Classifications

• • 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
• • 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
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
  • A01N43/82—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with three ring hetero atoms
• • 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
  • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
• • 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]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

• the present invention relates to a method for protecting a plant against pathogen attack through synergistic disease-resistance attained by applying a microbicide to an immunomodulated plant.
• 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 phloe 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. Plants that cannot accumulate SA due to treatment with specific inhibitors, epigenetic repression of phenylalanine ammonia-lyase, or transgenic expression of salicylate hydroxylase, which specifically degrades SA, also cannot induce either SAR gene expression or disease resistance (Gaffney et al., 1993; Delaney et al., 1994; Mauch-Mani and Slusarenko 1996; Maher et al., Proc. Natl. Acad. Sci. USA 91, 7802-7806 (1994), incorporated by reference herein in its entirety; Pallas et al., Plant J. 10, 281-293 (1996), incorporated by reference herein in its entirety).
• 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
• dm mutants Like Isd mutants and acd ⁇ , dm mutants have elevated SA and SAR gene expression and resistance, but in contrast to Isd or acd ⁇ , do not display detectable lesions on their leaves, cprl (constitutive expresser of PR genes) may be a type of dm mutant; however, because the presence of microscopic lesions on the leaves of cprl has not been ruled out, 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 ⁇ , 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).
• niml noninducible immunity
• P. parasitica i.e., causal agent of downy mildew disease
• INA treatment i.e., INA treatment
• niml 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.
• niml 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 niml and nprl phenotypes, respectively (Ryals etal., Plant Cell 9, 425-439 (1997), incorporated by reference herein in its entirety; Cao etal., 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 etal., 1997).
• Ryals etal., 1997 also report the isolation of five additional alleles of niml that show a range of phenotypes from weakly impaired in chemically induced PR-1 gene expression and fungal resistance to very strongly blocked. Transformation of the wild-type NPR1 gene into nprl mutants not only complemented the mutations, restoring the responsiveness of SAR induction with respect to PR-gene expression and disease resistance, but also rendered the transgenic plants more resistant to infection by P. syringae in the absence of SAR induction (Cao et al., 1997).
• NF- ⁇ B/l ⁇ B signal transduction 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, Cell 87, 13-20 (1996); Baldwin, Annu. Rev. Immunol. 14, 649-681 (1996)).
• IL-1 interleukin 1
• 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; Gaf ney 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 Arabidopsis.
• Drosophiia Bacterial infection of Drosophiia 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.
• a preferred aspect of the present invention pertains to a novel method of protecting plants from pathogen attack through synergistic disease resistance attained by applying a microbicide to immunomodulated plants.
• Immunomodulated plants are those in which SAR is activated, typically exhibiting greater-than-wild-type SAR gene expression, and are therefore referred to as "SAR-on" plants.
• Immunomodulated plants for use in the method of the invention may be obtained in at least three different ways: by applying to plants a chemical inducer of SAR such as BTH, INA, or SA; through a selective breeding program in which plants are selected based on constitutive expression of SAR genes and or a disease-resistant phenotype; or by genetically engineering plants by transforming them with one or more SAR genes such as a functional form of the NIM1 gene.
• the microbicide applied to the immunomodulated plants may be either a conventional microbicide such as the fungicide metalaxyl or, if applied to immunomodulated plants obtained through selective breeding or genetic engineering, the microbicide may be a chemical inducer of SAR such as BTH, INA, or SA.
• Immunomodulation provides a certain level of disease resistance in a plant.
• application of a microbicide to a plant provides a certain level of disease resistance.
• the expected result of combining immunomodulation with microbicide application would be a level of control reflecting the additive levels of control provided by the individual methods of providing disease resistance.
• the disease resistance is unexpectedly synergisticaliy enhanced; i.e., the level of disease resistance is greater than the expected additive levels of disease resistance.
• the present invention concerns the cultivation of immunomodulated plants and the application of a suitable amount of a conventional microbicide thereto.
• Especially preferred embodiments of the invention concern plants genetically engineered to contain and express a functional form of the NIM1 gene or a homologue or variant thereof.
• the method of the invention results in greater pathogen control than is achieved through either immunomodulation or microbicide application alone.
• Immunomodulation provides a certain level of disease resistance in a plant.
• application of a microbicide to a plant provides a certain level of disease resistance.
• the expected result of combining immunomodulation with microbicide application would be a level of control reflecting the additive levels of control provided by the individual methods of providing disease resistance.
• the control of pathogenic disease is unexpectedly synergisticaliy enhanced; i.e., the level of disease control is greater than the expected additive levels of disease resistance.
• another advantage of the present invention is that less microbicide is required to achieve the level of disease resistance provided by the method of the invention than is required for use with ordinary, wild-type plants.
• the result of this is both lower economic costs of microbicide, as well as less chance of adverse environmental consequences resulting from toxicity of some microbicides.
• the inventive method of protecting plants by combining the effects of immunomodulation and application of a microbicide results in a longer duration of antipathogenic action and altogether higher crop yields.
• Another advantage of this method is that because the two combined modes of action of pathogen control are completely different from one another, the threat of resistance developing is effectively prevented.
• the present invention relates to a method for protecting a plant from pathogen attack through synergistic disease resistance, comprising the steps of:
• dm mutant plant is selected from a population of plants according to the following steps:
• said immunomodulated plant is a lesion mimic mutant plant.
• said lesion mimic mutant plant is selected from a population of plants according to the following steps:
• immunomodulated plant is obtained by recombinant expression in a plant of an SAR gene.
• SAR gene is a functional form of a NIM1 gene. More preferred is method according to the invention, wherein said NIM1 gene encodes a NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants.
• NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO:2.
• NIM1 gene hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:1 : 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.
• NIM1 gene comprises the coding sequence set forth in SEQ ID NO:1 and all DNA molecules hybridizing therewith using moderate stringent conditions.
• SAR gene encodes an altered form of a NIM1 protein that acts as a dominant-negative regulator of the SAR signal transduction pathway.
• said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:8.
• said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:7 and all DNA molecules hybridizing therewith using moderate stringent conditions.
• a method according to the invention wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:7: 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.
• said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:10.
• said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:9 and all DNA molecules hybridizing therewith using moderate stringent conditions.
• a method according to the invention wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:9: 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.
• said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:12.
• said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:11 and all DNA molecules hybridizing therewith using moderate stringent conditions.
• a method according to the invention wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:11 : 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.
• said altered form of the NIM1 protein 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:2.
• said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:14.
• said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:13 and all DNA molecules hybridizing therewith using moderate stringent conditions.
• a method according to the invention wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:13: 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.
• said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID NO:2.
• said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:16.
• said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:15 and all DNA molecules hybridizing therewith using moderate stringent conditions.
• a method according to the invention wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:15: 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.
• target crops for the areas of indication disclosed herein comprise, without limitation, the following species of plants: cereals (maize, wheat, barley, rye, oats, rice, sorghum and related crops); beet (sugar beet and fodder beet); pomes, stone fruit and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries); leguminous plants (beans, lentils, peas, soybeans); oil plants (rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts); cucumber plants (marrows, cucumber, melons); fibre plants (cotton, flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit, mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, paprika); lauraceae (avocados, cinnamon, camphor); or plants such as tobacco, nuts, coffee, sugar can
• the method of the present invention can be used to confer resistance to a wide array of plant pathogens, which include, but are not limited to the following: viruses or viroids such as 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; Ascomycete fungi such as of the genera Venturia, Podosphaera, Erysiphe, Monolinia, Mycosphaerella, and Uncinula; Basidiomycete fungi such as from the genera Hemileia, Rhizoctonia, and P ⁇ ccinia; Fungi imperfecti such as the genera Botrytis, Helminthosporium, Rhynchosporium, Fusarium (i.e., F.
• viruses or viroids such as 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
• monoliforme monoliforme
• Septoria Cercospora
• Alternaria Pyricularia
• Pseudocercosporella i.e., P. herpotrichoides
• Oomycete fungi such as from the genera Phytophthora (i.e., P. parasitica), Peronospora (i.e, P.
• fungi such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora saccha ⁇ and Peronosclerospora maydis, Physopella zeae, Cercospora zeae-maydis, Colletotrichum graminicola, Gibberella zeae, Exserohilum turcicum, Kabatiellu zeae, and Bipolaris maydis; bacteria such as Pseudomonas syringae, Pseudomonas tabaci, and Erwinia stewartir, insects such as aphids, e.g. Myzus persicae; and lepidoptera such as Heliothus s
• a first route for obtaining immunomodulated plants involves applying to a plant a chemical capable of inducing SAR.
• Particularly potent chemical inducers of SAR are benzothiadiazoles such as benzo[1 ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (BTH).
• BTH benzothiadiazoles
• Derivatives of benzothiadiazoles that may further be used as regulators are described in U.S. Patent Nos. 5,523,311 and 5,614,395, both of which are incorporated herein by reference.
• BTH-induced SAR which supplies protection in the field against a broad spectrum of diseases in a variety of crops is described in detail in Freidrich et al., Plant Journal 10(1), 61-70 (1996); Lawton et al., Plant Journal 10(1), 71-82 (1996); and Goriach et al., Plant Cell 8, 629-643 (1996), each of which is inco ⁇ orated herein by reference.
• Other chemical inducers of SAR that may be used to obtain an immunomodulated plant for use in the method of the invention include isonicotinic acid compounds such as 2,6- dichloroisonicotinic acid (INA) and the lower alkyl esters thereof, as well as salicylic acid compounds (SA). Examples of suitable INA and SA compounds are described in U.S. Patent No. 5,614,395.
• a second route for obtaining immunomodulated plants is through a selective breeding program based on constitutive expression of SAR genes and or a disease-resistant phenotype.
• Considerable data shows a tight correlation between the expression of SAR genes and systemic acquired resistance itself (Ward et al. (1991); Uknes et al. (1992); Uknes et al. (1993); Lawton, et al. (1993); and Alexander et al. (1993) PNAS USA 90, 7327-7331, herein inco ⁇ orated by reference.
• examples of well characterized SAR genes are PR-1 , PR-2 and PR-5, with PR-1 expressed at the highest level with the lowest background.
• /so mutants jesion simulating disease
• cim Class I mutants form spontaneous lesions on the leaves, accumulated elevated concentrations of SA, high levels of PR-1 , PR-2 and PR-5 mRNA, and are resistant to fungal and bacterial pathogens (Dietrich et al., 1994; Weymann et al., 1995).
• cim mutants have all the characteristics of ted mutants except spontaneous lesions. That is, cim mutants are visibly phenotypically normal.
• plants that constitutively express SAR genes can be utilized in breeding programs to inco ⁇ orate constitutive expression of the SAR genes and resistance to pathogens into plant lines. Descendants for further crossing are selected based on expression of the SAR genes and disease resistance as well as for other characteristics important for production and quality according to methods well known to those skilled in the art of plant breeding. For example, because ted mutants display lesion formation and necrosis, dm mutants with their normal phenotypes are preferable for use in such breeding programs and in the method of the present invention, although ted mutants could be used if desired.
• a third route for obtaining immunomodulated plants is by transforming plants with an SAR gene, preferably a functional form of the NIM1 gene.
• NIM1 wild-type form of NIM1 (SEQ ID NO:1) gives rise to transgenic plants with a disease resistant phenotype. See, co-pending U.S. Patent Application Serial No. 08/880,179, inco ⁇ orated herein by reference.
• 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 expression of the NIM1 gene is at a level that is at least two-fold above the expression level of the NIM1 gene in wild-type plants and is more preferably at least tenfold above the wild-type expression level.
• Immunomodulated plants for use in the method of the present invention can also be created by recombinant expression of an altered form of the NIM1 gene, whereby the alteration of the NIM1 gene 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 ⁇ . See, co-pending PCT application "METHODS OF USING THE NIM1 GENE TO CONFER DISE ⁇ ASE RESISTANCE IN PLANTS" inco ⁇ orated herein by reference.
• the sequence of the NIM1 gene (SEQ ID NO:1) 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)). Pair- wise visual inspections between the NIM1 protein (SEQ ID NO:2) and 70 known ankyrin- containing proteins were carried out, and striking similarities were found to members of the IKB CC ciass of transcription regulators (Baeuerie and Baltimore 1996; Baldwin 1996).
• NIM1 protein shares significant homology with l ⁇ B ⁇ proteins from mouse, rat, and pig (SEQ ID NOs: 3, 4, and 5, 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 1), 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.
• there is homology between l ⁇ B ⁇ and NIM1 throughout the proteins with an overall similarity of 80%.
• 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 (Baeuerie 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 (Baeuerie and Baltimore, 1996).
• l ⁇ B ⁇ is phosphorylated at two serine residues (amino acids 32 and 36 of Mouse l ⁇ B ⁇ ). This programs ubiquitination at a double lysine (amino acids 21 and 22 of Mouse l ⁇ B ⁇ ).
• the NF- ⁇ B/l ⁇ B complex is routed through the proteosome where l ⁇ B ⁇ is degraded and NF- ⁇ B is released to the nucleus.
• NIM1 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 1). In contrast to l ⁇ B ⁇ , where the double lysine is located about 15 amino acids toward the N-terminus of the protein, in NIM1 a double lysine is located about 40 amino acids toward the C-terminal end. Furthermore, a high degree of homology exists between NIM1 and l ⁇ B ⁇ in the serine/threonine rich carboxy terminal region which has been shown to be important in basal turnover rate (Sun etal., Mol. Cell. Biol. 16, 1058-1065 (1996)). According to the present invention based on the analysis of structural homology and the presence of elements known to be important for l ⁇ B ⁇ function, NIM1 is expected to function like the l ⁇ B ⁇ , 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 (l ⁇ B homolog) or less phosphorylation of the NIM1 gene product (l ⁇ B 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.
• 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 niml mutant plants in that the plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and therefore a CIM phenotype; i.e, the transgenic plants are immunomodulated. 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.
• 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 (SEQ ID NO:2).
• 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 (SEQ ID NO:2).
• NIM1 Arabidopsis NIM1 amino acid sequence
• N-terminal/C-ter minal Deletion Chimera and Ankyrin Domains Altered forms of the NIM1 gene product may also be produced as a result of C- terminal and N-terminai segment deletions or chimeras.
• constructs comprising the ankyrin domains from the NIM 1 gene are provided.
• Immunomodulated plants for use in the method of the present invention can also be created by recombinant expression of various SAR genes such as those described in Ward et al. (1991 ). See, for example, U.S. Patent No. 5,614,395, which describes disease resistant plants created by overexpression of one or more PR-protein genes. Although it refers to recombinant expression of forms of the NIM1 gene particularly, the section below entitled “Recombinant DNA Technology” sets forth protocols that may also be used to recombinantly express other SAR genes such as PR-protein genes in transgenic plants at higher-than-wild-type levels.
• the wild-type or altered form of the NIM1 gene conferring disease resistance to plants by enhancing SAR gene expression can be incorporated into plant cells using conventional recombinant DNA technology. Generally, this involves inserting DNA molecule encoding the selected form of NIM1 described above into an expression system to which the DNA molecule is heterologous (i.e., not normally present) using standard cloning procedures known in the art.
• the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
• a large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses.
• Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems ⁇ gt , ⁇ gtIO and Charon 4; plasmid vectors such as pBI121 , pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; and other similar systems.
• the components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed.
• the expression systems described herein can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells can be regenerated into whole plants such that the chosen form of the NIM1 gene activates SAR in the transgenic plants.
• Gene sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants.
• the expression cassettes may also comprise any f uther sequences required or selected for the expression of the transgene.
• Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
• These expression cassettes can then be easily transferred to the plant transformation vectors described infra. The following is a description of various components of typical expression cassettes.
• Promoters The selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used. The following are non-limiting examples of promoters that may be used in the expression cassettes.
• pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone.
• a derivative of pCGN1761 is constructed which has a modified polylinker which includes ⁇ /of/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 pu ⁇ ose of their expression under the control of the 35S promoter in transgenic plants.
• the entire 35S promoter-gene sequence-fm/ terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites 5 * to the promoter and Xbal, BamHI and Bgl! sites 3' to the terminator for transfer to transformation vectors such as those described below.
• the double 35S promoter fragment can be removed by 5' excision with Hindlll, Sphl, Sail, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter.
• modifications around the cloning sites can be made by the introduction of sequences that may enhance translation. This is particularly useful when overexpression is desired.
• pCGN1761ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Patent No. 5,639,949, inco ⁇ orated herein by reference.
• the double 35S promoter in pCGN1761ENX may be replaced with any other promoter of choice that will result in suitably high expression levels.
• one of the chemically regulatable promoters described in U.S. Patent No. 5,614,395 may replace the double 35S promoter.
• the promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector.
• the chemically/pathogen regulatable tobacco PR- la promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0332 104, which is hereby inco ⁇ orated by reference) and transferred to plasmid pCGN1761ENX (Uknes et al., 1992).
• pCIB1004 is cleaved with Ncol and the resultant 3* overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase.
• the fragment is then cleaved with Hindlll and the resultant PR-1 a promoter- containing fragment is gel purified and cloned into pCGN1761ENX from which the double 35S promoter has been removed.
• Various chemical regulators may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395.
• actin promoter is a good choice for a constitutive promoter.
• the promoter from the rice Actl gene has been cloned and characterized (McElroy et al. 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)). d. Constitutive Expression, the Ubiquitin Promoter:
• 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 0342925 (to Lubrizol) which is herein inco ⁇ orated by reference. Taylor et al. (Plant Cell Rep.
• a vector that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment.
• the ubiquitin promoter is suitable for gene expression 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.
• a suitable root promoter is described by de Framond (FEBS 290: 103-106 (1991)) and also in the published patent application EP 0452269, which is herein inco ⁇ orated by reference. This promoter is transferred to a suitable vector such as pCGN1761ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.
• Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described (e.g. Xu etal. Plant Molec. Biol. 22: 573-588 (1993), Logemann etal. 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 etal. Plant J. 3: 191-201 (1993)) and all are suitable for use with the instant invention. Logemann etal. describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu et al.
• Patent Application WO 93/07278, which is herein inco ⁇ orated 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.
• transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
• intron sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.
• Various 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. See also, the section entitled “Expression With Chloroplast Targeting" in Example 37 of U.S. Patent No. 5,639,949.
• cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular protein bodies has been described by Rogers et al. (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 etal. 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 etal. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann et al. 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 that has an expression pattern different to that of the promoter from which the targeting signal derives.
• transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors.
• the selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al.
• vectors Suitable for Agrobacterium Transformation Many vectors are available for transformation using Agrobacterium t ⁇ mefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.
• the binary vectors pCIB200 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 EcoR Vfragment 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//-digested pTJS75kan to create pCIB200 (see also EP 0332 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 Agrobacterium-med aXe ⁇ transformation, the RK2-derived triA function for mobilization between E. coli and other hosts, and the Or/Tand Or/Vfunctions also from RK2.
• the pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
• the binary vector pCIBIO contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and inco ⁇ orates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein etal. (Gene 53: 153-161 (1987)).
• Various derivatives of pCIBIO are constructed which inco ⁇ orate the gene for hygromycin B phosphotransf erase described by Gritz etal. (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 that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for transformation is described.
• pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin).
• the plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278.
• the 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull.
• the new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site.
• the resultant derivative of pCIB246 is designated pCIB3025.
• the GUS gene is then excised from pCIB3025 by digestion with Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060.
• the plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the bargene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson etal. EMBO J 6: 2519-2523 (1987)).
• This generated pCIB3064 which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, Hindlll, and BamHI.
• This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
• pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate.
• DFR E. coli gene dihydrofolate reductase
• PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adhl gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250-bp fragment encoding the E.
• coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which comprises the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generates pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Pstl and EcoRI sites available for the cloning of foreign substances.
• MCMV Maize Chlorotic Mottle Virus
• the coding sequence of interest Once the coding sequence of interest has been cloned into an expression system, it is transformed into a plant cell.
• Methods for transformation and regeneration of plants are well known in the art.
• Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles.
• bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants.
• Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-base ⁇ techniques and techniques that do not require Agrobacterium.
• Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski etal., EMBO J 3: 2717-2722 (1984), Potrykus etal., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., 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 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 w ' 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 etal. Plant Cell 5: 159-169 (1993)).
• the binary vector carrying the foreign DNA of interest e.g. pCIB200 or pCIB2001
• an appropriate Agrobacterium strain which may depend of the complement of w ' 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 etal. 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. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain.
• the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (H ⁇ fgen & Willmitzer, 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. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford inco ⁇ oration within the interior thereof.
• the vector can be introduced into the eel! by coating the particles with the vector containing the desired gene.
• the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
• Biologically active particles e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced
• Transformation of most monocotyledon species has now also become routine.
• Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co- transformation) and both these techniques are suitable for use with this invention.
• Co- transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable.
• a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher etal. Biotechnology 4: 1093-1096 (1986)).
• Patent Applications EP 0292 435, EP 0 392225, 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)
• WO 93/07278 and Koziel et al. describe techniques for the transformation of elite inbred lines of maize by particle bombardment.
• This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment. Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for apon ca-types and / ⁇ d/ca-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988); Shimamoto etal. Nature 338: 274-277 (1989); Datta etal. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou etal. Biotechnology 9: 957-962 (1991)).
• Patent Application EP 0332581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil etal. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology H: 1553-1558 (1993)) and Weeks etal. (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 embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35).
• regeneration medium MS + 1 mg/liter NAA, 5 mg/liter GA
• selection agent 10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35.
• GA7s sterile containers which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.
• the immunomodulated plants obtained via tranformation with an SAR gene such as a form of the NIM1 gene can be any of a wide variety of plant species, including those of monocots and dicots; however, the immunomodulated plants used in the method of the invention are preferably selected from the list of agronomicaliy important target crops set forth supra.
• the expression of the chosen form of the NIM1 gene in combination with other characteristics important for production and quality can be inco ⁇ orated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981 ); Crop Breeding, Wood D. R.
• 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 descendants plants.
• said maintenance and propagation make use of known agricultural methods developed to fit specific pu ⁇ oses 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.
• 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 descendants plants.
• Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.
• the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow one to dispense with said methods due to their modified genetic properties.
• new crops with improved stress tolerance can be obtained, which, due to their optimized genetic "equipmenf , yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.
• Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD*), metaiaxyl (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 a form of a NIM 1 gene 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.
• the inventive method of protecting plants involves two steps: first, activating the SAR pathway to provide an immunomodulated plant, and second, applying a microbicide to such immunomodulated plants to attain synergisticaliy enhanced disease resistance.
• any commercial or conventional microbicide may be applied to immunomodulated plants obtained through any of the three above-described routes.
• suitable microbicides include, but are not limited to, the following fungicides: 4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]mo ⁇ holine ("dimethomo ⁇ h"), (reference: C. Tomlin (Editor): The Pesticide Manual, 10th edition, Famhan, UK, 1994, pages 351-352); 5-methyl-1,2,4-triazolo[3,4-b][1,3]benzothiazole ("tricyclazole”), (reference: C.
• the chosen microbicide is preferably applied to the immunomodulated plants to be protected in the form of a composition with further carriers, surfactants or other application- promoting adjuvants customarily employed in formulation technology.
• Suitable carriers and adjuvants can be solid or liquid and are the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders or fertilizers.
• a preferred method of applying a microbicidal composition is application to the parts of the plants that are above the soil, especially to the leaves (foliar application).
• the frequency and rate of application depend upon the biological and climatic living conditions of the pathogen.
• the microbicide can, however, also penetrate the plant through the roots via the soil or via the water (systemic action) if the locus of the plant is impregnated with a liquid formulation (e.g. in rice culture) or if the microbicide is introduced in solid form into the soil, e.g. in the form of granules (soil application).
• the microbicide can also be applied to the seeds (coating), either by impregnating the tubers or grains with a liquid formulation of the microbicide, or by coating them with an already combined wet or dry formulation.
• coating either by impregnating the tubers or grains with a liquid formulation of the microbicide, or by coating them with an already combined wet or dry formulation.
• other methods of application to plants are possible, for example treatment directed at the buds or the fruit trusses.
• the microbicide may be used in unmodified form or, preferably, together with the adjuvants conventionally employed in formulation technology, and is therefore formulated in known manner e.g. into emulsifiable concentrates, coatable pastes, directly sprayable or dilatable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granules, or by encapsulation in e.g. polymer substances.
• the methods of application such as spraying, atomising, dusting, scattering, coating or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.
• Advantageous rates of application of the microbicide are normally from 50 g to 2 kg a.i_ha, preferably from 100 g to 1000 g a.iVha, especially from 150 g to 700 g a.i7ha.
• the rates of application are from 0.5 g to 1000 g, preferably from 5 g to 100 g, a.i. per 100 kg of seed.
• the formulations are prepared in known manner, e.g. by homogeneously mixing and/or grinding the microbicide with extenders, e.g. solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).
• Suitable solvents are: aromatic hydrocarbons, preferably the fractions containing 8 to 12 carbon atoms, e.g. xylene mixtures or substituted naphthalenes, phthalates, such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons, such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol, ethylene glycol monomethyl or monoethyl ether, ketones, such as cyclohexanone, strongly polar solvents, such as N-methyl-2-pyrroiidone, dimethyl sulfoxide or dimethylformamide, as well as vegetable oils or epoxidised vegetable oils, such as epoxidised coconut oil or soybean oil; or water.
• aromatic hydrocarbons preferably the fractions containing 8 to 12 carbon atoms, e.g. xylene mixtures or substituted naphthalenes,
• the solid carriers used are normally natural mineral fillers, such as calcite, talcum, kaolin, montmorillonite or attapulgite.
• calcite talcum
• kaolin kaolin
• montmorillonite attapulgite
• highly dispersed silicic acid or highly dispersed absorbent polymers e.g., calcite, talcum, kaolin, montmorillonite or attapulgite.
• Suitable granulated adso ⁇ tive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite, and suitable nonsorbent carriers are, for example, calcite or sand.
• pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverised plant residues.
• suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties.
• surfactants will also be understood as comprising mixtures of surfactants.
• Particularly advantageous application-promoting adjuvants are also natural or synthetic phospholipids of the cephalin and lecithin series, e.g. phosphatidylethanolamine, phos- phatidylserine, phosphatidylglycerol and lysolecithin.
• phospholipids of the cephalin and lecithin series e.g. phosphatidylethanolamine, phos- phatidylserine, phosphatidylglycerol and lysolecithin.
• the agrochemical compositions generally comprise 0.1 to 99 %, preferably 0.1 to 95 %, active microbicidal ingredient, 99.9 to 1 %, preferably 99.9 to 5 %, of a solid or liquid adjuvant and 0 to 25 %, preferably 0.1 to 25 %, of a surfactant.
• the microbicide may altemately be a chemical inducer of SAR (plant activating microbicide) such as a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound, which are described in U.S. Patent Nos. 5,523,311 and 5,614,395.
• SAR plant activating microbicide
• a benzothiadiazole compound such as a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound
• two methods of immunomodulation are concurrently employed.
• transgenic immunomodulated plants overexpressing NIM1 responded much faster and to much lower doses of BTH, as shewn by PR-1 gene expression and resistance to P. parasitica, than wild-type plants. See, Example 35 and the Northern blots in Figure 3.
• Synergisticaliy enhanced disease resistance in NIM1- overexpressors can be achieved with only 10 ⁇ M BTH application, a concentration normally insufficient for any efficacy at all.
• Normally phytotoxic or otherwise undesirable concentrations of SAR-inducing chemicals can be avoided by taking advantage of this synergy.
• one can take advantage of the alteration of the time-course of SAR activation that occurs when SAR-inducing chemicals are applied to already- immunomodulated plants such as NIM 7-overexpressors.
• economic gains can be realized as a result of the decreased quantity of SAR-inducing chemicals required to provide a given level of protection to plants.
• both a conventional microbicide and a plant activating microbicide may be applied to immunomodulated plants obtained through either a selective breeding route or a genetic engineering route. This results in an even higher level of synergistic disease resistance compared to the level of disease resistance obtained through immunomodulation alone, through immunomodulation plus only one type of microbicide, or through the simultaneous application of both types of microbicides (conventional and plant activating). See, for example, Table 35 in Example 19.
• 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 (1993). Plants are watered, allowed to drain well, and then inoculated by applying 10 ml of a sporangium suspension (300 sporangia/ml) to the soil. Inoculated plants are kept in a greenhouse maintained at 23-25°C day temperature, and 20-22°C night temperature.
• a spore suspension of Cercospora nicotianae (ATCC #18366) (100,000-150,000 spores per ml) is sprayed to imminent run-off onto the surface of the leaves.
• the plants are maintained in 100% humidity for five days. Thereafter the plants are misted with water 5-10 times per day.
• Six individual plants are evaluated at each time point. Cercospora nicotianae is rated on a % leaf area showing disease symptoms basis.
• a T-test (LSD) is conducted on the evaluations for each day and the groupings are indicated after the Mean disease rating value. Values followed by the same letter on that day of evaluation are not statistically significantly different.
• Assays for resistance to Peronospora parasitica are performed on plants as described in Uknes et al, (1993). Plants are inoculated with a 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 etal., Trans. Br. Mycol. Soc. 74: 329-333 (1980)) for microscopic examination.
• FIGURE 1 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 h B ⁇ 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.
• the five ankyrin repeats in l ⁇ B ⁇ are indicated by the dashed lines under the sequence. Amino acids are numbered relative to the NIM1 protein with gaps introduced where appropriate. Plus signs (+) are placed above the
• FIGURE 2 is an amino acid sequence comparison of regions of the NIM1 protein (numbers correspond to amino acid positions in SEQ ID NO:2) and rice EST protein products (SEQ ID NOs: 17-24).
• FIGURE 3 presents the results of Northern analysis showing the time course of PR-1 gene expression in wild-type and ⁇ //Mf-overexpressing lines following treatment with water or BTH.
• RNA was prepared from treated plants and analyzed as described in the Examples.
• Ws is the wild-type Arabidopsis thaliana Ws ecotype.
• 3A”, “5B”, “6E”, and “7C” are individual NIM f-overexpressing plant lines produced according to Example 21.
• “0 BTH” is water treatment;
• 10 BTH is 10 ⁇ M BTH treatment;
• 100 BTH is 100 ⁇ M BTH treatment.
• “0” is day zero control samples;
• “1", "3", and "5" are samples at days 1 , 3, and 5.
• SEQ ID NO:1 is a 5655-bp genomic sequence comprising the coding region of the wild-type
• SEQ ID NO:2 is the amino acid sequence of the wild-type Arabidopsis thaliana NIM1 protein encoded by the coding region of SEQ ID NO:1.
• SEQ ID NO:3 is the mouse l ⁇ B ⁇ amino acid sequence from Figure 1.
• SEQ ID NO:4 is the rat hcB ⁇ amino acid sequence from Figure 1.
• SEQ ID NO:5 is the pig l ⁇ B ⁇ amino acid sequence from Figure 1.
• SEQ ID NO:6 is the cDNA sequence of the Arabidopsis thaliana NIM1 gene.
• SEQ ID NO's:7 and 8 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:9 and 10 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:11 and 12 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:13 and 14 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:15 and 16 are the DNA coding sequence and encoded amino acid sequence, respectively, of the ankyrin domain of NIML
• SEQ ID NO:17 is the Rice-1 AA sequence 33-155 from Figure 2.
• SEQ ID NO:18 is the Rice-1 AA sequence 215-328 from Figure 2.
• SEQ ID NO:19 is the Rice-2 AA sequence 33-155 from Figure 2.
• SEQ ID NO:20 is the Rice-2 AA sequence 208-288 from Figure 2.
• SEQ ID NO:21 is the Rice-3 AA sequence 33-155 from Figure 2.
• SEQ ID NO:22 is the Rice-3 AA sequence 208-288 from Figure 2.
• SEQ ID NO:23 is the Rice-4 AA sequence 33-155 from Figure 2.
• SEQ ID NO:24 is the Rice-4 AA sequence 215-271 from Figure 2.
• SEQ ID NOs:25 through 32 are oligonucleotide primers.
• 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 inco ⁇ orated 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). 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 etal., WO 94/12650 and Treco etal., WO 95/31560 which can be applied to increasing peroxidase activity in a monocot.
• regulatory regions ex. promoters
• Also included as recombinant DNA technology is the insertion of a peroxidase coding sequence lacking selected expression signals into a monocot and assaying the transgenic monocot plant for increased expression of peroxidase due to endogenous control sequences in the monocot. This would result in an increase in copy number of peroxidase coding sequences within the plant.
• the initial insertion of the recombinant DNA into the genome of the R° plant is not defined as being accomplished by traditional plant breeding methods but rather by technical methods as described herein. Following the initial insertion, transgenic descendants can be propagated using essentially traditional breeding methods.
• 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 Sequence 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. acd accelerated cell death mutant plant
• AFLP Amplified Fragment Length Polymo ⁇ hism avrRpt2: avirulence gene Rpt2, isolated from Pseudomonas syringae BAC: Bacterial Artificial Chromosome
• CIM Constitutive jMmunity phenotype (SAR is constitutively activated)
• dm constitutive immunity mutant plant
• cM centimorgans
• cprV constitutive expresser of PR genes mutant plant
• Col-O Arabidopsis ecotype Columbia
• Emwa Peronospora parasitica isolate compatible in the Ws-O ecotype of Arabidopsis
• 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 niml: mutant allele of NIM1, conferring disease susceptibility to the plant; also refers to mutant Arabidopsis thaliana plants having the niml mutant allele of NIM1
• SAR Systemic Acquired Resistance SAR-on: Immunomodulated plants in which SAR is activated, typically exhibiting greater- than-wild-type SAR gene expression and having a disease resistant phenotype
• SSLP Simple Sequence Length Polymo ⁇ hism
• SAR was induced in plants by application of a chemical inducer of SAR such as a benzothiadiazole.
• a chemical inducer of SAR such as a benzothiadiazole.
• conventional microbicides were applied to the plants. Plants were then subjected to disease pressure from various pathogens. The combination of both methods of combating pathogens (inducing chemical + microbicide) produced.
• SF synergy factor
• Example 2 Action Against Colletotrichum lagenarium On Cucumis sativus L.
• cucumber plants were sprayed with a spray mixture prepared from a wettable powder formulation of the test compound. After 3 to 4 days, the plants were infected with a spore suspension (1.0 x 10 5 spores/ml) of the fungus and incubated for 30 hours at high humidity and a temperature of 23°C. Incubation was then continued at normal humidity and 22°C to 23°C. Evaluation of protective action was made 7 to 10 days after infection and was based on fungus infestation.
• a spore suspension 1.0 x 10 5 spores/ml
• cucumber plants were treated by soil application with a spray mixture prepared from a wettable powder formulation of the test compound. After 3 to 4 days, the plants were infected with a spore suspension (1.5 x 10 s spores/ml) of the fungus and incubated for 30 hours at high humidity and a temperature of 23°C. Incubation was then continued at normal humidity and 22°C. Evaluation of protective action was made 7 to 10 days after infection and was based on fungus infestation.
• E ⁇ xample 3 Action against Cercospora nicotianae On Tobacco Plants Tobacco plants (6 weeks old) were sprayed with a formulated solution of the test compound (concentration: max. 0.02 % active ingredient). Four days after treatment, the plants were inoculated with a sporangia suspension of Cercospora nicotianae (150,000 spores/ml) and kept at high humidity for 4 to 5 days and then incubated further under a normal day/night sequence. Evaluation of the symptoms in the tests was based on the leaf surface infested with fungus.
• Rice plants about 2 weeks old were placed together with the soil around the roots in a container filled with spray mixture (max. 0.006 % active ingredient). 96 hours later, the rice plants were infected with a conidia suspension of the fungus. Fungus infestation was evaluated after incubating the infected plants for 5 days at 95-100 % relative humidity and about 24°C. Table 13
• Component I benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester
• Component II tricyclazole
• 7-day-old wheat plants were sprayed to drip point with a spray mixture prepared from a formulated active ingredient, or combination of active ingredients. After 4 days, the treated plants were infected with a conidia suspension of the fungus, and the treated plants were subsequently incubated for 2days at a relative atmospheric humidity of 90-100% and 20 C. 10 days post- infection, the fungus infestation was assessed.
• Component I benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester
• Component II propiconazole
• Component I benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester
• Component II propiconazole
• Component I benzo[1 ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester
• Component II cyprodinil
• Component I benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester
• Component II propiconazole
• Tomato plants on a 7m 2 plot were sprayed at 7-day intervals with a spray mixture prepared with a wettable powder of the active ingredient; in total 9 times. Infection was naturally. For evaluation, the leaf infested with the fungus was measured. The following results were obtained:
• Tomato plants cv. "Roter Gnom" were sprayed to drip point with a spray mixture prepared with the formulated active ingredient, or combination of active ingredients. After 4 days, the treated plants were sprayed with a sporangia suspension of the fungus and subsequently incubated in a cabinet for 2 days at 18-20 * C and a relative atmospheric humidity of 90-100%.5 days post-infection, the fungus infestation was assessed. The following results were obtained: Table 25
• Component I benzo[1 ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester
• Tobacco plants (6 weeks old) were sprayed with a formulated solution of the test compound. Four days after treatment, the plants were inoculated with a sporangia suspension of the fungus, kept at high humidity for 4 to 5 days and then incubated further under a normal day/ - night sequence. Evaluation of the symptoms in the tests was based on the leaf surface infested with fungus. The infestation of the untreated plants corresponded to 0 % action.
• Example 13 Action against Peronospora parasitica In Arabidopsis thaliana
• plants were inoculated with a Peronospora parasitica conidial suspension as described in Delaney etal. (1995). Ws plants were inoculated with the compatible P.
• Phosphor Imager olecular Dynamics, Sunnyvale, CA
• a high-throughput Northern blot screen was developed to identify mutant plants having high concentrations of PR-1 mRNA during normal growth, with the idea that these mutants also exhibit systemic acquired resistance.
• a number of mutants have been isolated using this screen and they have been shown to accumulate not only PR-1 but also PR-2 and PR-5 mRNAs (Lawton et al. (1993); Dietrich etal. (1994); and Weymann etal. (1995). These mutants also have elevated levels of SA and are resistant to pathogen infection, confirming that this approach can be used to isolate SAR signal transduction mutants.
• Isd mutants jesion simulating disease
• This class of mutants is also referred to as "cim Class I” as disclosed in WO 94/16077 the disclosure of which is hereby inco ⁇ orated by reference in its entirety.
• This Isd class (aka cim Class I) formed spontaneous lesions on the leaves, accumulated elevated concentrations of SA, high levels of PR-1 , PR-2 and PR-5 mRNA and was resistant to fungal and bacterial pathogens (Dietrich et al., 1994; Weymann et al., 1995).
• This second class (cim) corresponds to the "cim Class II" mutants discussed WO 94/16077.
• the cim3 mutant plant line described below falls into this cim class (cim Class II) and is a dominant mutation with wild-type appearance that expresses stable, elevated levels of SA, SAR gene mRNA and has broad spectrum disease resistance.
• Exampie 14 Isolation and Characterization of cim Mutants With Constitutive SAR Gene Expression
• EMS M2 mutagenized Arabidopsis plants were grown in Aracon trays (Lehle Seeds, Round Rock, TX) in sets of approximately 100. Plants were grown as described in Uknes et al., 1993, supra, with special attention given to avoid over-watering and pathogen infection. Briefly, Metro Mix 360 was saturated with water and autoclaved three times for 70 minutes in 10-liter batches. The potting mix was stirred thoroughly in between each autoclaving. Seeds were surface sterilized in 20% Clorox for 5 minutes and washed with seven changes of sterile water before sowing. Planted seeds were vernalized for 3-4 days followed by growth in chambers with a 9 hour day and 15 hour night at 22°C.
• RNA was isolated using a rapid, mini-RNA preparation (Verwoerd et al. (1989) Nuc. Acid Res. 17, 2362).
• PR-1 gene expression was analyzed by Northern blot analysis (Lagrimini et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7542-7546; Ward et al., 1991).
• Each set of plants also contained a non- treated A. thaliana Col-O and a 2-day INA-treated (0.25 mg/ml) control. All plants were maintained as described in Weymann et al., (1995).
• putative mutants accumulating elevated levels of PR-1 mRNA were identified. Following descendants testing, five were chosen for further characterization. Putative cim mutants displayed elevated SAR gene expression in the absence of pathogen or inducing treatment. Descendants testing of the putative cim mutants confirmed that constitutive PR-1 expression was heritable. Of the cim mutants, two, cim2 and cim3, with the highest, most stable expression of PR-1 were characterized further.
• cim3 When initially identified, cim3 also appeared slightly dwarfed with thin, distorted leaves. However, F2 plants resulting from a cross with ecotype Col-gl1 retained high SAR gene expression and could not be distinguished from wild-type plants. This suggested that the dwarfed, distorted-leaf phenotype was caused by an independent mutation that was not associated with constitutive SAR gene expression. The cim3 mutant phenotype was also observed when plants were grown in sterile conditions confirming that PR-1 mRNA accumulation was not caused by a pathogen.
• SAR gene expression varied between the descendants, but were always more than 10-fold higher than the untreated control and similar to the levels obtained following a resistance-inducing INA (0.25 mg/ml) treatment of wild-type plants.
• the glucose conjugate of SA was 13.1 -fold higher in cim3 than in non-infected wild-type Arabidopsis (4519 ⁇ 473 vs. 344 ⁇ 58 ng g fresh weight, respectively). These increased levels of SA and SAG are comparable to the levels that have been reported for either pathogen-infected tissue or the cpr mutant.
• cim3 was evaluated for resistance to Peronospora parasitica (NoCo2), the causal agent of downy mildew disease of Arabidopsis. Thirty cim3 (confirmed by PR-1 RNA expression) and thirty control plants (ecotype Columbia), each about 4 weeks old, were inoculated with P. parasitica, as described in Uknes, etal. 1992, supra. Seven days later, plants were analyzed for sporulation and stained with trypan blue to visualize fungal structures, as described in Keogh et al. (1980) Trans. Br. My ⁇ l. Soc.74, 329-333, and in Koch and Slusarenko (1990) Plant Cell2, 437-445.
• Wild-type (Col-O) plants support the growth of hyphae, conidia, and oospores, while wild type plants treated with INA (0.25 mg/ml) and cim3 plants showed no fungal growth.
• the cim3-mediated resistance is typically seen as a small group of dead cells at the site of pathogen infection. This type of resistance is similar to that seen in Isd mutants (Dietrich et al., 1994, supra; Weymann et al., 1995, supra), or in wild-type plants in which SAR has been induced (Uknes et al., 1992, supra). Occasionally, intermediate resistance phenotypes were observed, including trailing necrosis in the wake of the hyphal tip in cim3 plants.
• cultures of Pseudomonas syringae pv. tomato strain DC3000 were grown on King's B media (agar plates or liquid) plus rifampicin (50 ⁇ g/ ml) at 28°C (Walen et al. (1991) Plant Cell 3, 49-59).
• An overnight culture was diluted and resuspended in 10 mM MgCI 2 to a density of 2-5 x 10 s cells per ml and injected into Arabidopsis leaves. Injections were carried out by creating a small hole with a 28 gauge needle midway up the leaf and then injecting approximately 250 ⁇ l of the diluted bacterial solution with a 1 cc syringe.
• INA 2,6-Dichloroisonicotinic acid
• Col-nahG Arabidopsis carries a dominant kanamycin resistance gene in addition to the dominant nahG gene, so Col- nahG was used as the pollen donor.
• F1 seed was hydrated in water for 30 minutes and then surface sterilized in 10% Clorox, .05% Tween 20 for five minutes and washed thoroughly in sterile water.
• GM Murashige and Skoog medium containing 10gL sucrose buffered with 0.5 g/L 2-(N-mo ⁇ holino) ethanesulfonic acid, pH 5.7 with KOH
• Kanamycin resistant F t plants were transferred to soil after 18 days. The presence of the nahG gene and PR-1 expression was confirmed in all experiments by Northern blot analysis.
• Example 19 Synergistic Disease-Resistance Attained by Applying Microbicide and/or BTH to cim Mutants
• the data presented in Table 31 demonstrates that synergy is also achieved by applying a chemical inducer of systemic acquired resistance such as BTH to an immunomodulated (SAR-on) cim3 plant.
• a chemical inducer of systemic acquired resistance such as BTH
• SAR-on systemic acquired resistance
• a 0.03 mM concentration of BTH is normally insufficient to confer effective disease resistance, providing only 20.8% fungal growth inhibition.
• this normally inadequate concentration of BTH provided 73.1% fungal growth inhibition, which was nearly as high as the level of inhibition provided by 0.1 mM BTH, the recommended concentration for efficacy.
• the synergy factor of 2.2 calculated from the data in Table 31 clearly demonstrates the synergistic effect achieved by applying BTH to a plant that is already immunomodulated through other means.
• the NIM1 gene is a key component of the systemic acquired resistance (SAR) pathway in plants (Ryals et a/.,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 has been determined by molecular biological analysis of the genome of mutant plants known to carry the mutant niml 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:1).
• the wild-type NIM1 gene product (SEQ ID NO:2) 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 immunomodulated plants with a constitutive immunity (CIM) phenotype and therefore confers disease resistance in transgenic plants, 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. See, co-pending U.S. Application Serial No. 08/880,179, inco ⁇ orated herein by reference.
• NIM1 gene product has been shown to be a structural homologue of the mammalian signal transduction factor l ⁇ B subclass ⁇ (Ryals et al., 1997). Mutations of IKB have been described that act as super-repressors or dominant-negatives of the NF- ⁇ B/l ⁇ B regulation scheme. Thus, certain altered forms of NIM1 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 niml mutant; i.e., immunomodulated plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and a CIM phenotype. See, co-pending PCT application "METHODS OF USING THE NIM1 GENE TO CONFER DISEASE RESISTANCE IN PLANTS" inco ⁇ orated herein by reference.
• Cosmid D7 (deposited with the ATCC on September 25, 1996, as ATCC 97736) was generated from a clone spanning the NIM1 gene region and therefore includes the wild-type NIM1 gene (SEQ ID NO:1).
• Cosmid E1 was also generated from a clone spanning the NIM1 gene region and therefore also includes the wild-type NIM1 gene (SEQ ID NO:1).
• Cosmids D7 and E1 were moved into Agrobacterium tumefaciens AGL-1 through conjugative transfer in a tri-parental mating with helper strain HB101 (pRK2013) as described in the U.S. Patent Application No. 08/880,179.
• Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 300mM 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 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 niml plants were treated in the same way to serve as controls for each experiment.
• Peronospora parasitica isolated EmWa
• 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: 1 - 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: 6) 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 and IkB A multiple sequence alignment between the protein gene products of NIM1 and IkB was performed by which it was determined that the NIM 1 gene product is a homolog of l ⁇ B ⁇ ( Figure 1). 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:2
• mouse l ⁇ B ⁇ SEQ ID NO:3, GenBank Accession #: 1022734
• rat l ⁇ B ⁇ SEQ ID NO:4, GenBank accession Nos. 57674 and X63594; Tewari et al., Nucleic Acids Res. 20, 607 (1992)
• pig l ⁇ B ⁇ SEQ ID NO:5, GenBank accession No. Z21968; de Martin et al., EMBO J. 12, 2773-2779 (1993); GenBank accession No. 517193, de Martin et al., Gene 152, 253-255 (1995)).
• NIM1 contains 2 serines at amino acid positions 55 and 59; the serine at position 59 is in a context (D/ExxxxS) and position (N-terminal) consistent with a role in phosphorylation-dependent, ubiquitin-mediated, inducible degradation. All l ⁇ B ⁇ 's 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 IKBS.
• This altered form of l ⁇ B ⁇ 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:6, positions I92-226): 5'- CAA CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3' (SEQ ID NO:25) and 5'- CAT CCG GCG CGT CAA AGA CGG CTT CGA AGC TGT TG-3' (SEQ ID NO:26), 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:7 and 8 show the DNA coding sequence and encoded amino acid sequence, respectively, of this altered form of the NIM1 gene.
• a 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:6: 418 to 2011): 5'-gg aat tca-ATG GATTCG GTT GTG ACT GTT TTG-3' (SEQ ID NO:27) and 5'-gga att cTA CAA ATC TGT ATA CCA TTG G-3' (SEQ ID NO:28) 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 MgCla 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 pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal site of pCIB200.
• SEQ ID NO's:9 and 10 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM 1 gene having an N-terminal amino acid deletion.
• 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: 35x (94°C 30 sec: 52°C 30 sec: 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 Xbal and ligated into the Xbal site of dephosphorylated pCIB200.
• SEQ ID NO's:11 and 12 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.
• NIM 1 An N-terminal and C-terminal deletion form of NIM 1 is generated using a unique Kpnl restriction site at position 819 (SEQ ID NO:6).
• the N-terminal deletion form (Example 25) is restriction endonuclease digested with EcoRI/Kpn I and the 415 bp fragment corresponding to the modified N-terminus is recovered by gel electrophoresis.
• the C-terminal deletion form (Example 26) 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 24) fused to the C-terminal deletion (Example 26).
• 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:13 and 14 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.
• NIM1 exhibits homology to ankyrin motifs at approximately amino acids 103-362.
• the DNA sequence encoding the putative ankyrin domains (SEQ ID NO:1: 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:6: 349- 1128) using the following primers: 5'- ⁇ oaattcaATGGACTCCAACAACACCGCCGC-3' (SEQ ID NO:31) and 5'-ggaattcJCAACCTTCCAAAGTTGCTTCTGATG-3' (SEQ ID NO:32).
• 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 Xbal site of dephosphorylated pCIB200.
• SEQ ID NO's:15 and 16 show the DNA coding sequence and encoded amino acid sequence, respectively, of the ankyrin domain of NIML
• a 4407 bp Hindlll/BamHI fragment (SEQ ID NO:1: bases 1249-5655) and/or a 5655 bp EcoRV/BamHI fragment (SEQ ID NO:1: bases 1-5655) containing the NIM1 promoter and gene is used for the creation of the altered NIM1 forms in Examples 24-28 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 24-28 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-O 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 plantiets that are transformed 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 31 Assessment of CIM Phenotype in Plants Transformed with the Wild-Type NIM1 Gene or an Altered Form of the NIM1 Gene
• RNA blot analysis A leaf from each primary transformant is harvested, 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-O and Col-O 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 Iactophenol 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 Iactophenol 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 ndrl 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 ndrl 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 homologs are obtainable that hybridize under moderately stringent conditions either to the entire NIM1 gene from Arabidopsis or, preferably, to an oligonucleotide probe derived from the Arabidopsis NIM1 gene that comprises a contiguous portion of its coding sequence at least approximately 10 nucleotides in length.
• Factors that affect the stability of hybrids determine the stringency of the hybridization.
• One such factor is the melting temperature T m , which can be easily calculated according to the formula provided in DNA PROBES, George H. Keller and Mark M. Manak , Macmillan Publishers Ltd, 1993, Section one: Molecular Hybridization Technology; page 8 ff.
• the preferred hybridization temperature is in the range of about 25°C below the calculated melting temperature T m , 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 .
• NIM1 cDNA SEQ ID NO:6
• 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 E ⁇ xample 33. 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)).
• 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 NaPO 4 , pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 M sodium chloride) at 55°C for 18-24h] with ⁇ 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 NaCl, 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 can be used to isolate homologues.
• EST expressed sequence tags
• ESTs rice expressed sequence tags
• a multiple sequence alignment was constructed using Clustal V (Higgins, Desmond G. and Paul M. Sha ⁇ (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).
• Certain regions of the NIM1 protein are homologous in amino acid sequence to 4 different rice cDNA protein products. The homologies were identified using the NIM 1 sequences in a GenBank BLAST search.
• NIM1 and the rice cDNA products are shown in Figure 2 (See also, SEQ ID NO:2 and SEQ ID NO's:17-24).
• 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 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. 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-O 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 failing 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 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.
• Example 34 Synergistic Disease Resistance Attained by Applying A Conventional Microbicide to Transgenic Plants Overexpressing NIM1
• the plant lines used in this example (6E and 7C) were generated from transformation of wild-type Arabidopsis thaliana plants (ecotype Ws) with the BamHI-Hindlll NIM1 genomic fragment (SEQ ID NO:1 - bases 1249-5655), as described above in Example 21.
• the fungicides metalaxyl, fosetyl, and copper hydroxide, formulated as 25%, 80%, and 70% active ingredient (ai), respectively, with a wettable powder carrier, were applied as fine mist to leaves of three week-old transgenic Ws plants constitutively expressing the NIM 1 gene.
• the wettable powder alone was applied as a control.
• plants were inoculated with a Peronospora parasitica isolate Emwa conidial suspension (1-2 x 10 5 spores/ml), as described in Delaney etal. (1995). Following inoculation, plants were covered to maintain high humidity and were placed in a Percival growth chamber at 17°C with a 14-hr day/10-hr night cycle (Uknes et al., 1993). Tissue was harvested 8 days after inoculation.
• NIM1 wt wild-type Ws
• the synergy factor of 5.5 calculated from these data clearly demonstrates the synergistic effect achieved by applying a microbicide to an immunomodulated (SAR-on) plant.
• SAR-on immunomodulated
• EExample 35 Synergistic Disease Resistance Attained by Applying A Chemical Inducer Of SAR to Transgenic Plants Overexpressing NIM1 Transgenic plants containing the NIM1 genomic DNA fragment under its own promoter (Example 21) were also analyzed for response to different concentrations of BTH relative to the wild-type Ws line. Seeds from each line were sown and grown as previously described. At approximately three weeks post-planting, leaf samples were harvested from each line (day 0 controls), and the remaining plants were treated with H 2 0, 10 ⁇ M BTH, or 100 ⁇ M BTH. Additional samples were harvested at days 1, 3, and 5 following treatment. After harvesting the day 3 samples, a subset of plants for each line was removed and treated with Peronospora parasitica isolate Emwa as described above. RNA was prepared from the harvested tissue and Northern analysis was performed using the Arabidopsis PR-1 gene probe. Plants were scored for fungal resistance 8 days following infection.
• 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 TTCTTCCTTT AACCTTTTGT AACTCGAATT ACACAGCAAG TTAGTTTCAG GTCTAGAGAT 5286
• 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
• ORGANISM Arabidopsis thaliana
• GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390 Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu 105 110 115 ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438 He Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130
• GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC 726 Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu He He Val 215 220 225
• GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390 Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu 105 110 115
• GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC 726 Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu He He Val 215 220 225

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