathway. These altered forms of NIM1 confer the opposite phenotype in plants transformed therewith as the nim1 mutant; plants i.e., plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and a CIM phenotype.
  • DNA molecules that hybridize to a DNA molecule according to the invention as defined hereinbefore, but preferably to an oligonucleotide probe obtainable from said DNA molecule comprising a contiguous portion of the coding sequence for the said altered forms of NIM1 at least 10 nucleotides in length, under moderately stringent conditions.
  • T m melting temperature
  • the preferred hybridization temperature is in the range of about 25°C below the calculated melting temperature T m and preferably in the range of about 12-15°C below the calculated melting temperature T m and in the case of oligonucleotides in the range of about 5-10°C below the melting temperature T m .
  • the NIM1 gene is altered so that the encoded product has alanines instead of serines in the amino acid positions corresponding to positions 55 and 59 of the wild-type Arabidopsis NIM1 amino acid sequence (SEQ ID NO:3).
  • SEQ ID NO:3 An example of a preferred embodiment of this altered form of the NIM1 gene, which results in changes of these serine residues to alanine residues, is presented in SEQ ID NO:22.
  • An exemplary dominant-negative form of the NIM1 protein with alanines instead of serines at amino acid positions 55 and 59 is shown in SEQ ID NO:23.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderate stringent conditions to the coding sequence set forth in SEQ ID NO:22, especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:22 under the above conditions are altered so that the encoded product has alanines instead of serines in the amino acid positions that correspond to positions 55 and 59 of SEQ ID NO:22.
  • the NIM1 gene is altered so that the encoded product has an N-terminal truncation, which removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type protein.
  • An example of a preferred embodiment of this altered form of the NIM1 gene, which encodes a gene product having an N-terminal deletion, is presented in SEQ ID NO:24.
  • An exemplary dominant-negative form of the NIM1 protein with an N-terminal deletion is shown in SEQ ID NO:25.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderate stringent conditions to the coding sequence set forth in SEQ ID NO:24; especially preferred are the following conditions: hybridization in 1 %BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:24 under the above conditions are altered so that the encoded product has an N- terminal deletion that removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product.
  • the NIM1 gene is altered so that the encoded product has a C-terminal truncation, which is believed to result in enhanced intrinsic stability by blocking the constitutive phosporylation of serine and threonine residues in the C-terminus of the wild-type gene product.
  • An example of a preferred embodiment of this altered form of the NIM1 gene, which encodes a gene product having a C-terminal deletion, is presented in SEQ ID NO:26.
  • An exemplary dominant-negative form of the NIM1 protein with a C-terminal deletion is shown in SEQ ID NO:27.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderate stringent conditions to the coding sequence set forth in SEQ ID NO:26; especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1 ) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:26 under the above conditions are altered so that the encoded product has a C-terminal deletion that removes serine and threonine residues.
  • the NIM1 gene is altered so that the encoded product has both an N-terminal deletion and a C-terminal truncation, which provides the benefits of both the above-described embodiments of the invention.
  • a preferrred embodiment of the invention is an altered form of the NIM1 protein that has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1- 125 of SEQ ID NO:2 and a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO:3.
  • NIM1 gene which encodes a gene product having both an N-terminal and a C-terminal deletion
  • SEQ ID N0:28 An example of a preferred embodiment of this altered form of the NIM1 gene, which encodes a gene product having both an N-terminal and a C-terminal deletion, is presented in SEQ ID N0:28.
  • An exemplary dominant-negative form of the NIM1 protein with a C- terminal deletion is shown in SEQ ID NO:29.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the moderate stringent conditions to the coding sequence set forth in SEQ ID NO:28; especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:28 under the above conditions are altered so that the encoded product has both an N-terminal deletion, which removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product, as well as a C-terminal deletion, which removes serine and threonine residues.
  • the NIM1 gene is altered so that the encoded product consists essentially of only the ankyrin domains of the wild-type gene product.
  • An example of a preferred embodiment of this altered form of the NIM1 gene, which encodes the ankyrin domains, is presented in SEQ ID NO:30.
  • An exemplary dominant-negative form of the NIM1 protein consists essentially of only the ankyrin domains is shown in SEQ ID NO:31.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the moderate stringent conditions to the coding sequence set forth in SEQ ID NO:30; especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaP04, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:30 under the above conditions are altered so that the encoded product consists essentially of the ankyrin domains of the wild- type gene product.
  • the present invention concerns DNA molecules encoding altered forms of the NIM1 gene, such as those described above and all DNA molecules hybridizing therewith using moderate stringent conditions.
  • the present invention also encompasses a chimeric gene comprising a promoter active in plants operatively linked to one of the above-described altered forms of the NIM1 gene, a recombinant vector comprising such a chimeric gene, wherein the vector is capable of being stably transformed into a host cell, as well as a host cell stably transformed with such a vector.
  • the host cell is a plant, plant cells and the descendants thereof from, for example, one of the following agronomically important crops: rice, wheat, barley, rye, 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 crops rice, wheat, barley, rye, 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,
  • the present invention is also directed to a method of conferring a CIM phenotype to a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to one of the above-described altered forms of the NIM1 gene, wherein the encoded dominant-negative form of the NIM1 protein is expressed in the transformed plant and confers a CIM phenotype to the plant.
  • the present invention is directed to a method of activating systemic acquired resistance in a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to one of the above-described altered forms of the NIM1 gene, wherein the encoded dominant- negative form of the NIM1 protein is expressed in the transformed plant and activates systemic acquired resistance in the plant.
  • the present invention is directed to a method of conferring broad spectrum disease resistance to a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to one of the above-described altered forms of the NIM1 gene, wherein the encoded dominant-negative form of the NIM1 protein is expressed in the transformed plant and confers broad spectrum disease resistance to the plant.
  • the present invention is directed to a method of screening for a NIM1 gene involved in the signal transduction cascade leading to systemic acquired resistance in a plant, comprising probing a genomic or cDNA library from said plant with a NIM1 coding sequence that hybridizes under the following set of conditions to the coding sequence set forth in SEQ ID N0:2: hybridization in 1 %BSA; 520mM NaPO 4 , pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1 ) at 55°C.
  • An isolated DNA molecule according to the invention wherein said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID NO:3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:30: hybridization in 1%BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • An altered form of a NIM1 gene according to the invention which has been constructed by mutagenization.
  • a method of producing an altered form of a NIM1 gene is provided.
  • a method of producing transgenic descendants of a transgenic parent plant comprising an isolated DNA molecule encoding an altered form of a NIM1 protein according to the invention comprising transforming said parent plant with a recombinant vector molecule according to the invention and transferring the trait to the descendants of said transgenic parent plant involving known plant breeding techniques.
  • a method of producing a DNA molecule comprising a DNA portion containing a DNA portion encoding an altered form of a NIM1 protein
  • nucleotide probe capable of specifically hybridizing to an altered form of a NIM1 gene or mRNA, wherein said probe comprises a contiguous portion of the coding sequence for an altered form of a NIM1 of at least 10 nucleotides length;
  • step (b) probing for other altered forms of a NIM1 coding sequence in populations of cloned genomic DNA fragments or cDNA fragments from a chosen organism using the nucleotide probe prepared according to step (a); and (c) isolating and multiplying a DNA molecule comprising a DNA portion containing a
  • DNA portion encoding an altered form of a NIM1 protein isolated by A method of isolating a DNA molecule comprising a DNA portion containing an altered form of a NIM1 sequence comprising
  • nucleotide probe capable of specifically hybridizing to an altered form of a NIM1 gene or mRNA, wherein said probe comprises a contiguous portion of the coding sequence for an altered form of a NIM1 protein from a plant of at least 10 nucleotides length;
  • step (b) probing for other altered forms of NIM1 sequences in populations of cloned genomic DNA fragments or cDNA fragments from a chosen organism using the nucleotide probe prepared according to step (a);
  • the present invention relates to mutant plants, as well as genes isolated therefrom, which are defective in their normal response to pathogen infection in that they do not express genes associated with SAR.
  • These mutants are referred to as nim mutants (for non-inducible immunity) and are "universal disease susceptible” (UDS) by virtue of their being susceptible to many strains and pathotypes of pathogens of the host plant and also to pathogens that do not normally infect the host plant, but that normally infect other hosts.
  • UDS universalal disease susceptible
  • Such mutants can be selected by treating seeds or other biological material with mutagenic agents and then selecting descendant plants for the UDS phenotype by treating descendant plants with known chemical inducers (e.g. INA) of SAR and then infecting the plants with a known pathogen.
  • known chemical inducers e.g. INA
  • Non-inducible mutants develop severe disease symptoms under these circumstances, whereas wild type plants are induced by the chemical compound to systemic acquired resistance, nim mutants can be equally selected from mutant populations generated by chemical and irradiation mutagenesis, as well as from populations generated by T-DNA insertion and transposon-induced mutagenesis. Techniques of generating mutant plant lines are well known in the art. nim mutants provide useful indicators of the evaluation of disease pressure in field pathogenesis tests where the natural resistance phenotype of so-called wild type (i.e. non- mutant) plants may vary and therefore not provide a reliable standard of susceptibility. Furthermore, nim plants have additional utility for the testing of candidate disease resistance transgenes.
  • nim stock line as a recipient for transgenes, the contribution of the transgene to disease resistance is directly assessable over a base level of susceptibility. Furthermore, the nim plants are useful as a tool in the understanding of plant-pathogen interactions, nim host plants do not mount a systemic response to pathogen attack, and the unabated development of the pathogen is an ideal system in which to study its biological interaction with the host.
  • nim host plants may also be susceptible to pathogens outside of the host-range they normally fall, these plants also have significant utility in the molecular, genetic, and biological study of host-pathogen interactions.
  • the UDS phenotype of nim plants also renders them of utility for fungicide screening, nim mutants selected in a particular host have considerable utility for the screening of fungicides using that host and pathogens of the host.
  • the advantage lies in the UDS phenotype of the mutant, which circumvents the problems encountered by hosts being differentially susceptible to different pathogens and pathotypes, or even resistant to some pathogens or pathotypes.
  • nim mutants have further utility for the screening of fungicides against a range of pathogens and pathotypes using a heterologous host, i.e. a host that may not normally be within the host species range of a particular pathogen.
  • a heterologous host i.e. a host that may not normally be within the host species range of a particular pathogen.
  • the susceptibility of nim mutants of Arabidopsis to pathogens of other species facilitates efficacious fungicide screening procedures for compounds against important pathogens of crop plants.
  • nim1 noninducible immunity
  • P. parasitica i.e., causal agent of downy mildew disease
  • INA treatment i.e., INA treatment
  • nim1 can accumulate SA following pathogen infection, neither SAR gene expression nor disease resistance can be induced, 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).
  • nim1-1 This first Arabidopsis nim1 mutant (herein designated nim1-1) was isolated from 80,000 plants of a T-DNA tagged Arabidopsis ecotype Issilewskija (Ws-0) population by spraying two week old plants with 0.33 mM INA followed by inoculation with P. parasitica (Delaney et al., 1995). Plants that supported fungal growth after INA treatment were selected as putative mutants. Five additional mutants (herein designated nim1-2, nim1-3, nim1-4, nim1-5, and nim1-6) were isolated from 280,000 M 2 plants from an ethyl methanesulfonate (EMS)-mutagenized Ws-0 population.
  • EMS ethyl methanesulfonate
  • Ws-0 plants were used as pollen donors to cross to each of these mutants.
  • the F-i plants were then scored for their ability to support fungal growth following INA treatment.
  • Table 3 of the Examples all nim1-1, nim1-2, nim1-3, nim1-4, and nim1-6 F ⁇ plants were phenotypically wild type, indicating a recessive mutation in each line.
  • nim1-5 showed the nim phenotype in all 35 F-i plants, indicating that this particular mutant is dominant.
  • the reciprocal cross was carried out using nim1 -5 as the pollen donor to fertilize Ws-0 plants. In this case, all 18 F, plants were phenotypically nim, confirming the dominance of the nim1-5 mutation.
  • nim1-1 carried resistance to kanamycin
  • F-i descendants were identified by antibiotic resistance.
  • the kanamycin-resistant F ⁇ plants were nim, indicating they were all allelic to the nim1-1 mutant.
  • the nim1-5 mutant is dominant and apparently homozygous for the mutation, it was necessary to analyze nim1-1 complementation in the F 2 generation. If nim1-1 and nim1-5 were allelic, then the expectation would be that all F 2 plants have a nim phenotype.
  • nim 1 -5 carries a point mutation in the NIM1 gene (infra), it is considered to be a nim1 allele.
  • each mutant was analyzed for the growth of P. parasitica under normal growth conditions and following pretreatment with either SA, INA, or BTH.
  • nim1-1, nim1-2, nim1-3, nim1-4, and nim1-6 a ⁇ supported approximately the same rate of fungal growth, which was somewhat faster than the Ws-0 control.
  • the exception was the n/m7-5 plants, in which fungal growth was delayed by several days relative to both the other nim1 mutants and the Ws-0 control, but eventually all of the nim1-5 plants succumbed to the fungus.
  • nim1-4 and nim1-6 showed a relatively rapid fungal growth; nim1-1, nim1-2, nim1-3 p ⁇ an ⁇ s exhibited a somewhat slower rate of fungal growth; and fungal growth in /wn 7 -5 plants was even slower than in the untreated Ws-0 controls.
  • the mutants also seemed to fall into three classes where nim1-4 was the most severely compromised in its ability to restrict fungal growth following chemical treatment; nim1-1, nim1-2, nim1-3, and nim1-6were all moderately compromised; and nim1-5 was only slightly compromised.
  • Ws-0 did not support fungal growth following INA or BTH treatment.
  • the mutants fall into three classes with nim1-4 being the most severely compromised, nim1-1, nim1-2, nim1-3 and nim 1-6 showing an intermediate inhibition of fungus and nim 1-5 with only slightly impaired fungal resistance.
  • RNA gel blot in Figure 3 shows that PR-1 mRNA accumulated to high levels following treatment of wild-type plants with SA, INA, or BTH or infection by P. parasitica.
  • PR-1 mRNA accumulation was dramatically reduced relative to the wild type following chemical treatment.
  • PR-1 mRNA was also reduced following P. parasitica infection, but there was still some accumulation in these mutants.
  • PR-1 mRNA accumulation was more dramatically reduced than in the other alleles following chemical treatment (evident in longer exposures) and significantly less PR-1 mRNA accumulated following P. parasitica infection, supporting the idea that these could be particularly strong nim1 alleles.
  • nim 1-1 was found to lie about 8.2 centimorgans (cM) from nga128 and 8.2 cM from ngal 11 on the lower arm of chromosome 1.
  • nim1-1 was found to lie between ngal 1 1 and about 4 cM from the SSLP marker ATHGENEA.
  • nim plants from an F 2 population derived from a cross between nim1-1 and LerDP23 were identified based on both their inability to accumulate PR-1 mRNA and their ability to support fungal growth following INA treatment.
  • DNA was extracted from these plants and scored for zygosity at both ATHGENEA and ngal 11.
  • 93 recombinant chromosomes were identified between ATHGENEA and n/t ⁇ 77, giving a genetic distance of approximately 4.1 cM (93 of 2276), and 239 recombinant chromosomes were identified between nga111 and nim1, indicating a genetic distance of about 10.5 cM (239 of 2276).
  • AFLP amplified fragment length polymorphism
  • AFLPs were developed that were specific for BAC or P1 clones, and these were used to determine whether the NIM1 gene had been crossed. It was determined that NIM1 had been crossed when BAC and P1 clones were isolated that gave rise to both AFLP markers L84.6a and L84.8.
  • the AFLP marker L84.6a found on P1 clones P1-18, P1-17, and P1 -21 identified three recombinants and L84.8 found on P1 clones P1-20, P1- 22, P1-23, and P1-24 and BAC clones, BAC-04, BAC-05, and BAC-06 identified one recombinant.
  • nim1 was located between L84.Y1 and L84.8, representing a gap of about 0.09 cM.
  • a cosmid library was constructed in the Agrobacterium-compa ib ⁇ e T-DNA cosmid vector pCLD04541 using DNA from BAC-06, BAC-04, and P1-18.
  • a cosmid contig was developed using AFLP markers derived from these clones. Physical mapping showed that the physical distance between L84.Y1 and L84.8 was greater than 90 kb, giving a genetic to physical distance of roughly 1 megabase per cM. To facilitate the later identification of the NIM1 gene, the DNA sequence of BAC-04 was determined.
  • cosmids contained the NIM1 gene were transformed into nim1-1, and transformants were evaluated for their ability to complement the mutant phenotype.
  • Cosmids D5, E1 , and D7 were all found to complement nim1-1, as determined by the ability of the transformants to accumulate PR-1 mRNA following INA treatment.
  • the ends of these cosmids were sequenced and found to be located on the DNA sequence of BAC-04. There were 9,918 base pairs in the DNA region shared by D7 and D5 that contained the NIM1 gene. As shown in Figure 5D, four putative gene regions were identified in this 10-kb sequence.
  • Region 1 could potentially encode a protein of 19,105 D
  • region 3 could encode a protein of 44,554 D
  • region 4 could encode a protein of 52,797 D.
  • Region 2 had four open reading frames of various sizes located close together, suggesting a gene with three introns. Analysis using the NetPlantGene program (Hebsgaard et al., 1996) indicated a high probability that the open reading frames could be spliced together to form a large open reading frame encoding a protein of 66,039 D.
  • the DNA sequence from each of the four gene regions was determined for each of the nimi alleles and compared with the corresponding gene region from Ws-0. No mutations were detected between Ws-0 and the mutant alleles in either gene regions 3 or 4 and only a single change was found in gene region 1 in the nim 1-6 mutant. However, a single base pair mutation was found in each of the alleles relative to Ws-0 for region 2.
  • the DNA sequence of gene region 2 is shown in Figure 6. As shown in Table 5 and Figure 6, in nim 1 -1, a single adenosine is inserted at position 3579 that causes a frameshift resulting in a change in seven amino acids and a deletion of 349 amino acids.
  • nim1-2 there is a cytidine-to-thymidine transition at position 3763 that changes a histidine to a tyrosine.
  • nim1-3 a single adenosine is deleted at position 3301 causing a frameshift that altered 10 amino acids and deleted 412 from the predicted protein.
  • both nim1-4 and nim 1-5 have a guanosine-to-adenosine transition at position 4160 changing an arginine to a lysine
  • nim1-6 there is a cytosine-to-thymidine transition resulting in a stop codon causing the deletion of 255 amino acids from the predicted protein.
  • RT-PCR analysis indicates that this mutation does not lead to an alteration of mRNA splicing (data not shown).
  • the gene region 2 DNA sequence was used in a Blast search (Altschul et al., 1990) and identified an exact match with the Arabidopsis expressed sequence tag (EST) T22612 and significant matches to the rice ESTs S2556, S2861 , S3060 and S3481 (see Figure 8).
  • a DNA probe covering base pairs 2081 to 3266 was used to screen an Arabidopsis cDNA library, and 14 clones were isolated that correspond to gene region 2. From the cDNA sequence, we could confirm the placement of the exon/intron borders shown in Figure 6. Rapid amplification of cDNA ends by polymerase chain reaction (RACE) was carried out using RNA from INA-treated Ws-0 plants and the likely transcriptional start site was determined to be the A at position 2588 in Figure 6.
  • RACE polymerase chain reaction
  • homologs of Arabidopsis NIM1 can be identified and isolated through screening genomic or cDNA libraries from different plants such as, but not limited to following crop plants: rice, wheat, barley, rye, 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.
  • Standard techniques for accomplishing this include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g. Sambrook et al., Molecular Cloning , eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers (see, e.g. Innis et al., PCR Protocols, a Guide to Methods and Applications eds., Academic Press (1990)).
  • Homologues identified are genetically engineered into the expression vectors listed below and transformed into the above listed crops. Transformants are evaluated for enhanced disease resistance using relevant pathogens of the crop plant being tested.
  • NIM1 homologs in the genomes of cucumber, tomato, tobacco, maize, wheat and barley have been detected by DNA blot analysis.
  • Genomic DNA was isolated from cucumber, tomato, tobacco, maize, wheat and barley, restriction digested with the enzymes BamHI, Hindlll, Xbal, or Sail, electrophoretically separated on 0.8% agarose gels and transferred to nylon membrane by capillary blotting.
  • the membrane was hybridized under low stringency conditions [(1%BSA; 520mM NaP0 4 , pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 55°C for 18-24h] with 3 P-radiolabelled >4rab/dops s t/7a//a ⁇ a NIM1 cDNA.
  • the blots were washed under low stringency conditions [6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C; 1XSSC is 0.15M NaCI, 15mM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIML
  • expressed sequence tags identified with similarity to the NIM1 gene such as the rice EST's described above can also be used to isolate homologues.
  • the rice EST's may be especially useful for isolation of NIM1 homologues from other monocots.
  • Homologues may also be obtained by PCR. In this method, comparisons are made between known homologues (e.g., rice and Arabidopsis). Regions of high amino acid and DNA similarity or identity are then used to make PCR primers. Once a suitable region is identified, primers for that region are made with a diversity of substitutions in the 3 rd codon position.
  • the PCR reaction is performed from cDNA or genomic DNA under a variety of standard conditions. When a band is apparent, it is cloned and/or sequences to determine if it is a NIM1 homologue.
  • the present invention also concerns the production of transgenic plants that express higher-than-wild-type levels of the NIM1 gene, or functional variants and mutants thereof, and thereby have broad spectrum disease resistance.
  • the expression of the NIM1 gene is at a level which is at least two-fold above the expression level of the NIM1 gene in wild-type plants and is preferably tenfold above the wild-type expression level.
  • Overexpression of the NIM1 gene mimics the effects of inducer compounds in that it gives rise to plants with a constitutive immunity (CIM) phenotype.
  • CIM constitutive immunity
  • a first method is selecting transformed plants that have high-level expression of NIM1 and therefore a CIM phenotype due to insertion site effect.
  • Table 6 shows the results of testing of various transformants for resistance to fungal infection. As can be seen from this table, a number of transformants showed less than normal fungal growth and several showed no visible fungal growth at all.
  • RNA was prepared from collected samples and analyzed as described in Delaney et al, 1995. Blots were hybridized to the Arabidopsis gene probe PR-1 (Uknes et al, 1992). Three lines showed early induction of PR-1 gene expression in that PR-1 mRNA was evident by 24 or 48 hours following fungal treatment. These three lines also demonstrated resistance to fungal infection.
  • plant transformation vectors comprising a constitutive plant-active promoter, such as the CaMV 35S promoter, operatively linked to a coding region that encodes an active NIM1 protein.
  • a constitutive plant-active promoter such as the CaMV 35S promoter
  • High levels of the active NIM1 protein produce the same disease-resistance effect as chemical induction with inducing chemicals such as BTH, INA, and SA.
  • the NIM1 Gene Is A Homolog Of l ⁇ B ⁇
  • the NIM1 gene is a key component of the systemic acquired resistance (SAR) pathway in plants (Ryals et al., 1996).
  • SAR systemic acquired resistance
  • the NIM1 gene is associated with the activation of SAR by chemical and biological inducers and, in conjunction with such inducers, is required for SAR and SAR gene expression.
  • the location of the NIM1 gene was determined by molecular biological analysis of the genome of mutant plants known to carry the mutant nimi gene, which gives the host plants extreme sensitivity to a wide variety of pathogens and renders them unable to respond to pathogens and chemical inducers of SAR.
  • the wildtype NIM1 gene of Arapidopsis has been mapped and sequenced (SEQ ID NO:2).
  • the wild-type NIM1 gene product (SEQ ID NO:3) is involved in the signal transduction cascade leading to both SAR and gene-for-gene disease resistance in Arabidopsis (Ryals et al., 1997).
  • Recombinant overexpression of the wild-type form of NIM1 gives rise to plants with a constitutive immunity (CIM) phenotype and therefore confers disease resistance in transgenic plants.
  • CCM constitutive immunity
  • Increased levels of the active NIM1 protein produce the same disease- resistance effect as chemical induction with inducing chemicals such as BTH, INA, and SA.
  • the sequence of the NIM1 gene (SEQ ID NO:2) was used in BLAST searches, and matches were identified based on homology of one rather highly conserved domain in the NIM1 gene sequence to ankyrin domains found in a number of proteins such as spectrins, ankyrins, NF- ⁇ B and l ⁇ B (Michaely and Bennett, Trends Cell Biol. 2, 127-129 (1992)). Beyond the ankyrin motif, however, conventional computer analysis did not detect other strong homologies, including homology to IKBCC.
  • NIM1 protein SEQ ID NO:3
  • 70 known ankyrin-containing proteins were carried out, and striking similarities were found to members of the l ⁇ B ⁇ class of transcription regulators (Baeuerle and Baltimore 1996; Baldwin 1996).
  • the NIM1 protein shares significant homology with l ⁇ B ⁇ proteins from mouse, rat, and pig (SEQ ID NOs: 18, 19, and 20, respectively).
  • NIM1 contains several important structural domains of l ⁇ B ⁇ throughout the entire length of the protein, including ankyrin domains (indicated by the dashed underscoring in Figure 9), 2 amino-terminal serines (amino acids 55 and 59 of NIM1) , a pair of lysines (amino acids 99 and 100 in NIM1 ) and an acidic C-terminus.
  • NIM1 and l ⁇ B ⁇ share identity at 30% of the residues and conservative replacements at 50% of the residues.
  • l ⁇ B ⁇ protein functions in signal transduction is by binding to the cytosolic transcription factor NF- ⁇ B and preventing it from entering the nucleus and altering transcription of target genes (Baeuerle and Baltimore, 1996; Baldwin, 1996).
  • the target genes of NF- ⁇ B regulate (activate or inhibit) several cellular processes, including antiviral, antimicrobial and cell death responses (Baeuerle and Baltimore, 1996).
  • l ⁇ B ⁇ is phosphorylated at two serine residues (amino acids 32 and 36 of Mouse IKBOC). This programs ubiquitination at a double lysine (amino acids 21 and 22 of Mouse IKBCC).
  • the NF- ⁇ B/l ⁇ B complex is routed through the proteosome where l ⁇ B ⁇ is degraded and NF- ⁇ B is released to the nucleus.
  • the phosphorylated serine residues important in l ⁇ B ⁇ function are conserved in NIM1 within a large contiguous block of conserved sequence from amino acids 35 to 84 ( Figure 9).
  • a double lysine is located about 40 amino acids toward the C-terminal end.
  • NIM1 is expected to function like the IKBCC, having analogous effects on plant gene regulation.
  • Plants containing the wild-type NIM1 gene when treated with inducer chemicals are predicted to have more NIM1 gene product (IKB homolog) or less phosphorylation of the NIM1 gene product (IKB homolog).
  • IKB homolog the result is that the plant NF- ⁇ B homolog is kept out of the nucleus, and SAR gene expression and resistance responses are allowed to occur.
  • a non-functional NIM1 gene product is present. Therefore, in accordance with this model, the NF- ⁇ B homolog is free to go to the nucleus and repress resistance and SAR gene expression.
  • the present invention encompasses altered forms of NIM1 that act as dominant-negative regulators of the SAR signal transduction pathway.
  • Plants transformed with these dominant negative forms of NIM1 have the opposite phenotype as nimi mutant plants in that the plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and therefore a CIM phenotype. Because of the position the NIM1 gene holds in the SAR signal transduction pathway, it is expected that a number of alterations to the gene, beyond those specifically disclosed herein, will result in constitutive expression of SAR genes and, therefore, a CIM phenotype.
  • NIM1 Based on the amino acid sequence comparison between NIM1 and IKB shown in Figure 9, serines 55 (S55) and 59 (S59) in NIM1 (SEQ ID NO:3) are homologous to S32 and S36 in human l ⁇ B ⁇ .
  • serines 55 (S55) and 59 (S59) in NIM1 are homologous to S32 and S36 in human l ⁇ B ⁇ .
  • the serines at amino acid positions 55 and 59 are mutagenized to alanine residues.
  • the NIM1 gene is altered so that the encoded product has alanines instead of serines in the amino acid positions corresponding to positions 55 and 59 of the Arabidopsis NIM1 amino acid sequence.
  • the present invention also encompasses disease-resistant transgenic plants transformed with such an altered form of the NIM1 gene, as well as methods of using this altered form of the NIM1 gene to confer disease resistance and activate SAR gene expression in plants transformed therewith.
  • the NIM1 gene is altered so that the encoded product is missing approximately the first 125 amino acids compared to the native Arabidopsis NIM1 amino acid sequence.
  • the present invention also encompasses disease- resistant transgenic plants transformed with such an altered form of the NIM1 gene, as well as methods of using this altered form of the NIM1 gene to confer disease resistance and activate SAR gene expression in plants transformed therewith. Deletion of amino acids 261 -317 of human IkBa may result in enhanced intrinsic stability by blocking constitutive phosphorylation of serine and threonine residues in the C- terminus. This altered form of l ⁇ B ⁇ is expected to function as a dominant-negative form.
  • the NIM1 gene is altered so that the encoded product is missing approximately its C-terminal portion, including amino acides 522-593, compared to the native Arabidopsis NIM1 amino acid sequence.
  • the present invention also encompasses disease-resistant transgenic plants transformed with such an altered form of the NIM1 gene, as well as methods of using this altered form of the NIM1 gene to confer disease resistance and activate SAR gene expression in plants transformed therewith.
  • altered forms of the NIM1 gene product are produced as a result of C-terminal and N-terminal segment deletions or chimeras.
  • constructs comprising the ankyrin domains from the NIM1 gene are provided.
  • the present invention encompasses disease-resistant transgenic plants transformed with such NIM1 chimera or ankyrin constructs, as well as methods of using these variants of the NIM1 gene to confer disease resistance and activate SAR gene expression in plants transformed therewith.
  • the present invention concerns DNA molecules encoding altered forms of the NIM1 gene such as those described above, expression vectors containing such DNA molecules, and plants and plant cells transformed therewith.
  • the invention also concerns methods of activating SAR in plants and conferring to plants a CIM phenotype and broad spectrum disease resistance by transforming the plants with DNA molecules encoding altered forms of the NIM1 gene product.
  • the present invention additionally concerns plants transformed with an altered form of the NIM1 gene.
  • the overexpression of the wild-type NIM1 gene in plants and the expression of altered forms of the NIM1 gene in plants results 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. Phythophthora parasitica and Peronospora tabacina; bacteria, e.g. Pseudomonas syringae and Pseudomonas tabacr ' , 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. Phythophthora parasitica and Peron
  • the vectors and methods of the invention are useful against a number of disease organisms 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, Exseroh
  • the methods of the present invention can be utilized to confer disease resistance to a wide variety of plants, including gymnosperms, monocots, and dicots.
  • 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, 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.
  • Transformed cells can be regenerated into whole plants such that the gene imparts disease resistance to the intact transgenic plants.
  • the expression system can be modified so that the disease resistance gene is
  • the NIM1 DNA molecule or gene fragment conferring disease resistance to plants by allowing induction of SAR gene expression or the altered form of the NIM1 gene conferring disease resistance to plants by enhancing SAR gene expression can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule comprised within SEQ ID NO:1 or a functional variant thereof or a molecule encoding one of the altered forms of NIM1 described above into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • 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.
  • 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
  • NIM1 coding sequence and the altered NIM1 coding sequences described herein can be cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Laboratory, Cold Spring Harbor, New York (1982).
  • a promoter that will result in a sufficient expression level or constitutive expression must be present in the expression vector.
  • RNA polymerase normally binds to the promoter and initiates transcription of a gene. 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.
  • the components of the expression cassette may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. Plant cells transformed with such modified expression systems, then, exhibit overexpression or constitutive expression of genes necessary for activation of SAR.
  • transformation vectors are available for plant transformation, and the genes of 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. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bargene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl.
  • 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 is described.
  • 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, J. Bacteriol.
  • Xhol linkers are ligated to the Ec ⁇ /Wfragment 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., Gene 53: 153-161 (1987)), and the Xhol- digested fragment are cloned into Sa/7-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 >4gro.3acter/ ' /vt77-mediated transformation, the RK2-derived trfA 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 pCIB10 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. (Gene 53: 153-161 (1987)).
  • Various derivatives of pCIB10 are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).
  • 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 which 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. a. pCIB3064: 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 bat-gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)).
  • This generated pCIB3064 which comprises the bargene 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 which 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 Adh1 gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOGIO. 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.
  • MCMV Maize Chlorotic Mottle Virus
  • 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.
  • Gene sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable high expression level promoter and upstream of a suitable transcription terminator. These expression cassettes can then be easily transferred to the plant transformation vectors described above.
  • Promoter Selection 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 NIM1 gene product or altered NIM1 gene product. Alternatively, the selected promoter may drive expression of the gene under a light-induced or other temporally regulated promoter.
  • pCGN1761 contains the "double" 35S promoter and the tm/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 gene 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-gene sequence-tm/ 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 above.
  • 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.
  • pCGN1761 ENX is cleaved with Sphl, treated with T4 DNA polymerase and religated, thus destroying the Sphl site located 5' to the double 35S promoter. This generates vector pCGN1761 ENX/Sph-.
  • pCGN1761 ENX/Sph- is cleaved with EcoRI, and ligated to an annealed molecular adaptor of the sequence 5'-AATTCTAAAGCATGCCGATCGG-375'- AATTCCGATCGGCATGCTTTA-3' (SEQ ID NO's: 12 and 13).
  • This generates the vector pCGNSENX, which incorporates the uasAoptimized plant translational initiation sequence TAAA-C adjacent to the ATG which is itself part of an Sphl site which is suitable for cloning heterologous genes at their initiating methionine. Downstream of the Sphl site, the EcoRI, Notl, and Xhol sites are retained.
  • An alternative vector is constructed which utilizes an Ncol site at the initiating ATG.
  • This vector designated pCGN1761 NENX is made by inserting an annealed molecular adaptor of the sequence 5'-AATTCTAAACCATGGCGATCGG-3'/5'-
  • the vector includes the qivasAoptimized sequence TAAACC adjacent to the initiating ATG which is within the Ncol site. Downstream sites are EcoRI, Notl, and Xhol. Prior to this manipulation, however, the two Ncol sites in the pCGN1761 ENX vector (at upstream positions of the 5' 35S promoter unit) are destroyed using similar techniques to those described above for Sphl or alternatively using "inside-outside” PCR. Innes et al. PCR Protocols: A guide to methods and applications. Academic Press, New York (1990). This manipulation can be assayed for any possible detrimental effect on expression by insertion of any plant cDNA or reporter gene sequence into the cloning site followed by routine expression analysis in plants.
  • the double 35S promoter in pCGN1761 ENX may be replaced with any other promoter of choice which will result in suitably high expression levels.
  • a chemically regulated PR-1 promoter which is described in U.S. Patent No. 5,614,395, which is hereby incorporated by reference in its entirety, 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 which carry appropriate terminal restriction sites.
  • the chemically/pathogen regulatable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (see EP 0 332 104, example 21 for construction which is hereby incorporated by reference) 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-la-promoter- containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and blunting with T4 polymerase, followed by cleavage with Hindlll and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-1a promoter and the tml terminator and an intervening polylinker with unique EcoRI and Notl sites. Selected NIM1 genes can be inserted into this vector, and the fusion products (i.e. promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described in this application.
  • chemical regulators may be employed to induce expression of the NIM1 coding sequence in the plants transformed according to the present invention.
  • “chemical regulators” include chemicals known to be inducers for the PR-1 promoter in plants, or close derivatives thereof.
  • a preferred group of regulators for the PR-1 promoter is based on the benzo-1 ,2,3-thiadiazole (BTH) structure and includes, but is not limited to, the following types of compounds: benzo-1 ,2,3- thiadiazolecarboxylic acid, benzo-1 ,2,3-thiadiazolethiocarboxylic acid, cyanobenzo-1 ,2,3- thiadiazole, benzo-1 ,2,3-thiadiazolecarboxylic acid amide, benzo-1 ,2,3-thiadiazolecarboxylic acid hydrazide, benzo-1 ,2,3-thiadiazole-7-carboxylic acid, benzo-1 ,2,3-thiadiazole-7- thiocarboxylic acid, 7-cyanobenzo-1 ,2,3-thiadiazole, benzo-1 ,2,3-thiadiazolecarboxylate in which the alkyl group contains one to six carbon atoms, methyl benzo-1 ,2,3-thiadiazole
  • Other chemical inducers may include, for example, benzoic acid, salicylic acid (SA), polyacrylic acid and substituted derivatives thereof; suitable substituents include lower alkyl, lower alkoxy, lower alkylthio, and halogen.
  • SA salicylic acid
  • Still another group of regulators for the chemically inducible DNA sequences of this invention is based on the pyridine carboxylic acid structure, such as the isonicotinic acid structure and preferably the haioisonicotinic acid structure. Preferred are dichloroisonicotinic acids and derivatives thereof, for example the lower alkyl esters.
  • Suitable members of this class of regulator compounds are, for example, 2,6-dichloroisonicotinic acid (INA), and the lower alkyl esters thereof, especially the methyl ester.
  • INA 2,6-dichloroisonicotinic acid
  • actin promoter is a good choice for a constitutive promoter.
  • the promoter from the rice >Act/gene has been cloned and characterized (McElroy etal. Plant Cell 2: 163-171 (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 et al. Mol. Gen. Genet. 231 : 150-160 (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. Plant Cell Rep. 12: 506-509 (1993)).
  • Ubiquitin is another 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 al. Plant Science 79: 87-94 (1991) and maize - Christensen et al. Plant Molec. Biol. 12: 619-632 (1989)).
  • 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) which is herein incorporated by reference. Taylor et al. (Plant Cell Rep.
  • a vector which comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment.
  • the ubiquitin promoter is suitable for the expression of cellulase genes in transgenic plants, especially monocotyledons.
  • 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.
  • NIM1 gene of the instant invention Another pattern of expression for the NIM1 gene of the instant invention is root expression.
  • a suitable root promoter is described by de Framond (FEBS 290: 103-106 (1991)) and also in the published patent application EP 0 452 269 (to Ciba-Geigy) which is herein incorporated by reference.
  • This promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of a cellulase 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 expression of NIM1 genes of the invention.
  • Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191 -201 (1993)) and all are suitable for use with the instant invention.
  • Patent Application WO 93/07278 (to Ciba-Geigy) which is herein incorporated by reference 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 (Plant Molec Biol 12: 579-589 (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.
  • the Dral-Sphl fragment extends from -58 relative to the initiating rbcS ATG to, and including, the first amino acid (also a methionine) of the mature peptide immediately after the import cleavage site, whereas the Tsp509l-Sphl fragment extends from -8 relative to the initiating rbcS ATG to, and including, the first amino acid of the mature peptide.
  • these fragments can be appropriately inserted into the polylinker of any chosen expression cassette generating a transcriptional fusion to the untranslated leader of the chosen promoter (e.g.
  • the 5' Tsp509l site may be rendered blunt by T4 polymerase treatment, or may alternatively be ligated to a linker or adaptor sequence to facilitate its fusion to the chosen promoter.
  • the 3' Sphl site may be maintained as such, or may alternatively be ligated to adaptor of linker sequences to facilitate its insertion into the chosen vector in such a way as to make available appropriate restriction sites for the subsequent insertion of a selected NIM1 gene.
  • the ATG of the Sphl site is maintained and comprises the first ATG of the selected NIM1 gene.
  • Chen & Jagendorf provide consensus sequences for ideal cleavage for chloroplast import, and in each case a methionine is preferred at the first position of the mature protein. At subsequent positions there is more variation and the amino acid may not be so critical.
  • fusion constructions can be assessed for efficiency of import in vitro using the methods described by Bartlett et al. (In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982)) and Wasmann et al. (Mol. Gen. Genet. 205: 446-453 (1986)).
  • the best approach may be to generate fusions using the selected NIM1 gene or altered form of the NIM1 gene with no modifications at the amino terminus, and only to incorporate modifications when it is apparent that such fusions are not chloroplast imported at high efficiency, in which case modifications may be made in accordance with the established literature (Chen & Jagendorf; Wasman et al.; Ko & Ko, J. Biol. Chem 267.: 13910-13916 (1992)).
  • a preferred vector is constructed by transferring the Dral-Sphl transit peptide encoding fragment from prbcS-8B to the cloning vector pCGN1761 ENX/Sph-.
  • This plasmid is cleaved with EcoRI and the termini rendered blunt by treatment with T4 DNA polymerase.
  • Plasmid prbcS-8B is cleaved with Sphl and ligated to an annealed molecular adaptor of the sequence 5 * -CCAGCTGGAATTCCG-3'/5'-CGGAATTCCAGCTGGCATG-3' (SEQ ID NO's: 16 and 17).
  • the resultant product is 5'-termirially phosphorylated by treatment with T4 kinase.
  • DNA sequences are transferred to pCGN1761/CT in frame by amplification using PCR techniques and incorporation of an Sphl, NSphl, or a/// site at the amplified ATG, which following restriction enzyme cleavage with the appropriate enzyme is ligated into Sp/V-cleaved pCGN1761/CT.
  • it may be required to change the second amino acid of the product of the cloned gene; however, in almost all cases the use of PCR together with standard site directed mutagenesis will enable the construction of any desired sequence around the cleavage site and first methionine of the mature protein.
  • a further preferred vector is constructed by replacing the double 35S promoter of pCGN1761 ENX with the BamHI-Sphl fragment of prbcS-8A which contains the full-length, light-regulated rbcS-8A promoter from -1038 (relative to the transcriptional start site) up to the first methionine of the mature protein.
  • the modified pCGN1761 with the destroyed Sphl is cleaved with Pstl and EcoRI and treated with T4 DNA polymerase to render termini blunt.
  • prbcS-8A is cleaved with Sphl and ligated to the annealed molecular adaptor of the sequence described above.
  • the resultant product is 5'-terminally phosphorylated by treatment with T4 kinase. Subsequent cleavage with BamHI releases the promoter-transit peptide containing fragment which is treated with T4 DNA polymerase to render the BamHI terminus blunt.
  • the promoter-transit peptide fragment thus generated is cloned into the prepared pCGN1761 ENX vector, generating a construction comprising the rbcS- ⁇ A promoter and transit peptide with an Sphl site located at the cleavage site for insertion of heterologous genes. Further, downstream of the Sphl site there are EcoRI (re-created), Notl, and Xhol cloning sites. This construction is designated pCGN1761 rbcS/CT.
  • 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 which 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.
  • 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., Genes Develop. 1: 1183-1200 (1987)).
  • the intron from the maize bronzel gene had a similar effect in enhancing expression.
  • Intron sequences have been routinely incorporated 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.
  • 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. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).
  • sequences have been characterized which cause the targeting of gene products to other cell compartments.
  • Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (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. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann etal. Mol. Gen. Genet. 205: 446-453 (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 which has an expression pattern different to that of the promoter from which the targeting signal derives.
  • Plant tissues suitable for transformation include leaf tissues, root tissues, meristems, and protoplasts.
  • the present system can be utilized in any plant which can be transformed and regenerated. Such methods for transformation and regeneration are well known in the art. Methodologies for the construction of plant expression cassettes as well as the introduction of foreign DNA into plants is generally described in the art. Generally, for the introduction of foreign DNA into plants, Ti plasmid vectors have been utilized for the delivery of foreign DNA. Also utilized for such delivery have been direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. Such methods had been published in the art.
  • Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system Plant Physiol. 81 :301-305, 1986; Fry, J., Barnason, A., and Horsch, R.B. Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Pl.Cell Rep. 6:321-325, 1987; Block, M.d. Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor.appi. genet.
  • Agrobacterium Tumefaciens Mediated Gene Transfer in Peanut (Arachis Hypogaea L). Pl.Cell Rep. 13:582-586, 1994; Hartman, C.L., Lee, L., Day, P.R., and Turner, N.E. Herbicide Resistant Turfgrass (Agrostis Palustris Huds) by Biolistic Transformation. Bid-Technology 12:919923, 1994; Howe, G.T., Goldfarb, B., and Strauss, S.H. Agrobacterium Mediated Transformation of Hybrid Poplar Suspension Cultures and Regeneration of Transformed Plants. Plant Cell Tissue & Orgart Culture 36:59-71 , 1994; Konwar, B.K.
  • Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogens. Agrobacterium tumefaciens (e.g., strains LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants.
  • Transformation of Dicotyledons Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques which do not require Agrobacterium.
  • Hon-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., EMBO J 3: 2717-2722 (1984), Potrykus etal., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001 -1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
  • Agrobacterium-medlated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species.
  • the many crop species which are alfalfa and poplar EP 0 317 511 (cotton), EP 0 249 432 (tomato, to Calgene), WO 87/07299 (Brassica, to Calgene), US 4,795,855 (poplar)).
  • 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 rgenes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)).
  • the transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E.
  • the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (H ⁇ fgen & Willmitzer, Nucl. Acids Res. 16: 9877 (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 all to Sanford et al.
  • 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 incorporation 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
  • 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. Biotechnology 4: 1093-1096 (1986)). Patent Applications EP 0 292 435 ([1280/1281] to Ciba-Geigy), EP 0 392 225 (to
  • Ciba-Geigy 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 et al. Plant Cell 2: 603-618 (1990)
  • Fromm et al. Biotechnology 8: 833-839 (1990)
  • application WO 93/07278 to Ciba-Geigy
  • Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment.
  • Protoplast-mediated transformation has been described for Japonica-types and /nd/ca-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).
  • Patent Application EP 0 332 581 (to Ciba-Geigy) 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. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus.
  • a preferred technique for wheat transformation 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, Physiologia Plantarum 15: 473-497 (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
  • 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 isolated gene fragment of the present invention or altered forms of the NIM1 gene can be utilized to confer disease resistance to a wide variety of plant cells, including those of gymnosperms, monocots, and dicots.
  • the gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, 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.
  • a further embodiment of the present invention is a method of producing transgenic descendants of a transgenic parent plant comprising an isolated DNA molecule encoding an altered form of a NIM1 protein according to the invention comprising transforming said parent plant with a recombinant vector molecule according to the invention and transferring the trait to the descendants of said transgenic parent plant involving known plant breeding techniques.
  • 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 descendant 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 descendant plants. Depending on the desired properties 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 which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow 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 which 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.
  • Comprised by the present invention is a commercial bag comprising seed of a transgenic plant comprising at least one altered form of a NIM1 protein or a NIM1 protein that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with lable 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., Proc. Natl. Acad. Sci. USA 90: 7327-7331. 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.
  • C. 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.
  • D. Peronospora parasitica Resistance Assays for resistance to Peronospora parasitica are performed on plants as described in Uknes et al, (1993). Plants are inoculated with a combatible 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., Trans. Br. Mycol. Soc. 74: 329-333 (1980)) for microscopic examination. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGURE 1 shows the effect of chemical inducers on the induction of SAR gene expression in wild-type and nimi plants. Chemical induction of SAR genes is diminished in nimi plants. Water, SA, INA, or BTH is applied to wild type (WT) and t7/ ' m7 plants. After 3 days, RNA is prepared from these plants and examined for expression of PR-1 , PR-2, and PR-5.
  • FIGURE 2 depicts PR-1 gene expression in pathogen-infected Ws-O and nimi plants.
  • Pathogen induction of PR-1 is diminished in nimi plants.
  • Wild type (WT) and nimi plants were spray-inoculated with the Emwa race of P. parasitica. Samples were collected at days 0, 1 , 2, 4, and 6 and RNA is analyzed by blot hybridization with an A. thaliana PR-1 cDNA probe to measure PR-1 mRNA accumulation.
  • FIGURE 3 shows the accumulation of PR-1 mRNA in t7/ ' t ⁇ 7 mutants and wild-type plants after pathogen infection or chemical treatment.
  • Plants containing nimi alleles ⁇ /tn - 1, -2, -3, -4, -5, and -6 and Ws-0 (Ws) were treated with water (C), SA, INA, or BTH 3 days before RNA isolation.
  • the Emwa sample consists of RNA isolated from plants 14 days post-inoculation with the Emwa isolate of P. parasitica. Blots were hybridized using an Arabidopsis PR-1 cDNA as a probe (Uknes etal., 1992).
  • FIGURE 4 shows the levels of SA accumulation in Ws-0 and nimi plants infected with P. syringae. nimi plants accumulate SNA following pathogen exposure. Leaves of wild type and nimi plantsare infiltrated with Pst DC3000(avrRpt2) or carrier medium (10 mM MgCI 2 ) alone. After 2 days, samples were collected from untreated, MgCI 2 -treated, and DC3000(awflpf2)-treated plants. Bacteria-treated samples were separated into primary (infiltrated) and secondary (noninfiltrated) leaves. Free SA and total SA following hydrolysis with ⁇ -glucosidase were quantified by HPLC. Error bars indicate SD of three replicate samples.
  • FIGURES 5A-D present a global map at increasing levels of resolution of the chromosomal region centered on NIM1 with recombinants indicated, including, BACs, YACs and Cosmids in NIM1 region.
  • A Map position of NIM1 on chromosome 1. The total number of gametes scored is 2276.
  • B Yeast artificial chromosome (striped), bacterial artificial chromosome (BAC), and P1 clones used to clone NIML
  • (C) Cosmid clones that cover the NIM1 locus. The three cosmids that complement nim1-1 are shown as thicker lines.
  • (D) The four putative gene regions on the smallest fragment of complementing genomic DNA. The four open reading frames that comprise the NIM1 gene are indicated by the open bars. The arrows indicate the direction of transcription. Numbering is relative to the first base of Arabidopsis genomic DNA present in cosmid D7.
  • FIGURE 6 shows the nucleic acid sequence of the NIM1 gene and the amino acid sequence of the NIM1 gene product, including changes in the various alleles.
  • This nucleic acid sequence which is on the opposite strand as the 9.9 kb sequence presented in SEQ ID NO:1 , is also presented in SEQ ID NO:2, and the amino acid sequence of the NIM1 gene product is also presented in SEQ ID NO:3.
  • FIGURE 7 shows the accumulation of NIM1 induced by INA, BTH, SA and pathogen treatment in wild type plants and mutant alleles of nimL
  • the RNA gel blots in Figure 3 were probed for expression of RNA by using a probe derived from 2081 to 3266 in the sequence shown in Figure 6.
  • FIGURE 8 is an amino acid sequence comparison of Expressed Sequence Tag regions of the NIM1 protein and cDNA protein products of 4 rice gene sequences (SEQ ID NOs: 4-11 ); numbers correspond to amino acid positions in SEQ ID NO:3).
  • FIGURE 9 is a sequence alignment of the NIM1 protein sequence with l ⁇ B ⁇ from mouse, rat, and pig.
  • Vertical bars (I) above the sequences indicate amino acid identity between NIM1 and the l ⁇ B ⁇ sequences (matrix score equals 1.5); double dots (:) above the sequences indicate a similarity score >0.5; single dots (.) above the sequences indicate a similarity score ⁇ 0.5 but >0.0; and a score ⁇ 0.0 indicates no similarity and has no indicia above the sequences (see Examples).
  • Locations of the mammalian IKBCC ankyrin domains were identified according to de Martin et al., Gene 152, 253-255 (1995). The dots within a sequence indicate gaps between NIM1 and l ⁇ B ⁇ proteins.
  • Plasmid BAC-04 was deposited with ATCC on May 8, 1996 as ATCC 97543. Plasmid P1-18 was deposited with ATCC on June 13, 1996 as ATCC 97606. Cosmid D7 was deposited with ATCC on September 25, 1996 as ATCC 97736.
  • SEQ ID NO: 2 5655-bp genomic sequence in Rgure 6 (opposite strand from SEQ ID NO:1 ). comprising the coding region of the wild-type Arabidopsis thaliana NIM1 gene.
  • SEQ ID NO: 3 - AA sequence of wild-type NIM1 protein encoded by eds of SEQ ID N02.
  • SEQ ID NO: 12 OligonucleotJde.
  • SEQ ID NO: 13 Oiigonucleotide.
  • SEQ ID NO: 17 Oiigonucleotide.
  • SEQ ID NO: 18 is the mouse l ⁇ B ⁇ amino acid sequence from Figure 8.
  • SEQ ID NO: 19 is the rat l ⁇ B ⁇ amino acid sequence from Figure 8.
  • SEQ ID NO: 20 is the pig IKBCC amino acid sequence from Figure 8.
  • SEQ ID NO: 21 is the cDNA sequence of the Arabidopsis thaliana NIM1 gene.
  • SEQ ID NO's: 22 and 23 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having alanine residues instead of serine residues at amino acid positions 55 and 59.
  • SEQ ID NO's: 24 and 25 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having an N-terminal deletion.
  • SEQ ID NO's: 26 and 27 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having a C-terminal deletion.
  • SEQ ID NO's: 28 and 29 are the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having both N-terminal and C- terminal amino acid deletions.
  • SEQ ID NO's: 30 and 31 are the DNA coding sequence and encoded amino acid sequence, respectively, of the ankyrin domain of NIML SEQ ID NOs:32 through 39 are oiigonucleotide primers.
  • AFLP Amplified Fragment Length Polymorphism avrRpt2: avirulence gene Rpt2, isolated from Pseudomonas syringae
  • CIM Constitutive IMmunity phenotype (SAR is constitutively activated)
  • cim constitutive immunity mutant plant
  • cM centimorgans
  • cpr7 constitutive expresser of PR genes mutant plant
  • Col-O Arabidopsis ecotype Columbia
  • Emwa Peronospora parasitica isolate compatible in the Ws-0 ecotype of
  • NahG Arabidopsis line transformed with nahG gene ndr. non-race-specific disease resistance mutant plant nim: non-inducible immunity mutant plant
  • NIM1 the wild type gene, involved in the SAR signal transduction cascade
  • NIM1 Protein encoded by the wild type NIM1 gene nimi: mutant allele of NIM1, conferring disease susceptibility to the plant; also refers to mutant Arabidopsis thaliana plants having the nimi mutant allele of
  • SSLP Simple Sequence Length Polymorphism
  • UDS Universal Disease Susceptible phenotype
  • Ws-O Arabidopsis ecotype Issilewskija
  • Arabidopsis thaliana ecotype Isilewskija (Ws-O; stock number CS 2360) and fourth- generation (T 4 ) seeds from T-DNA-transformed lines were obtained from the Ohio State University Arabidopsis Biological Resource Center (Columbus, OH).
  • Second generation (M- 2 ) seeds from ethyl methane sulfonate (EMS) mutagenized Ws-0 plants were obtained from Lehle Seeds (Round Rock, TX).
  • Pseudomonas syringae pv. Tomato (Pst) strain DC3000 containing the cloned avrRpt2 gene [DC3000(awPpf2)] was obtained from B. Staskawicz, University of California, Berkeley.
  • P. parasitica pathovars and their sources were as follows: Emwa from E. Holub and I.R. Crute, Horticultural Research Station, East Mailing, Kent; Wela from A. Slusarenko and B. Mauch-Mani, Institut fur Organbiologie, Zurich, Switzerland; and Noco from J. Parker, Sainsbury Laboratory, Norwich, England. Fungal cultures were maintained by weekly culturing on Arabidopsis ecotype Ws-O, Weiningen, and Col-O, for P. parasitica pathovars Emwa, Wela, and Noco, respectively.
  • Example 2 Mutant Screens
  • M 2 or T 4 seeds were grown on soil for 2 weeks under 14 hr of light per day, misted with 0.33 mM INA (0.25 mg/ml made from 25% INA in wettable powder; Ciba, Basel, Switzerland), and inoculated 4 days later by spraying a P. parasitica conidial suspension containing 5-10 x 10 4 conidiospores per ml of water.
  • This fungus is normally virulent on the Arabidopsis Ws-O ecotype, unless resistance is first induced in these plants with isonicotinic acid (INA) or a similar compound. Plants were kept under humid conditions at 18°C for 1 week and then scored for fungal sporulation. Plants that supported fungal growth after INA treatment were selected as putative mutants.
  • INA isonicotinic acid
  • nim mutant plants were isolated from the flat, placed under low humidity conditions and allowed to set seed. Plants derived from this seed were screened in an identical manner for susceptibility to the fungus Emwa, again after pretreatment with INA. The descendant plants that showed infection symptoms were defined as nim mutants. Six nim lines were thus identified. One line (nim1-1) was isolated from the T-DNA population and five (nim1-2, nim1-3, nim1-4, nim1-5, and nim1-6) from the EMS population.
  • BTH broad spectrum disease resistance
  • SAR broad spectrum disease resistance
  • P. parasitica isolate 'Emwa' is a P.p. isolate that is compatible in the Ws ecotype. Compatible isolates are those that are capable of causing disease on a particular host.
  • the P. parasitica isolate 'Noco' is incompatible on Ws but compatible on the Columbia ecotype. Incompatible pathogens are recognized by the potential host, eliciting a host response that prevents disease development.
  • Wild-type seeds and seeds for each of the nimi alleles were sown onto MetroMix 300 growing media, covered with a transparent plastic dome, and placed at 4°C in the dark for 3 days. After 3 days of 4°C treatment, the plants were moved to a phytotron for 2 weeks. By approximately 2 weeks post-planting, germinated seedlings had produced 4 true leaves. Plants were then treated with H 2 0, 5mM SA, 300 ⁇ M BTH ,or 300 ⁇ M INA. Chemicals were applied as a fine mist to completely cover the seedlings using a chromister.
  • Water control plants were returned to the growing phytotron while the chemically treated plants were held in a separate but identical phytotron.
  • water and chemically treated plants were inoculated with the compatible 'Emwa' isolate.
  • 'Noco' inoculation was conducted on water treated plants only. Following inoculation, plants were covered with a clear plastic dome to maintain high humidity required for successful P. parasitica infection and placed in a growing chamber with 19°C day/17° C night temperatures and 8h light/16h dark cycles.
  • each mutant was microscopically analyzed at various timepoints after inoculation for the growth of P. parasitica under normal growth conditions and following pretreatment with either SA, INA, or BTH. Under magnification, sporulation of the fungus could be observed at very early stages of disease development. The percentage of plants/pot showing sporulation at 5d, 6d, 7d, 11d and 14d after inoculation was determined and the density of sporulation was also recorded.
  • Table 1 shows, for each of the nimi mutant plant lines, the percent of plants that showed some surface conidia on at least one leaf after infection with the Emwa race of P. parasitica. P. parasitica was inoculated onto the plants three days after water or chemical treatment. The table indicates the number of days after infection that the disease resistance was rated.
  • nim1-4 and nim 1-6 showed a relatively rapid fungal growth; nim1-1, nim1-2, nim1-3 plants exhibited a somewhat slower rate of fungal growth; and fungal growth in t7/ ' m7-5 plants was even slower than in the untreated Ws-0 controls.
  • the mutants also fell into three classes where nim1-4 was the most severely compromised in its ability to restrict fungal growth following chemical treatment; nim1-1, nim1-2, nim1-3, and nim 1-6 were all moderately compromised; and nim 1-5 was only slightly compromised. In these experiments, Ws-0 did not support fungal growth following INA or BTH treatment.
  • nim1-4 being the most severely compromised
  • nim1-1, nim1-2, nim1-3 and nim 1-6 showing an intermediate inhibition of fungus and t7//777-5with only slightly impaired fungal resistance.
  • Table 2 shows the disease resistance assessment via infection rating of the various nimi alleles as well as of NahG plants at 7 and 11 days after innoculation with Peronospora parasitica.
  • WsWT indicates the Ws wild type parent line in which the nimi alleles were found.
  • the various nimi alleles are indicated in the table and the NahG plant is indicated also.
  • NahG Arabidopsis is also described in U.S. Patent Application Serial No. 08/454,876, incorporated by reference herein.
  • nahG is a gene from Pseudomonas putida encoding a salicylate hydroxylase that converts salicylic acid to catechol, thereby eliminating the accumulation of salicylic acid, a necessary signal transduction component for SAR in plants.
  • NahG Arabidopsis plants do not display normal SAR, and they show much greater susceptibility in general to pathogens.
  • the NahG plants still respond to the chemical inducers INA and BTH.
  • NahG plants therefore serve as a kind of universal susceptibility control. Table 2
  • NahG 0 0 From Table 2 it can be seen that the nim1-4 and n/m 7 -6 alleles had the most severe Peronospora parasitica infections; this was most easily observable at the earlier time points. In addition, the n/tn -5 allele showed the greatest response to both INA and BTH and therefore was deemed the weakest t7/ ' m allele. The NahG plants showed very good response to both INA and BTH and looked very similar to the nim1-5 allele.
  • nimi plants' lack of responsiveness to the SAR-inducing chemicals SA, INA, and BTH implies that the mutation is downstream of the entry point(s) for these chemicals in the signal transduction cascade leading to systemic acquired resistance.
  • SAR gene mRNA was also used as a criterion to characterize the different t7/ ' m alleles. Wild-type seeds and seeds for each of the nimi alleles (nim 1-1, -2, -3, -4, -5, -6) were sown onto MetroMix 300 growing media, covered with a transparent plastic dome, and placed at 4°C in the dark for 3 days. After 3 days of 4°C treatment, the plants were moved to a phytotron for 2 weeks.
  • Figures 1-3 present various RNA gel blots that indicate that SA, INA and BTH induce neither SAR nor SAR gene expression in nimi plants.
  • Figure 1 replicate blots were hybridized to Arabidopsis gene probes PR-1 , PR-2 and PR-5 as described in Uknes et al. (1992). In contrast to the case in wild type plants, the chemicals did not induce RNA accumulation from any of these 3 SAR genes in nim1-1 plants.
  • pathogen infection Emwa
  • PR-1 gene expression was not induced until 6 days after infection and the level was reduced relative to the wild type at that time.
  • PR-1 gene expression in nim1-1 plants was delayed and reduced relative to the wild type.
  • RNA gel blot in Figure 3 shows that PR-1 mRNA accumulates to high levels following treatment of wild-type plants with SA, INA, or BTH or infection by P. parasitica.
  • PR-1 mRNA accumulation was dramatically reduced relative to the wild type following chemical treatment.
  • PR-1 mRNA was also reduced following P. parasitica infection, but there was still some accumulation in these mutants.
  • PR-1 mRNA accumulation was more dramatically reduced than in the other alleles following chemical treatment (evident in longer exposures) and significantly less PR-1 mRNA accumulated following P. parasitica infection, supporting the idea that these are particularly strong nimi alleles.
  • PP-7 mRNA accumulation was elevated in the n/t-7- mutant, but only mildly induced following chemical treatment or P. parasitica infection. Based on both Pfi-7 mRNA accumulation and fungal infection, the mutants have been determined to fall into three classes: severely compromised alleles (nim1-4 and niml- 6); moderately compromised alleles (nim1-1, nim1-2, and nim1-3); and a weakly compromised allele (nim1-5).
  • the leaves were harvested 2 days later for SA analysis as described by Delaney et al, 1995, PNAS 92, 6602-6606.
  • This analysis showed that the nimi plants accumulated high levels of SA in infected leaves, as shown in Figure 4.
  • Uninfected leaves also accumulated SA, but not to the same levels as the infected leaves, similar to what has been observed in wild-type Arabidopsis. This indicates that the nim mutation maps downstream of the SA marker in the signal transduction pathway.
  • nim1-2, -3, -4, -5, -6 was not complemented by the nim1-1; these plants all fall within the same complementation group and are therefore allelic.
  • Wild type denotes the wild type Ws-0 strain.
  • nim1-1 was determined to lie about 8.2 centimorgans (cM) from nga128 and 8.2 cM from ngal 11 on the lower arm of chromosome 1. In addition, nim1-1 was determined to lie between ngal 11 and about 4 cM from the SSLP marker ATHGENEA.
  • nim plants from an F 2 population derived from a cross between nim 1-1 and LerDP23 were identified based on both their inability to accumulate PR-1 mRNA and their ability to support fungal growth following INA treatment.
  • DNA was extracted from these plants and scored for zygosity at both ATHGENEA and ngal 11.
  • 93 recombinant chromosomes were identified between ATHGENEA and nim1-1, giving a genetic distance of approximately 4.1 cM (93 of 2276), and 239 recombinant chromosomes were identified between nga111 and nim1-1, indicating a genetic distance of about 10.5 cM (239 of 2276).
  • Informative recombinants in the ATHGENEA to ngal 11 interval were further analyzed using amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995).
  • AFLP amplified fragment length polymorphism
  • AFLP markers between ATHGENEA and ngal 11 were identified and were used to construct a low resolution map of the region ( Figures 5A and 5B).
  • AFLP markers W84.2 (1 cM from nim1-1) and W85.1 (0.6 cM from nim1-1) were used to isolate yeast artificial chromosome (YAC) clones from the CIC (for Centre d'Etude du Polymorphisme Humain, INRA and CNRS) library (Creusot et al., 1995).
  • YAC clones, CIC12H07 and CIC12F04 were identified with W84.2 and two YAC clones CIC7E03 and CIC10G07 were identified with the W85.1 marker.
  • Figure 5B To bridge the gap between the two sets of flanking YAC clones, bacterial artificial chromosome (BAC) and P1 clones that overlapped CIC12H07 and CIC12F04 were isolated and mapped, and sequential walking steps were carried out extending the BAC/P1 contig toward NIM1 ( Figure 5C; Liu et al., 1995; Chio et al., 1995). New AFLP's were developed during the walk that were specific for BAC or P1 clones, and these were used to determine whether the NIM1 gene had been crossed. NIM1 had been crossed when BAC and P1 clones were isolated that gave rise to both AFLP markers L84.6a and L84.8.
  • BAC bacterial artificial chromosome
  • P1 clones that overlapped CIC12H07 and CIC12F04 were isolated and mapped, and sequential walking steps were carried out extending the BAC/P1 contig toward NIM1 ( Figure 5C; Liu et al., 1995; Chio
  • a cosmid library of the NIM1 region was constructed in the Agrobacterium-compaWb ⁇ e
  • T-DNA cosmid vector pCLD04541 using CsCI-purified DNA from BAC-06, BAC-04, and P1- 18.
  • the DNAs of the three clones were mixed in equimolar quantities and were partially digested with the restriction enzyme Sau3A.
  • the 20-25 kb fragments were isolated using a sucrose gradient, pooled and filled in with dATP and dGTP.
  • Plasmid pCLD04541 was used as T-DNA cosmid vector. This plasmid contains a broad host range pRK290-based replicon, a tetracycline resistance gene for bacterial selection and the nptll gene for plant selection.
  • the vector was cleaved with Xhol and filled in with dCTP and dTTP. The prepared fragments were then ligated into the vector . The ligation mix was packaged and transduced into E. coli strain XL1-blue MR (Stratagene). Resulting transformants were screened by hybridization with the BAC04, BAC06 and P1-18 clones and positive clones isolated. Cosmid DNA was isolated from these clones and template DNA was prepared using the ECs EcoRI/Msel and Hindlll/Msel. The resulting AFLP fingerprint patterns were analyzed to determine the order of the cosmid clones.
  • Cosmids generated from clones spanning the NIM1 region were moved into Agrobacterium tumefaciens AGL-1 through conjugative transfer in a tri-parental mating with helper strain HB101 (pRK2013). These cosmids were then used to transform a kanamycin- sensitive nim1-1 Arabidopsis line using vacuum infiltration (Bechtold et al., 1993; Mindrinos et al., 1994). Seed from the infiltrated plants was harvested and allowed to germinate on GM agar plates containing 50 mg/ml kanamycin as a selection agent. Only plantlets that were transformed with cosmid DNA could detoxify the selection agent and survive.
  • Seedlings that survived the selection were transferred to soil approximately two weeks after plating and tested for the nimi phenotype as described below. Transformed plants that no longer had the nimi phenotype identified cosmid(s) containing a functional NIM1 gene.
  • Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 300 ⁇ m INA was applied as a fine mist to completely cover the plants using a chromister. After two days, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate Emwa) and grown under high humidity conditions in a growing chamber with 19°C day/17 C C night temperatures and 8h light/16h dark cycles. Eight to ten days following fungal infection, plants were evaluated and scored positive or negative for fungal growth. Ws and t7/ ' m7 plants were treated in the same way to serve as controls for each experiment.
  • BAC04 DNA (25 ug, obtained from KeyGene) was the source of DNA used for sequence analysis, as this BAC was the clone completely encompassing the region that complemented the t?/m7 mutants.
  • BAC04 DNA was randomly sheared in a nebulizer to generate fragments with an average length of about 2 kb. Ends of the sheared fragments were repaired, and the fragments were purified.
  • Prepared DNA was ligated with EcoRV- digested pBRKanF4 (a derivative of pBRKan F ⁇ (Bhat 1993)). Resulting kanamycin-resistant colonies were selected for plasmid isolation using the Wizard Plus 9600 Miniprep System (Promega). Plasmids were sequenced using dye terminator chemistry (Applied
  • a region of approximately 9.9 kb defined by the overlap of cosmids E1 and D7 was identified by complementation analysis to contain the n/t777 region. Primers that flanked the insertion site of the vector and that were specific to the cosmid backbone were designed using Oligo 5.0 Primer Analysis Software (National Biosciences, Inc.). DNA was isolated from cosmids D7 and E1 using a modification of the ammonium acetate method (Traynor, P.L., 1990. BioTechniques 9(6): 676.) This DNA was directly sequenced using Dye Terminator chemistry above. The sequence obtained allowed determination of the endpoints of the complementing region. The region defined by the overlap of cosmids E1 and D7 is presented as SEQ ID NO:1.
  • a truncated version of the BamHI-EcoRV fragment was also constructed, resulting in a construct that contained none of the "Gene 3" region (Fig. 5D).
  • the following approach was necessary due the presence of Hindlll sites in the Bam-Spe region of the DNA.
  • the BamHI-EcoRV construct was completely digested with Spel, then was split into two separate reactions for double digestion. One aliquot was digested with BamHI, the other Hindlll.
  • a BamHI-Spel fragment of 2816 bp and a Hindlll-Spel fragment of 1588 bp were isolated from agarose gels (QiaQuick Gel extraction kit) and were ligated to BamHI-Hindlll- digested pSGCGOI .
  • DH5a was transformed with the ligation mix. Resulting colonies were screened for the correct insert by digestion with Hindlll following preparation of DNA using Wizard Magic MiniPreps (Promega).
  • a clone containing the correct construct was electroporated into Agrobacterium strain GV3101 for transformation of Arabidopsis plants.
  • Example 12 Sequence Analysis and Subcloning of the NIM1 Region
  • the 9.9 kb region containing the NIM1 gene was analyzed for the presence of open reading frames in all six frames using Sequencher 3.0 and the GCG package.
  • Four regions containing large ORF's were identified as possible genes (Gene Regions 1-4 in Figure 5D). These four regions were PCR amplified from DNA of the wild-type parent and the six different nimi allelic variants nim1-1, -2, -3, -4, -5, and -6. Primers for these amplifications were selected using Oligo 5.0 (National Biosciences, Inc.) and were synthesized by Integrated DNA Technologies, Inc. PCR products were separated on 1.0% agarose gels and were purified using the QIAquick Gel Extraction Kit. The purified genomic PCR products were directly sequenced using the primers used for the initial amplification and with additional primers designed to sequence across any regions not covered by the initial primers. Average coverage for these gene regions was approximately 3.5 reads/base.
  • Sequences were edited and were assembled using Sequencher 3.0. Base changes specific to various t?/m alleles were identified only in the region designated Gene Region 2, as shown below in Table 5, which shows sequence variations among all six of the nimi alleles.
  • NIM1 gene lies within Gene Region 2, because there are amino acid changes or alterations of sequence within the open reading frame of Gene Region 2 in all six nimi alleles. At the same time, at least one of the nimi alleles shows no changes in the open reading frames within Gene Regions 1 , 3 and 4. Therefore, the only gene region within the 9.9 kb region that could contain the NIM1 gene is Gene Region 2.
  • the Ws section of Table 5 indicates the changes in the Ws ecotype of Arabidopsis relative to the Columbia ecotype of Arabidopsis.
  • the sequences presented herein relate to the Columbia ecotype of Arabidopsis, which contains the wild type gene in the experiments described herein. The changes are listed as amino acid changes within Gene Region 2 (the NIM1 region) and are listed as changes in base pairs in the other regions.
  • the cosmid region containing the nimi gene was delineated by a BamHI-EcoRV restriction fragment of -5.3 kb.
  • Cosmid DNA from D7 and plasmid DNA from pBlueScriptll(pBSII) were digested with BamHI and with EcoRV (NEB).
  • the 5.3 kb fragment from D7 was isolated from agarose gels and was purified using the QIAquick gel extraction kit (# 28796, Qiagen). The fragment was ligated overnight to the Bam-EcoRV-digested pBSII and the ligation mixture was transformed into E. coli strain DH5a. Colonies containing the insert were selected, DNA was isolated, and confirmation was made by digestion with Hindlll. The Bam-EcoRV fragment was then engineered into a binary vector (pSGCGOI) for transformation into Arabidopsis.
  • pSGCGOI binary vector
  • RNA samples isolated from water-, SA-, BTH- and INA-treated Ws and t7/tr?7 lines as previously described in Delaney, et al. (1995). These blots were hybridized with PCR products generated from the four gene regions identified in the 9.9 kb NIM1 gene region (SEQ ID NO:1). Only the gene region containing the NIM1 gene (Gene Region 2) had detectable hybridization with the RNA samples, indicating that only the NIM1 region contains a detectable transcribed gene ( Figure 5D and Table 5).
  • Gene Region 2 (Fig. 5D) was also demonstrated to contain the functional NIM1 gene by doing additional complementation experiments.
  • a BamHI/Hindlll genomic DNA fragment containing Gene Region 2 was isolated from cosmid D7 and was cloned into the binary vector pSGCGOI containing the gene for kanamycin resistance.
  • the resulting plasmid was transformed into the Agrobacterium strain GV3101 and positive colonies were selected on kanamycin. PCR was used to verify that the selected colony contains the plasmid. Kanamycin-sensitive nim1-1 plants were infiltrated with this bacteria as previously described. The resulting seed was harvested and planted on GM agar containing 50 ⁇ g/ml kanamycin.
  • Plants surviving selection were transferred to soil and tested for complementation.
  • Transformed plants and control Ws and nimi plants were sprayed with 300 ⁇ m INA. Two days later, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate Emwa) and grown as previously described. Ten days following fungal infection, plants were evaluated and scored positive or negative for fungal growth. All of the 15 transformed plants, as well as the Ws controls, were negative for fungal growth following INA treatment, while the nimi controls were positive for fungal growth. RNA was extracted and analyzed as described above for these transformants and controls. Ws controls and all 15 transformants showed PR-1 gene induction following INA treatment, while the nimi controls did not show PR-1 induction by INA.
  • PNAS 88, 1731-1735 was plated and plaque lifts were performed. Filters were hybridized with a 32 P-labeled PCR product generated from Gene Region 2 (Figure 5D). 14 positives were identified from a screen of approximately 150,000 plaques. Each plaque was purified and plasmid DNA was recovered. cDNA inserts were digested out of the vector using EcoRI, agarose-gel-purified and sequenced. Sequence obtained from the longest cDNA is indicated in SEQ ID NO:2 and Figure 6. To confirm that the 5' end of the cDNA had been obtained, a Gibco BRL 5' RACE kit was used following manufacturer's instructions. The resulting RACE products were sequenced and found to include the additional bases indicated in Figure 6.
  • RNA samples produced in the induction studies were also probed with the NIM1 gene using a full-length cDNA clone as a probe.
  • Figure 7 it can be seen that INA induced the NIM1 gene in the wild type Ws allele.
  • the nim1-1 mutation allele showed a lower basal level expression of the NIM1 gene, and it was not inducible by INA. This was similar to what was observed in the nim1-3 allele and the nim1-6 allele.
  • the nim1-2 allele showed approximately normal levels in the untreated sample and showed similar induction to that of the wild type sample, as did the nim1-4 allele.
  • the /7/m7- 5 allele seemed to show higher basal level expression of the NIM1 gene and much stronger expression when induced by chemical inducers.
  • D NIM1 Homologues
  • NIM1 protein is homologous in amino acid sequence to 4 different rice cDNA protein products.
  • the homologies were identified using the NIM1 sequences in a GenBank BLAST search. Comparisons of the regions of homology in NIM1 and the rice cDNA products are shown in Figure 8 (See also, SEQ ID NO:3 and SEQ ID NO's: 4-11).
  • the NIM1 protein fragments show from 36 to 48% identical amino acid sequences with the 4 rice products.
  • homologs of Arabidopsis NIM1 are identified through screening genomic or cDNA libraries from different crops such as, but not limited to those listed below in Example 22. Standard techniques for accomplishing this include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g. Sambrook et al., Molecular Cloning , eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oiigonucleotide primers (see, e.g. Innis et al., PCR Protocols, a Guide to Methods and Applications eds., Academic Press (1990)). Homologs identified are genetically engineered into the expression vectors herein and transformed into the above listed crops. Transformants are evaluated for enhanced disease resistance using relevant pathogens of the crop plant being tested.
  • NIM1 homologs in the genomes of cucumber, tomato, tobacco, maize, wheat and barley have been detected by DNA blot analysis.
  • Genomic DNA was isolated from cucumber, tomato, tobacco, maize, wheat and barley, restriction digested with the enzymes BamHI, Hindlll, Xbal, or Sail, electrophoretically separated on 0.8% agarose gels and transferred to nylon membrane by capillary blotting.
  • the membrane was hybridized under low stringency conditions [(1%BSA; 520mM NaP0 4 , pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 55°C for 18-24h] with 32 P-radiolabelled Arabidopsis thaliana NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C; 1XSSC is 0.15M NaCI, 15mM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIML
  • expressed sequence tags identified with similarity to the NIM1 gene such as the rice EST's described in Example 16 can also be used to isolate homologues.
  • the rice EST's may be especially useful for isolation of NIM1 homologues from other monocots.
  • Homologues may be obtained by PCR. In this method, comparisons are made between known homologues (e.g., rice and Arabidopsis). Regions of high amino acid and DNA similarity or identity are then used to make PCR primers. Regions rich in M and W are best followed by regions rich in F, Y, C, H, Q, K and E because these amino acids are encoded by a limited number of codons. Once a suitable region is identified, primers for that region are made with a diversity of substitutions in the 3 rd codon position. This diversity of substitution in the third position may be constrained depending on the species that is being targeted. For example, because maize is GC rich, primers are designed that utilize a G or a C in the 3 rd position, if possible.
  • the PCR reaction is performed from cDNA or genomic DNA under a variety of standard conditions. When a band is apparent, it is cloned and/or sequenced to determine if it is a NIM1 homologue.
  • Example 10/Table 4 To determine if any of the transformants described above in Example 10/Table 4 had overexpression of NIM1 due to insertion site effect, primary transformants containing the D7, D5 or E1 cosmids (containing the NIM1 gene) were selfed and the T2 seed collected. Seeds from one E1 line, four D5 lines and 95 D7 lines were sown on soil and grown as described above. When the T2 plants had obtained at least four true leaves, a single leaf was harvested separately for each plant. RNA was extracted from this tissue and analyzed for PR-1 and NIM1 expression. Plants were then inoculated with P. parasitica (Emwa) and analyzed for fungal growth at 3-14 days, preferably 7-12 days, following infection. Plants showing higher than normal NIM1 and PR-1 expression and displaying fungal resistance demonstrated that overexpression of NIM1 confers a CIM phenotype.
  • P. parasitica Emwa
  • Table 6 shows the results of testing of various transformants for resistance to fungal infection. As can be seen from the table, a number of transformants showed less than normal fungal growth and several showed no visible fungal growth at all.
  • RNA was prepared from collected samples and analyzed as previously described (Delaney et al, 1995). Blots were hybridized to the Arabidopsis gene probe PR-1 (Uknes et al, 1992). Lines D7-74, D5-6 and E1-1 showed early induction of PR-1 gene expression, whereby PR- 1 mRNA was evident by 24 or 48 hours following fungal treatment. These three lines also demonstrated resistance to fungal infection.
  • Plants constitutively expressing the NIM1 gene were generated from transformation of Ws wild type plants with the BamHI-Hindlll NIM1 genomic fragment (SEQ ID NO: 2 - bases 1249-5655) containing 1.4 kb of promoter sequence. This fragment was cloned into pSGCGOI and transformed into the Agrobacterium strain GV3101 (pMP90, Koncz and Schell (1986) Mol. Gen. Genet. 204:383-396). Ws plants were infiltrated as previously described. The resulting seed was harvested and plated on GM agar containing 50 ⁇ g/ml kanamycin. Surviving plantlets were transferred to soil and tested as described above for resistance to Peronospora parasitica isolate Emwa.
  • Selected plants were selfed and selected for two subsequent generations to generate homozygous lines. Seeds from several of these lines were sown in soil and 15-18 plants per line were grown for three weeks and tested again for Emwa resistance without any prior treatment with an inducing chemical. Approximately 24 hours, 48 hours, and five days after fungal treatment, tissue was harvested, pooled and frozen for each line. Plants remained in the growth chamber until ten days after inoculation when they were scored for resistance to Emwa.
  • the full-length NIM1 cDNA (SEQ ID NO: 21) was cloned into the EcoRI site of pCGN1761 ENX (Comai et al. (1990) Plant Mol. Biol. 15, 373-381). From the resulting plasmid, an Xbal fragment containing an enhanced CaMV 35S promoter, the NIM1 cDNA in the correct orientation for transcription, and a tml 3' terminator was obtained. This fragment was cloned into the binary vector pCIB200 and transformed into GV3101. Ws plants were infiltrated as previously described. The resulting seed was harvested and plated on GM agar containing 50 ⁇ g/ml kanamycin.
  • NIM1 gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, 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.
  • crop plant cells such as rice, wheat, barley, rye, 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,
  • the expression of the NIM1 gene is at a level which is at least two-fold above the expression level of the NIM1 gene in wild type plants and is preferably ten-fold above the wild type expression level.
  • Example 22 The Use of nim Mutants in Disease Testing
  • nim mutants are challenged with numerous pathogens and found to develop larger lesions more quickly than wild-type plants.
  • This phenotype is referred to as UDS (i.e. universal disease susceptibility) and is a result of the mutants failing to express SAR genes to effect the plant defense against pathogens.
  • UDS i.e. universal disease susceptibility
  • the UDS phenotype of nim mutants renders them useful as control plants for the evaluation of disease symptoms in experimental lines in field pathogenesis tests where the natural resistance phenotype of so-called wild type lines may vary (i.e. to different pathogens and different pathotypes of the same pathogen).
  • nim mutants are used as host plants for the transformation of transgenes to facilitate their assessment for use in disease resistance.
  • an Arabidopsis nim mutant line characterized by its UDS phenotype, is used for subsequent transformations with candidate genes for disease resistance thus enabling an assessment of the contribution of an individual gene to resistance against the basal level of the UDS nim mutant plants.
  • nim mutants are useful for the understanding of plant pathogen interactions, and in particular for the understanding of the processes utilized by the pathogen for the invasion of plant cells. This is so because nim mutants do not mount a systemic response to pathogen attack, and the unabated development of the pathogen is an ideal scenario in which to study its biological interaction with the host. Of futher significance is the observation that a host nim mutant may be susceptible to pathogens not normally associated with that particular host, but instead associated with a different host. For example, an Arabidopsis nim mutant such as nim1-1, -2, -3, -4, -5, or -6 is challenged with a number of pathogens that normally only infect tobacco, and found to be susceptible. Thus, the nim mutation causing the UDS phenotype leads to a modification of pathogen-range susceptibility and this has significant utility in the molecular, genetic and biochemical analysis of host-pathogen interaction.
  • nim mutants are particularly useful in the screening of new chemical compounds for fungicide activity
  • nim mutants selected in a particular host have considerable utility for the screening of fungicides using that host and pathogens of the host.
  • the advantage lies in the UDS phenoytpe of the mutant that circumvents the problems encountered by the host being differentially susceptible to different pathogens and pathotypes, or even resistant to some pathogens or pathotypes.
  • nim mutants in wheat could be effectively used to screen for fungicides to a wide range of wheat pathogens and pathotypes as the mutants would not mount a resistance response to the introduced pathogen and would not display differential resistance to different pathotypes that might otherwise require the use of multiple wheat lines, each adequately susceptible to a particular test pathogen.
  • Wheat pathogens of particular interest include (but are not limited to) Erisyphe graminis (the causative agent of powdery mildew), Rhizoctonia solani (the causative agent of sharp eyespot), Pseudocercosporella herpotrichoides (the causative agent of eyespot), Puccinia spp.
  • nim mutants of corn would be highly susceptible to corn pathogens and therefore useful in the screening for fungicides with activity against corn diseases.
  • nim mutants have further utility for the screening of a wide range of pathogens and pathotypes in a heterologous host i.e. in a host that may not normally be within the host species range of a particular pathogen and that may be particularly easily to manipulate (such as Arabidopsis).
  • a heterologous host i.e. in a host that may not normally be within the host species range of a particular pathogen and that may be particularly easily to manipulate (such as Arabidopsis).
  • the heterologous host is susceptible to pathogens of other plant species, including economically important crop plant species.
  • the same Arabidopsis nim mutant could be infected with a wheat pathogen such as Erisyphe graminis (the causative agent of powdery mildew) or a corn pathogen such as Helminthosporium maydis and used to test the efficacy of fungicide candidates.
  • a wheat pathogen such as Erisyphe graminis (the causative agent of powdery mildew) or a corn pathogen such as Helminthosporium maydis and used to test the efficacy of fungicide candidates.
  • NIM1 and IkB A multiple sequence alignment between the protein gene products of NIM1 and IkB was performed by which it was determined that the NIM1 gene product is a homolog of IKB ⁇ ( Figure 9). Sequence homology searches were performed using BLAST (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The multiple sequence alignment was constructed using Clustal V (Higgins et al., CABIOS 5, 151 -153 (1989)) as part of the Lasergene Biocomputing Software package from DNASTAR (Madison, Wl).
  • NIM1 SEQ ID NO:3
  • mouse l ⁇ B ⁇ SEQ ID NO:18, GenBank Accession #: 1022734
  • rat IKBCC SEQ ID NO:19, GenBank accession Nos. 57674 and X63594; Tewari et al.
  • NIM1 contains 2 serines at amino acid positions 55 and 59, the serine at position 59 is in a context (D/ExxxxxS) and position (N-terminal) consistent with a role in phosphorylation-dependent, ubiquitin-mediated, inducible degradation. All IKBS have these N-terminal serines and they are required for inactivation of IKB and subsequent release of NF- ⁇ B. NIM1 has ankyrin domains (amino acids 262-290 and 323-371).
  • NIM1 has some homology to a QL-rich region (amino acids 491-499) found in the C-termini of some l ⁇ Bs.
  • This altered form of IKBCC functions as a dominant negative form by retaining NF- ⁇ B in the cytoplasm, thereby blocking downstream signaling events.
  • serines 55 (S55) and 59 (S59) of NIM1 are homologous to S32 and S36 in human l ⁇ B ⁇ .
  • the serines at amino acid positions 55 and 59 are mutagenized to alanine residues. This can be done by any method known to those skilled in the art, such as, for example, by using the QuikChange Site Directed Mutagenesis Kit (#200518:Strategene).
  • the mutagenized construct can be made per the manufacturer's instructions using the following primers (SEQ ID NO:21 , positions I92-226): 5'-CAA CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3' (SEQ ID NO:32) and 5'- CAT CCG GCG CGT CAA AGA CGG CTT CGA AGC TGT TG-3' (SEQ ID NO:33), where the underlined bases denote the mutations.
  • the strategy is as follows: The NIM1 cDNA cloned into vector pSE936 (Elledge et al., Proc. Nat. Acad. Sci. USA 88:1731-1735 (1991)) is denatured and the primers containing the altered bases are annealed. DNA polymerase (Pfu) extends the primers by nonstrand-displacement resulting in nicked circular strands. DNA is subjected to restriction endonuclease digestion with Dpnl, which only cuts methylated sites (nonmutagenized template DNA). The remaining circular dsDNA is transformed into E.coli strain XL1-Blue.
  • Plasmids from resulting colonies are extracted and sequenced to verify the presence of the mutated bases and to confirm that no other mutations occurred.
  • the mutagenized NIM1 cDNA is digested with the restriction endonuclease EcoRI and cloned into pCGN1761 under the transcriptional regulation of the double 35S promoter of the cauliflower mosaic virus.
  • the transformation cassette including the 35S promoter, NIM1 cDNA and tml terminator is released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal and ligated into the Xbal site of dephosphorylated pCIB200.
  • SEQ ID NO's:22 and 23 show the DNA coding sequence and encoded amino acid sequence, respectively, of this altered form of the NIM1 gene.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:22: hybridization in 1%BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO: 22 under these conditions are altered so that the encoded product has alanines instead of serines in the amino acid positions that correspond to positions 55 and 59 of SEQ ID NO: 22.
  • NIM1 form may be generated in which DNA encoding approximately the first 125 amino acids is deleted.
  • the following primers produce a 1612- bp PCR product (SEQ ID NO:21 : 418 to 2011): 5'-gg aat tca-ATG GAT TCG GTT GTG ACT GTT TTG-3' (SEQ ID NO:34) and 5'-gga att cTA CAA ATC TGT ATA CCA TTG G-3' (SEQ ID NO:35) in which the synthetic start codon is underlined (ATG) and EcoRI linker sequence is in lower case.
  • Amplification of fragments utilizes a reaction mixture comprising 0.1 to 100 ng of template DNA, 10mM Tris pH 8.3/50mM KCI/2 mM MgCI 2 /0.001 % gelatin/0.25 mM each dNTP/0.2 mM of each primer and 1 unit rTth DNA polymerase in a final volume of 50 mL and a Perkin Elmer Cetus 9600 PCR machine.
  • PCR conditions are as follows: 94°C 3min: 35x (94°C 30 sec: 52°C 1 min: 72°C 2 min): 72°C 10 min.
  • the PCR product is cloned directly into the pCR2.1 vector (Invitrogen).
  • the PCR-generated insert in the PCR vector is released by restriction endonuclease digestion using EcoRI and ligated into the EcoRI site of dephosphorylated pCGN1761 , under the transcriptional regulation of the double 35S promoter.
  • the construct is sequenced to verify the presence of the synthetic starting ATG and to confirm that no other mutations occurred during PCR.
  • the transformation cassette including the 35S promoter, modified NIM1 cDNA and tml terminator is released from pCGN1761ENX by partial restriction digestion with Xbal and ligated into the Xbal site of pCIB200.
  • SEQ ID NO's:24 and 25 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having an N-terminal amino acid deletion.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:24: hybridization in 1%BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:24 under these conditions are altered so that the encoded product has an N-terminal deletion that removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product.
  • the deletion of amino acids 261-317 of human l ⁇ B ⁇ is believed to result in enhanced intrinsic stability by blocking the constitutive phosphorylation of serine and threonine residues in the C-terminus.
  • a region rich in serine and threonine is present at amino acids 522-593 in the C-terminus of NIML
  • the C-terminal coding region of the NIM1 gene may be modified by deleting the nucleotide sequences which encode amino acids 522-593. Using the method of Ho et al.
  • PCR reaction components are as previously described and cycling parameters are as follows: 94°C 3 min: 30x (94°C 30 sec: 52°C 1 min: 72°C 2 min); 72°C 10 min].
  • the PCR product is cloned directly into the pCR2.1 vector (Invitrogen).
  • the PCR-generated insert in the PCR vector is released by restriction endonuclease digestion using EcoRI and ligated into the EcoRI site of dephosphorylated pCGN1761 , which contains the double 35S promoter.
  • the construct is sequenced to verify the presence of the synthetic in-frame stop codon and to confirm that no other mutations occurred during PCR.
  • the transformation cassette including the promoter, modified NIM1 cDNA, and tml terminator is released from pCGN1761 by partial restriction digestion with Xbaland ligated into the Xbal site of dephosphorylated pCIB200.
  • SEQ ID NO's:26 and 27 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having a C-terminal amino acid deletion.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:26: hybridization in 1%BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1 ) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:26 under the above conditions are altered so that the encoded product has a C-terminal deletion that removes serine and threonine residues.
  • NIM1 NIM1 is generated using a unique Kpnl restriction site at position 819 (SEQ ID NO:21).
  • the N-terminal deletion form (Example 28) is restriction endonuclease digested with EcoRI/Kpnl and the 415 bp fragment corresponding to the modified N-terminus is recovered by gel electrophoresis.
  • the C-terminal deletion form (Example 29) is restriction endonuclease digested with EcoRI/Kpnl and the 790 bp fragment corresponding to the modified C-terminus is recovered by gel electrophoresis.
  • the fragments are ligated at 15°C, digested with EcoRI to eliminate EcoRI concatemers and cloned into the EcoRI site of dephosphorylated pCGN1761.
  • the N/C- terminal deletion form of NIM1 is under the transcriptional regulation of the double 35S promoter.
  • a chimeric form of NIM1 is generated which consists of the S55/S59 mutagenized putative phosphorylation sites (Example 27) fused to the C-terminal deletion (Example 29).
  • the construct is generated as described above. The constructs are sequenced to verify the fidelity of the start and stop codons and to confirm that no mutations occurred during cloning.
  • the respective transformation cassettes including the 35S promoter, NIM1 chimera and tml terminator are released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal site of dephosphorylated pCIB200.
  • SEQ ID NO's:28 and 29 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having both N-terminal and C- terminal amino acid deletions.
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:28: hybridization in 1%BSA; 520mM NaP ⁇ 4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:28 under the above conditions are altered so that the encoded product has both an N-terminal deletion, which removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product, as well as a C-terminal deletion, which removes serine and threonine residues.
  • NIM1 exhibits homology to ankyrin motifs at approximately amino acids 103-362.
  • the DNA sequence encoding the putative ankyrin domains (SEQ ID NO:2: 3093-3951) is PCR amplified (conditions: 94°C 3 min:35x (94°C 30 sec: 62°C 30 sec: 72°C 2 min): 72°C 10 min) from the NIM1 cDNA (SEQ ID NO:21: 349- 1128) using the following primers: 5'-ggaattcaATGGACTCCAACAACACCGCCGC-3' (SEQ ID NO:38) and 5' ggaattcICAACCTTCCAAAGTTGCTTCTGATG-3' (SEQ ID NO:39).
  • the resulting product is restriction endonuclease digested with EcoRI and then spliced into the EcoRI site of dephosphorylated pCGN1761 under the transcriptional regulation of the double 35S promoter.
  • the construct is sequenced to verify the presence of the synthetic start codon (ATG), an in-frame stop codon (TGA) and to confirm that no other mutations occurred during PCR.
  • the transformation cassette including the 35S promoter, ankyrin domains, and tml terminator is released from pCGN1761 by partial restriction digestion with Xbal and ligated into the XbalsWe of dephosphorylated pCIB200.
  • SEQ ID NO's:30 and 31 show the DNA coding sequence and encoded amino acid sequence, respectively, of the ankyrin domain of NIML
  • the present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:30: hybridization in 1%BSA; 520mM NaP04, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.
  • alleles of NIM1 hybridizing to SEQ ID NO:30 under the above conditions are altered so that the encoded product consists essentially of the ankyrin domains of the wild- type gene product.
  • Example 32 Construction Of Chimeric Genes
  • a 4407 bp Hindlll/BamHI fragment (SEQ ID NO:2: bases 1249-5655) and/or a 5655 bp EcoRV/BamHI fragment (SEQ ID NO:2: bases 1-5655) containing the NIM1 promoter and gene is used for the creation of the altered NIM1 forms in Examples 27-31 above.
  • the construction steps may differ, the concepts are comparable to the examples previously described herein. Strong overexpression of the altered forms may potentially be lethal.
  • the altered forms of the NIM1 gene described in Examples 27-31 may be placed under the regulation of promoters other than the endogenous NIM1 promoter, including but not limited to the nos promoter or small subunit of Rubisco promoter.
  • the altered NIM1 forms may be expressed under the regulation of the pathogen-responsive promoter PR-1 (U.S. Pat. No. 5,614,395). Such expression permits strong expression of the altered NIM1 forms only under pathogen attack or other SAR- activating conditions.
  • constructs generated are moved into Agrobacterium tumefaciens by electroporation into strain GV3101. These constructs are used to transform Arabidopsis ecotypes Col-0 and Ws-0 by vacuum infiltration (Mindrinos et al., Cell 78, 1089- 1099 (1994)) or by standard root transformation. Seed from these plants is harvested and allowed to germinate on agar plates with kanamycin (or another appropriate antibiotic) as selection agent. Only plantlets that are transformed with cosmid DNA can detoxify the selection agent and survive. Seedlings that survive the selection are transferred to soil and tested for a CIM (constitutive immunity) phenotype. Plants are evaluated for observable phenotypic differences compared to wild type plants. • Example 34: Assessment Of CIM Phenotype In Plants Transformed With Altered Forms Of
  • RNA is isolated (Verwoerd et al., 1989, Nuc Acid Res, 2362) and tested for constitutive PR-1 expression by RNA blot analysis (Uknes et al., 1992). Each transformant is evaluated for an enhanced disease resistance response indicative of constitutive SAR expression analysis (Uknes et al., 1992).
  • Conidial suspensions of 5-10x10 4 spores/ml from two compatible P. parasitica isolates, Emwa and Noco i.e. these fungal strains cause disease on wildtype Ws-0 and Col-0 plants, respectively
  • transformants are sprayed with the appropriate isolate depending on the ecotype of the transformant.
  • Inoculated plants are incubated under high humidity for 7 days. Plants are disease rated at day 7 and a single leaf is harvested for RNA blot analysis utilizing a probe which provides a means to measure fungal infection. Transformants that exhibit a CIM phenotype are taken to the T1 generation and homozygous plants are identified. Transformants are subjected to a battery of disease resistance tests as described below. Fungal infection with Noco and Emwa is repeated and leaves are stained with lactophenol blue to identify the presence of fungal hyphae as described in Dietrich et al., (1994).
  • Transformants are infected with the bacterial pathogen Pseudomonas syringae DC3000 to evaluate the spectrum of resistance evident as described in Uknes et al. (1993). Uninfected plants are evaluated for both free and glucose-conjugated SA and leaves are stained with lactophenol blue to evaluate for the presence of microscopic lesions. Resistant plants are sexually crossed with SAR mutants such as NahG (U.S. Pat. No. 5,614,395) and ⁇ dr7 to establish the epistatic relationship of the resistance phenotype to other mutants and evaluate how these dominant-negative mutants of NIM1 may influence the SA-dependent feedback loop.
  • SAR mutants such as NahG (U.S. Pat. No. 5,614,395) and ⁇ dr7 to establish the epistatic relationship of the resistance phenotype to other mutants and evaluate how these dominant-negative mutants of NIM1 may influence the SA-dependent feedback loop.
  • NIM1 cDNA SEQ ID NO:21
  • homologs of Arabidopsis NIM1 are identified through screening genomic or cDNA libraries from different crops such as, but not limited to those listed below in Example 36. Standard techniques for accomplishing this include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g. Sambrook et al., Molecular Cloning , eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oiigonucleotide primers (see, e.g. Innis etal., PCR
  • Homologs identified are genetically engineered into the expression vectors herein and transformed into the above listed crops. Transformants are evaluated for enhanced disease resistance using relevant pathogens of the crop plant being tested.
  • NIM1 homologs in the genomes of cucumber, tomato, tobacco, maize, wheat and barley have been detected by DNA blot analysis.
  • Genomic DNA was isolated from cucumber, tomato, tobacco, maize, wheat and barley, restriction digested with the enzymes BamHI, Hindlll, Xbal, or Sail, electrophoretically separated on 0.8% agarose gels and transferred to nylon membrane by capillary blotting.
  • the membrane was hybridized under low stringency conditions [(1 %BSA; 520mM NaP0 4 , pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 55°C for 18-24h] with 32 P-radiolabelled Arabidopsis thaliana NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min. (X3) 3XSSC for 15 min.
  • expressed sequence tags identified with similarity to the NIM1 gene can be used to isolate homologues.
  • EST expressed sequence tags
  • a multiple sequence alignment was constructed using Clustal V (Higgins, Desmond G. and Paul M. Sharp (1989), Fast and sensitive multiple sequence alignments on a microcomputer, CABIOS 5:151-153) as part of the DNA * (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994).
  • NIM1 protein is homologous in amino acid sequence to 4 different rice cDNA protein products.
  • the homologies were identified using the NIM1 sequences in a GenBank BLAST search. Comparisons of the regions of homology in NIM1 and the rice cDNA products are shown in Figure 8 (See also, SEQ ID NO:3 and SEQ ID NO's:4-11).
  • the NIM1 protein fragments show from 36 to 48% identical amino acid sequences with the 4 rice products. These rice EST's may be especially useful for isolation of NIM1 homologues from other monocots.
  • Homologues may be obtained by PCR. In this method, comparisons are made between known homologues (e.g., rice and Arabidopsis). Regions of high amino acid and DNA similarity or identity are then used to make PCR primers. Regions rich in amino acid residues M and W are best followed by regions rich in amino acid residues F, Y, C, H, Q, K and E because these amino acids are encoded by a limited number of codons. Once a suitable region is identified, primers for that region are made with a diversity of substitutions in the 3 rd codon position. This diversity of substitution in the third position may be constrained depending on the species that is being targeted.
  • known homologues e.g., rice and Arabidopsis.
  • primers are designed that utilize a G or a C in the 3 rd position, if possible.
  • the PCR reaction is performed from cDNA or genomic DNA under a variety of standard conditions. When a band is apparent, it is cloned and/or sequenced to determine if it is a NIM1 homologue.
  • Those constructs conferring a CIM phenotype in Col-0 or Ws-0 are transformed into crop plants for evaluation.
  • altered native NIMI genes isolated from crops in the preceding example are put back into the respective crops.
  • the NIM1 gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, 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.
  • the expression of the altered form of the NIM1 gene is at a level which is at least two-fold above the expression level of the native NIM1 gene in wild type plants and is preferably ten-fold above the wild type expression level.
  • the CIC library a large insert YAC library for genome mapping in Arabidopsis thaliana. Plant J. 8, 763-770.
  • Mindrinos M., Katagiri, F., Yu, G. L. and Ausubel, F. (1994)
  • the Arabidopsis thaliana disease resistance gene rps2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats. Cell 78, 1089-1099.
  • Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal reduction. Plant Cell 6, 959-965.
  • AFLP a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407-4414.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • AACCCACTCT AACAGCAGAG TTGAAAAGTT TGGTGACATG CTTAAAACTT CAAAGCTGCG 360
  • CTGCATTTCA CTCATCTAAT GGGCTACTTG TGGACTGCAA TATGAGCTTT TCCCTAATCC 480
  • AAACTTCG CACGCAAAAG TTCTGAGATT CCGAGTCATA CCAGGCGATT TCGAAAGCCT 8520
  • ATCTCTCCTC TCATGGAAAA AACTGGTATC AAGTTTGTAT CCTCTTTCGT AGCGTTCTAG 8820
  • GTGTTTCCTT TTCAATCAAC ATCCATTTTC TTTAAAAATT AGCAAGTTTG TTCTTATATC 9480 ATCATTCAGC AGATTTCTTA ATTAAACTTA GTGATTTCCA TTTTGCACCT ATATGTTTCT 9540
  • MOLECULE TYPE DNA (genomic)
  • GGT GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 4866
  • AAAAGAATAT TCAAGTTCCC TGAACTTCTG GCAACATTCA TGTTATATGT ATCTTCCTAA 5226
  • Phe Lys lie Pro Glu Leu lie Thr Leu Tyr Gin Arg His Leu Leu Asp 180 185 190
  • Val Val Asp Lys Val Val lie Glu Asp Thr Leu Val lie Leu Lys Leu 195 200 205
  • Glu lie lie Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser 225 230 235 240
  • Glu lie Ala Glu Met Lys Gly Thr Cys Glu Phe lie Val Thr Ser Leu 450 455 460

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Abstract

L'invention concerne la localisation et la caractérisation d'un gène (appelé NIM1). Ledit gène est un élément clé de la voie SAR qui, combiné avec des inducteurs chimiques et biologiques, permet d'induire chez des plantes l'expression du gène SAR et une résistance à un large éventail de maladies. Le produit génique NIM1 est un homologue structurel du facteur (sous-classe α de IλB) de transduction de signal chez le mammifère. On exploite cette découverte de l'invention pour fournir des formes modifiées de NIM1 agissant comme régulateurs négatifs dominants de la voie de transduction du signal de l'immunisation systémique acquise (SAR). Ces formes modifiées de NIM1 confèrent le phénotype opposé comme le mutant nim1 chez les plantes transformées avec les formes modifiées de NIM1, par exemple les plantes transgéniques montrent une expression du gène constitutif SAR et un phénotype d'immunité constitutive (CIM). L'invention concerne aussi des vecteurs de transformation et des procédés de surexpression du gène NIM1 chez des plantes. Les plantes transgéniques ainsi créées présentent une résistance à un large éventail de maladies. L'invention concerne aussi des molécules d'ADN codant des formes modifiées du gène NIM1, des vecteurs d'expression contenant lesdites molécules d'ADN, ainsi que des plantes et cellules de plantes ainsi modifiées. L'invention concerne aussi des vecteurs de transformation et des procédés de surexpression du gène NIM1 chez des plantes. On décrit des vecteurs et des procédés pour induire une surexpression du gène NIM1 chez des plantes. L'invention concerne en outre des procédés pour activer l'immunisation systémique acquise chez des plantes et pour conférer à des plantes un phénotype CIM et une résistance à un large éventail de maladies, par modification des plantes avec des molécules codant des formes modifiées du produit génique NIM1.
EP97952940A 1996-12-13 1997-12-12 PROCEDE D'UTILISATION DU GENE $i(NIM1) POUR CONFERER A DES PLANTES UNE RESISTANCE AUX MALADIES Withdrawn EP0944728A1 (fr)

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US3317796P 1996-12-13 1996-12-13
US33177P 1996-12-13
US3437996P 1996-12-27 1996-12-27
US3438296P 1996-12-27 1996-12-27
US34379P 1996-12-27
US34382P 1996-12-27
US3502297P 1997-01-10 1997-01-10
US3502197P 1997-01-10 1997-01-10
US3473097P 1997-01-10 1997-01-10
US35021P 1997-01-10
US34730P 1997-01-10
US35022P 1997-01-10
US08/880,179 US6091004A (en) 1996-06-21 1997-06-20 Gene encoding a protein involved in the signal transduction cascade leading to systemic acquired resistance in plants
PCT/EP1997/007012 WO1998026082A1 (fr) 1996-12-13 1997-12-12 Procede d'utilisation du gene nim1 pour conferer a des plantes une resistance aux maladies
US880179 2004-06-29

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HUP0104392A3 (en) * 1996-08-09 2003-12-29 Univ Durham Acquired resistance npr genes and uses thereof
TR199901491T2 (xx) * 1996-12-27 1999-08-23 Novartis Ag Bitkileri korumak i�in y�ntem.
JP2001516588A (ja) 1997-09-15 2001-10-02 インスティテュート・オブ・モレキュラー・アグロバイオロジー 耐病害性に関連したイネ由来アンキリン反復体含有ペプチドであるｒａｎｋ１
US6528702B1 (en) 1999-03-09 2003-03-04 Syngenta Participations Ag Plant genes and uses thereof
TR200102718T2 (tr) * 1999-03-09 2002-03-21 Syngenta Participations Ag Yeni bitki genleri ve bunlarìn kullanìmlarì
US6504084B1 (en) 1999-04-23 2003-01-07 Pioneer Hi-Bred International, Inc. Maize NPR1 polynucleotides and methods of use
CA2372654A1 (fr) 1999-05-13 2000-11-23 Monsanto Technology Llc Gene de resistance acquise dans des plantes
US6706952B1 (en) 1999-12-15 2004-03-16 Syngenta Participations Ag Arabidopsis gene encoding a protein involved in the regulation of SAR gene expression in plants
US7199286B2 (en) 1999-12-15 2007-04-03 Syngenta Participations Ag Plant-derived novel pathogen and SAR-induction chemical induced promoters, and fragments thereof
US20020042113A1 (en) * 1999-12-21 2002-04-11 Crane Edmund H. NPR1-interactors and methods of use
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
US20090119793A1 (en) 2005-01-26 2009-05-07 Washington State University Research Foundation Plant Defense Signal Peptides
EP2802215B1 (fr) 2012-01-11 2021-09-15 The Australian National University Procédé pour moduler l'architecture racinaire d'une plante
AR098055A1 (es) 2013-10-16 2016-04-27 The Australian Nat Univ Método para modular el crecimiento de las plantas
CN115011567A (zh) * 2022-06-24 2022-09-06 安徽农业大学 (+)-新薄荷醇合酶、合酶基因及其在茶树抗病中的应用

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